Secondary literature sources for RA

The following references were automatically generated.

Song C et al.
Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras.
J Biol Chem. 2001; 276: 2752-7
Display abstract
Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes beta, gamma, and delta, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLCepsilon, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLCepsilon specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for Ha-Ras is estimated to be approximately 40 nm, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLCepsilon induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLCepsilon is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLCepsilon is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLCepsilon.

Barbieri MA, Gumusboga A, Roberts RL, Stahl PD
Measurement of Rab5 protein kinase B/akt and regulation of ras-activated endocytosis.
Methods Enzymol. 2001; 329: 145-56

Boettner B, Herrmann C, Van Aelst L
Ras and Rap1 interaction with AF-6 effector target.
Methods Enzymol. 2001; 332: 151-68

Becker CF, Hunter CL, Seidel RP, Kent SB, Goody RS, Engelhard M
A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface.
Chem Biol. 2001; 8: 243-52
Display abstract
BACKGROUND: The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras.GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras.GTP but not to Ras.GDP. RESULTS: The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras.GTP binding surface. Additionally, a C-terminal tag consisting of His6 was introduced. The Kd values for binding of the site-specifically modified proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras.GTP in the 100 nM range. Likewise, Ras.GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras.GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. CONCLUSIONS: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

Riese MJ, Wittinghofer A, Barbieri JT
ADP ribosylation of Arg41 of Rap by ExoS inhibits the ability of Rap to interact with its guanine nucleotide exchange factor, C3G.
Biochemistry. 2001; 40: 3289-94
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ExoS is a bifunctional type III cytotoxin that is secreted by Pseudomonas aeruginosa. The N-terminal domain comprises a RhoGAP activity, while the C-terminal domain comprises a ADP-ribosyltransferase activity. Previous studies showed that ExoS ADP ribosylated Ras at Arg41 which interfered with the ability of Ras to interact with its guanine nucleotide exchange factor. Rap and Ras share considerable primary amino acid homology, including Arg41. In this study, we report that ExoS ADP ribosylates Rap1b at Arg41 and that ADP ribosylation of Arg41 inhibits the ability of C3G to stimulate guanine nucleotide exchange. The mechanism responsible for this inhibition is one in which ADP-ribosylated Rap binds inefficiently to C3G, relative to wild type Rap. This identifies a second member of the Ras GTPase subfamily that can be ADP ribosylated by ExoS and indicates that ExoS can inhibit both Ras and Rap signaling pathways in eukaryotic cells.

Senga T, Iwamoto T, Kitamura T, Miyake Y, Hamaguchi M
JAK/STAT3-dependent activation of the RalGDS/Ral pathway in M1 mouse myeloid leukemia cells.
J Biol Chem. 2001; 276: 32678-81
Display abstract
The Ras-related GTPase (Ral) is converted to the GTP-bound form by Ral guanine nucleotide dissociation stimulator (RalGDS), a putative effector protein of Ras. Recently, it was proven that Ral regulates c-Src activity and subsequent phosphorylation of its substrate, STAT3. Here, we show that STAT3 inversely regulates activation of Ral through induction of expression of RalGDS. To identify new leukemia inhibitory factor-induced genes, we have performed representational difference analysis using M1 mouse myeloid leukemia cells and cloned RalGDS. The expression of RalGDS and subsequent activation of RalA were clearly suppressed by a dominant negative form of STAT3 and a JAK inhibitor, JAB/SOCS1/SSI-1, indicating that RalGDS/RalA signaling requires the activation of the JAK/STAT3 pathway. An experiment using a Ras inhibitor demonstrated that full activation of RalA also requires activation of Ras. These results suggest a novel cross-talk between JAK/STAT3 and the Ras/RalGDS/Ral signaling pathways through gp130.

Kelley GG, Reks SE, Ondrako JM, Smrcka AV
Phospholipase C(epsilon): a novel Ras effector.
EMBO J. 2001; 20: 743-54
Display abstract
Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.

Spoerner M, Herrmann C, Vetter IR, Kalbitzer HR, Wittinghofer A
Dynamic properties of the Ras switch I region and its importance for binding to effectors.
Proc Natl Acad Sci U S A. 2001; 98: 4944-9
Display abstract
We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)(.)GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. (31)P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. (31)P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

Rebhun JF, Chen H, Quilliam LA
Identification and characterization of a new family of guanine nucleotide exchange factors for the ras-related GTPase Ral.
J Biol Chem. 2000; 275: 13406-10
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Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner.

Chen JM, Rijhwani K, Friedman FK, Hyde MJ, Pincus MR
Identification, using molecular dynamics, of an effector domain of the ras-binding domain of the raf-p74 protein that is uniquely involved in oncogenic ras-p21 signaling.
J Protein Chem. 2000; 19: 545-51
Display abstract
By comparing the average structures, computed using molecular dynamics, of the ras-binding domain of raf (RBD) bound to activated wild-type ras-p21 and its homologous inhibitory protein, rap-1A, we formerly identified three domains of the RBD that changed conformation between the two complexes, residues 62-76, 97-110, and 111-121. We found that one synthetic peptide, corresponding to RBD residues 97-110, selectively inhibited oncogenic ras-p21-induced oocyte maturation. In this study, we performed molecular dynamics on the Val 12-ras-p21-RBD complex and compared its average structure with that for the wild-type protein. We find that there is a large displacement of a loop involving these residues when the structures of the two complexes are compared. This result corroborates our former finding that the RBD 97-110 peptide inhibits only signal transduction by oncogenic ras-p21 and suggests that oncogenic p21 uses this loop to interact with raf in a unique manner.

Shields JM, Pruitt K, McFall A, Shaub A, Der CJ
Understanding Ras: 'it ain't over 'til it's over'.
Trends Cell Biol. 2000; 10: 147-54
Display abstract
Since 1982, Ras has been the subject of intense research scrutiny, focused on determining the role of aberrant Ras function in human cancers and defining the mechanism by which Ras mediates its actions in normal and neoplastic cells. The long-term goal has been to develop antagonists of Ras as novel approaches for cancer treatment. Although impressive strides have been made in these endeavours, and our knowledge of Ras is quite extensive, it appears that we are at the beginning, rather than at the end, of fully understanding Ras function. This review highlights new issues that have further complicated our efforts to understand Ras.

Oertli B et al.
The effector loop and prenylation site of R-Ras are involved in the regulation of integrin function.
Oncogene. 2000; 19: 4961-9
Display abstract
The closely related small GTP-binding proteins H-Ras and R-Ras have opposing effects on the regulation of integrin cell adhesion receptors. To gain insight into the properties of R-Ras with respect to the regulation of integrin function and interactions with downstream effectors we performed an analysis of R-Ras variants containing mutations in the effector binding domain and C-terminal prenylation site. We found that the activation of the downstream effector PI 3-kinase was sensitive to mutations in the effector binding domain, as was the binding to the effectors, Ral-GDS, Raf-1 and the novel effector Nore1. Furthermore, specific mutations in the effector binding loop and C-terminal prenylation motif impaired the ability of R-Ras to regulate integrin function in CHO cells. However, the ability of the R-Ras effector loop mutants to bind, and activate known effectors did not correlate with their ability to regulate integrin function. Thus, the known R-Ras effectors are not critical for regulating integrin activation, at least in CHO cells. Consequently, these studies provide insight into the structural basis of the interactions between R-Ras and its candidate effectors and suggest the existence of novel mechanisms through which this GTPase could regulate cell adhesion.

Hernandez-Munoz I, Malumbres M, Leonardi P, Pellicer A
The Rgr oncogene (homologous to RalGDS) induces transformation and gene expression by activating Ras, Ral and Rho mediated pathways.
Oncogene. 2000; 19: 2745-57
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The effects of the 5'-truncated Rgr oncogene, a previously shown specific guanine exchange factor for Ral in vitro, in stimulating proliferation, cell transformation and gene expression were investigated. We have established TetRgr cell lines in which expression of Rgr can be inhibited by the presence of tetracycline in the medium. Using this system, we show that Rgr overexpressing cells are morphologically transformed and grow in a disorganized manner. At the transcriptional level, Rgr enhances the activity of the serum response element and c-Jun. Rgr induces phosphorylation of ERKs, p38 and JNK kinases, and increases the levels of the GTP-bound forms of Ral and Ras. Ras activation could account for the broad spectra of effects displayed by Rgr. The important role of these pathways is confirmed by experiments in which the transcriptional activation events can be blocked by dominant negative versions of Ras, Ral and Rho. Among all the Rgr-induced pathways, the Ras-Raf-MEK-ERK cascade is essential for the transforming properties of Rgr. Additional analysis has shown that the activation of this pathway by Rgr is not due to a feed back mechanism mediated by the Grb2 adaptor protein. Oncogene (2000).

Inoue K et al.
Pressure-induced local unfolding of the Ras binding domain of RalGDS.
Nat Struct Biol. 2000; 7: 547-50
Display abstract
The reliable prediction of the precise three-dimensional structure of proteins from their amino acid sequence is a major, still unresolved problem in biochemistry. Pressure is a parameter that controls folding/unfolding transitions of proteins through the volume change DeltaV of the protein-solvent system. By varying the pressure from 30 to 2,000 bar we detected using 15N/ 1H 2D NMR spectroscopy a unique equilibrium unfolding intermediate I in the Ras binding domain of the Ral guanine nucleotide dissociation stimulator (Ral GDS). It is characterized by a local melting of specific structural elements near hydrophobic cavities while the overall folded structure is maintained.

Ohba Y et al.
Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3.
J Biol Chem. 2000; 275: 20020-6
Display abstract
We studied the regulation of three closely related members of Ras family G proteins, R-Ras, TC21 (also known as R-Ras2), and M-Ras (R-Ras3). Guanine nucleotide exchange of R-Ras and TC21 was promoted by RasGRF, C3G, CalDAG-GEFI, CalDAG-GEFII (RasGRP), and CalDAG-GEFIII both in 293T cells and in vitro. By contrast, guanine nucleotide exchange of M-Ras was promoted by the guanine nucleotide exchange factors (GEFs) for the classical Ras (Ha-, K-, and N-), including mSos, RasGRF, CalDAG-GEFII, and CalDAG-GEFIII. GTPase-activating proteins (GAPs) for Ras, Gap1(m), p120 GAP, and NF-1 stimulated all of the R-Ras, TC21, and M-Ras proteins, whereas R-Ras GAP stimulated R-Ras and TC21 but not M-Ras. We did not find any remarkable difference in the subcellular localization of R-Ras, TC21, or M-Ras when these were expressed with a green fluorescent protein tag in 293T cells and MDCK cells. In conclusion, TC21 and R-Ras were regulated by the same GEFs and GAPs, whereas M-Ras was regulated as the classical Ras.

Esters H, Alexandrov K, Constantinescu AT, Goody RS, Scheidig AJ
High-resolution crystal structure of S. cerevisiae Ypt51(DeltaC15)-GppNHp, a small GTP-binding protein involved in regulation of endocytosis.
J Mol Biol. 2000; 298: 111-21
Display abstract
Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a central role in the coordination, activation and regulation of vesicle-mediated transport in eukaryotic cells. We present the 1.5 A high-resolution crystal structure of Ypt51 in its active, GppNHp-bound conformation. Ypt51 is an important regulator involved in the endocytic membrane traffic of Saccharomyces cerevisiae. The structure reveals small but significant structural differences compared with H-Ras p21. The effector loop and the catalytic loop are well defined and stabilized by extensive hydrophobic interactions. The switch I and switch II regions form a well-defined epitope for hypothetical effector protein binding. Sequence comparisons between the different isoforms Ypt51, Ypt52 and Ypt53 provide the first insights into determinants for specific effector binding and for fine-tuning of the intrinsic GTP-hydrolysis rate.

de Bruyn KM et al.
RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral.
J Biol Chem. 2000; 275: 29761-6
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Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.

Shao H, Andres DA
A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras.
J Biol Chem. 2000; 275: 26914-24
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The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (Ral GEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras.

Weber CK, Slupsky JR, Herrmann C, Schuler M, Rapp UR, Block C
Mitogenic signaling of Ras is regulated by differential interaction with Raf isozymes.
Oncogene. 2000; 19: 169-76
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In the mitogenic signaling cascade interaction of Ras with Raf represents a critical step for the regulation of cell growth and differentiation. The major effector of Ras, the serine/threonine kinase Raf exists as three isoforms with different tissue distributions. We demonstrate that transient transfection of oncogenic Ha-Ras leads to a preferential activation of endogenous c-Raf-1 in HEK 293 cells as opposed to A-Raf. In vitro binding studies using purified Ras binding domains of Raf as well as in vivo bindings tests with full length molecules reveals significantly lower binding affinities of A-Raf to Ha-Ras as compared to other Raf isoforms. The Ras-binding interface of c-Raf differs from A-Raf by a conservative Arg to Lys exchange at residue 59 or 22 respectively. Mutational analysis reveals that this residue represents a point of isozyme discrimination: c-Raf-R59K binds Ha-Ras weaker than the wildtype, likewise A-Raf-K22R increases its affinity to Ha-Ras in vivo and in vitro. Differential binding affinities are reflected in downstream signaling. Immunecomplex kinase assays reveal that Ha-Ras mediated Raf activation is decreased for c-Raf-R59K and increased for A-Raf-K22R when compared to the respective wildtype forms. Thus our observations introduce a new level of isoform discrimination in Ras/Raf signaling as a functional consequence of a conservative amino acid exchange in the Ras binding domains.

Fridman M et al.
Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras.
J Biol Chem. 2000; 275: 30363-71
Display abstract
A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.

Hart MJ, Roscoe W, Bollag G
Activation of Rho GEF activity by G alpha 13.
Methods Enzymol. 2000; 325: 61-71

Shao H, Kadono-Okuda K, Finlin BS, Andres DA
Biochemical characterization of the Ras-related GTPases Rit and Rin.
Arch Biochem Biophys. 1999; 371: 207-19
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We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.

Serebriiskii I, Khazak V, Golemis EA
A two-hybrid dual bait system to discriminate specificity of protein interactions.
J Biol Chem. 1999; 274: 17080-7
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Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.

Matsubara K, Kishida S, Matsuura Y, Kitayama H, Noda M, Kikuchi A
Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation.
Oncogene. 1999; 18: 1303-12
Display abstract
In COS cells, Ral GDP dissociation stimulator (RalGDS)-induced Ral activation was stimulated by RasG12V or a Rap1/Ras chimera in which the N-terminal region of Rap1 was ligated to the C-terminal region of Ras but not by Rap1G12V or a Ras/Rap1 chimera in which the N-terminal region of Ras was ligated to the C-terminal region of Rap1, although RalGDS interacted with these small GTP-binding proteins. When RasG12V, Ral and the Rap1/Ras chimera were individually expressed in NIH3T3 cells, they localized to the plasma membrane. Rap1Q63E and the Ras/Rap1 chimera were detected in the perinuclear region. When RalGDS was expressed alone, it was abundant in the cytoplasm. When coexpressed with RasG12V or the Rap1/Ras chimera, RalGDS was detected at the plasma membrane, whereas when coexpressed with Rap1Q63E or the Ras/Rap1 chimera, RalGDS was observed in the perinuclear region. RalGDS which was targeted to the plasma membrane by the addition of Ras farnesylation site (RalGDS-CAAX) activated Ral in the absence of RasG12V. Although RalGDS did not stimulate the dissociation of GDP from Ral in the absence of the GTP-bound form of Ras in a reconstitution assay using the liposomes, RalGDS-CAAX could stimulate it without Ras. RasG12V activated Raf-1 when they were coexpressed in Sf9 cells, whereas RasG12V did not affect the RalGDS activity. These results indicate that Ras recruits RalGDS to the plasma membrane and that the translocated RalGDS induces the activation of Ral, but that Rap1 does not activate Ral due to distinct subcellular localization.

Linnemann T et al.
Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily.
J Biol Chem. 1999; 274: 13556-62
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Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.

Liao Y et al.
RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans.
J Biol Chem. 1999; 274: 37815-20
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A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.

Nancy V, Wolthuis RM, de Tand MF, Janoueix-Lerosey I, Bos JL, de Gunzburg J
Identification and characterization of potential effector molecules of the Ras-related GTPase Rap2.
J Biol Chem. 1999; 274: 8737-45
Display abstract
In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.

Voice JK, Klemke RL, Le A, Jackson JH
Four human ras homologs differ in their abilities to activate Raf-1, induce transformation, and stimulate cell motility.
J Biol Chem. 1999; 274: 17164-70
Display abstract
Human cells contain four homologous Ras proteins, but it is unknown whether each of these Ras proteins participates in distinct signal transduction cascades or has different biological functions. To directly address these issues, we assessed the relative ability of constitutively active (G12V) versions of each of the four Ras homologs to activate the effector protein Raf-1 in vivo. In addition, we compared their relative abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell migration. We found a distinct hierarchy between the four Ras homologs in each of the parameters studied. The hierarchies were as follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>> N-Ras > Ha-Ras; for focus formation, Ha-Ras >/= Ki-Ras 4A >>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras 4A >/= N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras 4A = no migration. Our results indicate that the four Ras homologs significantly differ in their abilities to activate Raf-1 and induce distinctly different biological responses. These studies, in conjunction with our previous report that demonstrated that the Ras homologs can be differentially activated by upstream guanine nucleotide exchange factors (Jones, M. K., and Jackson, J. H. (1998) J. Biol. Chem. 273, 1782-1787), indicate that each of the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also suggest for the first time that the distinct and likely cooperative biological functions of the Ki-ras-encoded Ki-Ras 4A and Ki-Ras 4B proteins may help explain why constitutively activating mutations of Ki-ras, but not N-ras or Ha-ras, are frequently detected in human carcinomas.

Ichiba T et al.
Activation of C3G guanine nucleotide exchange factor for Rap1 by phosphorylation of tyrosine 504.
J Biol Chem. 1999; 274: 14376-81
Display abstract
C3G is a guanine nucleotide exchange factor for Rap1 and is activated by the expression of Crk adaptor proteins. We found that expression of CrkI in COS cells induced significant tyrosine phosphorylation of C3G. To understand the mechanism by which C3G is phosphorylated and activated by Crk, we constructed a series of deletion mutants. Deletion of the amino terminus of C3G to amino acid 61 did not remarkably affect either tyrosine phosphorylation or Crk-dependent activation of C3G. When C3G was truncated to amino acid 390, C3G was still phosphorylated on tyrosine but was not effectively activated by CrkI. Deletion of the amino terminus of C3G to amino acid 579 significantly reduced the Crk-dependent tyrosine phosphorylation of C3G and increased GTP-bound Rap1 irrespective of the presence of CrkI. We substituted all seven tyrosine residues in this region, amino acids 391-579, for phenylalanine for identification of the phosphorylation site. Among the substitution mutants, the C3G-Y504F mutant, in which tyrosine 504 was substituted by phenylalanine, was remarkably less activated and phosphorylated than the wild type. All the other substitution mutants were activated and tyrosyl-phosphorylated by the expression of CrkI. Thus, CrkI activates C3G by the phosphorylation of tyrosine 504, which represses the cis-acting negative regulatory domain outside the catalytic region.

Arava Y, Seger R, Walker MD
GRFbeta, a novel regulator of calcium signaling, is expressed in pancreatic beta cells and brain.
J Biol Chem. 1999; 274: 24449-52
Display abstract
By screening for genes expressed differentially in pancreatic beta cells, we have isolated a cDNA encoding GRFbeta, a novel 178-amino acid protein whose N terminus is identical to that of GRF1, a calcium-dependent guanine nucleotide exchange factor, and whose C terminus is unrelated to known proteins. We show that both GRF1 and GRFbeta are expressed selectively in beta cell lines, pancreatic islet cells and brain. Treatment of beta cell lines (betaTC1 and HIT) with calcium ionophore led to a significant elevation in activity of the Ras signal transduction pathway, as determined by phosphorylation of extracellular signal-related kinase (ERK). Transfection of beta cells with a plasmid encoding a dominant negative variant of GRF1 led to 70% reduction in ERK phosphorylation, consistent with a role for GRF1 in calcium-dependent Ras signaling in these cells. To examine the possible function of GRFbeta, cultured cells were transfected with a GRFbeta expression vector. This led to a significant reduction in both GRF1-dependent ERK phosphorylation and AP1-dependent reporter gene activity. The results suggest that GRF1 plays a role in mediating calcium-dependent signal transduction in beta cells and that GRFbeta represents a novel dominant negative modulator of Ras signaling.

de Rooij J, Boenink NM, van Triest M, Cool RH, Wittinghofer A, Bos JL
PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2.
J Biol Chem. 1999; 274: 38125-30
Display abstract
The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain.

Zeng J, Fridman M, Maruta H, Treutlein HR, Simonson T
Protein-protein recognition: an experimental and computational study of the R89K mutation in Raf and its effect on Ras binding.
Protein Sci. 1999; 8: 50-64
Display abstract
Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vivo. The binding to [gamma-35S]GTP-Ras of a fusion protein between the Raf-binding domain (RBD) of Raf and GST was reduced at least 175-fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9+/-1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein-protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 A away, namely residues 37-41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2-3 specific water molecules. This analysis will provide insights into the large experimental database of Ras-Raf mutations.

Azuma Y, Renault L, Garcia-Ranea JA, Valencia A, Nishimoto T, Wittinghofer A
Model of the ran-RCC1 interaction using biochemical and docking experiments.
J Mol Biol. 1999; 289: 1119-30
Display abstract
RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.

Wojcik J, Girault JA, Labesse G, Chomilier J, Mornon JP, Callebaut I
Sequence analysis identifies a ras-associating (RA)-like domain in the N-termini of band 4.1/JEF domains and in the Grb7/10/14 adapter family.
Biochem Biophys Res Commun. 1999; 259: 113-20
Display abstract
RA (RalGEF/AF6 or Ras-associating) domains are found in a wide variety of proteins, several of which are known to be Ras-GTP effectors. The three dimensional structure of the RA domain has been experimentally determined in Ral-guanine nucleotide exchange factor (Ral-GEF) and found to be similar to that of the Ras-binding domain of c-Raf1, in spite of a very low level of sequence identity. Using various approaches of sequence analysis, including automatic procedures such as BLAST2, profilescan, and hidden Markov models (HMM), as well as the bidimensional hydrophobic cluster analysis (HCA), here we found that a region with a similar structure is also present at the N-terminus of the band 4.1/JEF domain of KIAA0316 (a human cDNA open reading frame) and H09G03.2 (a related protein sequence predicted from C. elegans genome cloning), as well as in a particular class of adapter proteins including Grb7, Grb10, Grb14, MIG-10, and PRP48. Although the structural conservation of this motif does not necessarily imply a conservation of its ability to bind small GTPases of the Ras superfamily, several proteins with a band 4.1/JEF domain and adapters of the Grb7 group have close functional relationships with such small GTPases. Thus, our finding raises the intriguing possibility of a direct interaction between members of these two groups of proteins and Ras-like GTP-binding proteins.

Yamamoto T, Taya S, Kaibuchi K
Ras-induced transformation and signaling pathway.
J Biochem (Tokyo). 1999; 126: 799-803
Display abstract
Ras is a signal-transducing, guanine nucleotide-binding protein for various membrane receptors including tyrosine kinase receptors. Ras participates in the regulation of cell proliferation, differentiation, and morphology. Activated ras oncogenes have been identified in various forms of human cancer including epithelial carcinomas of the lung, colon, and pancreas. The cells of these cancers, as well as those that have been experimentally transformed by the activated ras gene, exhibit abnormal growth, morphological changes and alterations of cell adhesions. Although the main effector protein has been thought to be Raf serine/threonine kinase, research has revealed that the Ras-induced signaling pathway is mediated by multiple effector proteins and has the crosstalk with various factors containing other small GTPases. In this review, we summarize the involvement of each effector protein for Ras and the crosstalk with other small GTPases in Ras-induced transformation.

Bauer B et al.
Effector recognition by the small GTP-binding proteins Ras and Ral.
J Biol Chem. 1999; 274: 17763-70
Display abstract
The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

Sawamoto K et al.
Ectopic expression of constitutively activated Ral GTPase inhibits cell shape changes during Drosophila eye development.
Oncogene. 1999; 18: 1967-74
Display abstract
The small GTP-binding protein Ral is activated by RalGDS, one of the effector molecules for Ras. Active Ral binds to a GTPase activating protein for CDC42 and Rac. Although previous studies suggest a role for Ral in the regulation of CDC42 and Rac, which are involved in arranging the cytoskeleton, its in vivo function is largely unknown. To examine the effect of overexpressing Ral on development, transgenic Drosophila were generated that overexpress wild-type or mutated Ral during eye development. While wild-type Ral caused no developmental defects, expression of a constitutively activated protein resulted in a rough eye phenotype. Activated Ral did not affect cell fate determination in the larval eye discs but caused severe disruption of the ommatidial organization later in pupal development. Phalloidin staining showed that activated Ral perturbed the cytoskeletal structure and cell shape changes during pupal development. This phenotype is similar to that caused by RhoA overexpression. In addition, the phenotype was synergistically enhanced by the coexpression of RhoA. These results suggest that Ral functions to control the cytoskeletal structure required for cell shape changes during Drosophila development.

Tu Y, Wu C
Cloning, expression and characterization of a novel human Ras-related protein that is regulated by glucocorticoid hormone.
Biochim Biophys Acta. 1999; 1489: 452-6
Display abstract
Ras proteins are a family of guanine nucleotide (GDP and GTP)-binding proteins that play central roles in essential signal transduction pathways. We have isolated in a yeast two-hybrid screen a human cDNA encoding a new protein that is highly homologous (98% identical at the protein level) to mouse DexRas1, a member of the Ras superfamily. The human DexRas1 is expressed in a variety of tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, with the strongest expression in the heart. Using human fibrosarcoma HT-1080 cells as a model system, we show that the expression of human DexRas1 is stimulated by dexamethasone, suggesting a role of human DexRas1 in dexamethasone-induced alterations in cell morphology, growth and cell-extracellular matrix interactions.

Mello LV, van Aalten DM, Findlay JB
Dynamic properties of the guanine nucleotide binding protein alpha subunit and comparison of its guanosine triphosphate hydrolase domain with that of ras p21.
Biochemistry. 1998; 37: 3137-42
Display abstract
The dynamic properties of the alpha-subunit of bovine transducin (Galphat) were studied using molecular dynamics simulations and essential dynamics analyses. The helical domain of transducin seems to move toward the guanosine triphosphate hydrolase (GTPase) domain. Our studies suggest that this movement is facilitated by a hinge bending motion that is centered on residues Gly56 and Gly179 and that this motion may be involved in GDP release and GTP hydrolysis. The dynamic properties of the GTPase domain of Galphat-GDP were compared to those of ras p21 and reveal a significant degree of similarity, indicating common dynamic properties for an equivalent domain in two different proteins.

Li W, Melnick M, Perrimon N
Dual function of Ras in Raf activation.
Development. 1998; 125: 4999-5008
Display abstract
The small guanine nucleotide binding protein p21(Ras) plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21(Ras )in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21(Ras) (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.

Barnard D, Sun H, Baker L, Marshall MS
In vitro inhibition of Ras-Raf association by short peptides.
Biochem Biophys Res Commun. 1998; 247: 176-80
Display abstract
Seven amino acid peptides were tested as in vitro inhibitors of oncogenic Ras-Raf association. The sequences of these peptides were derived from the H-Ras effector region (amino acids 25 to 51) and the Ras binding domain of Raf-1 (amino acids 64 to 105). Eleven out of the twenty-one Ras 7-mers tested inhibited formation of the Ras-Raf complex by at least 20% at 100 microM. The most potent of these inhibitory peptides contained the effector residues 32 to 37 or 40 to 45. Of the Raf-1 peptides tested, only the 94-ECCAVFR-100 and 95-CCAVFRL-101 peptides were significant inhibitors of Ras-Raf binding. The 95-101 Raf peptide had an IC50 value of 7 microM and also inhibited Ras-RalGDS binding. Analysis of the 95-101 peptide showed that its inhibitory activity required at least one cysteine followed by several hydrophobic residues. Our results demonstrate the feasibility of using small molecules as inhibitors of Ras protein-protein interactions.

Kawasaki H et al.
A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia.
Proc Natl Acad Sci U S A. 1998; 95: 13278-83
Display abstract
Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082-1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

Pai EF
The alpha and beta of turning on a molecular switch.
Nat Struct Biol. 1998; 5: 259-63
Display abstract
The crystal structures of RCC1 and the Sec7 domain of human Arno, nucleotide exchange factors for the Ras-related GTPases Ran and ARF, reveal two very different folds, the former a seven-bladed beta-propeller, the latter a capped right-handed superhelix. Both are also unrelated to the folds of Mss4 and elongation factor Ts, nucleotide exchange factors for Rab and elongation factor Tu.

Ohnishi M, Yamawaki-Kataoka Y, Kariya K, Tamada M, Hu CD, Kataoka T
Selective inhibition of Ras interaction with its particular effector by synthetic peptides corresponding to the Ras effector region.
J Biol Chem. 1998; 273: 10210-5
Display abstract
Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17-44 competitively inhibited Ras association with all the three effectors at the Ki values of 1 approximately 10 microM, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function.

Shirouzu M et al.
Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS.
J Biol Chem. 1998; 273: 7737-42
Display abstract
The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.

Exton JH
Small GTPases minireview series.
J Biol Chem. 1998; 273: 19923-19923

Esser D, Bauer B, Wolthuis RM, Wittinghofer A, Cool RH, Bayer P
Structure determination of the Ras-binding domain of the Ral-specific guanine nucleotide exchange factor Rlf.
Biochemistry. 1998; 37: 13453-62
Display abstract
Ral-specific guanine nucleotide exchange factors RalGDS, Rgl, and Rlf have been suggested to function as intermediates between Ras and Ral pathways by being able to bind Ras proteins through their C-terminal Ras-binding domains (RBD). The RBDs of RalGDS and of the Ser/Thr kinase c-Raf-1 have been shown to have the same tertiary structure. In contrast to the RBDs of Raf and RalGDS, which bind either Ras or Rap with high affinity, Rlf-RBD has a similar affinity for both GTP-binding proteins. To be able to compare these RBDs on a structural level, we have solved the three-dimensional structure of Rlf-RBD by NMR spectroscopy. The overall tertiary structure of Rlf-RBD shows the betabetaalphabetabetaalphabeta-fold of the ubiquitin superfamily and is very similar to that of RalGDS-RBD. The binding interface of Rlf-RBD to Ras was mapped using chemical shift analysis and indicated a binding mode similar to that in the case of Rap.Raf-RBD. However, comparison of the putatively interacting regions revealed structural differences which are proposed to be responsible for the different substrate affinities of Rlf-, RalGDS-, and Raf-RBD.

Umezawa Y, Nishio M
CH/pi interactions as demonstrated in the crystal structure of guanine-nucleotide binding proteins, Src homology-2 domains and human growth hormone in complex with their specific ligands.
Bioorg Med Chem. 1998; 6: 493-504
Display abstract
The CH/pi interaction is a weak attractive molecular force occurring between CH groups and pi-systems. Possibility has been examined for the role of CH/pi interaction, by use of a computer program, in the crystallographic data of several guanine-nucleotide binding proteins, src homology-2 domains and human growth hormone complexed with their specific ligands. Short CH/pi contacts have been found in every case where cohesive forces are expected. Comparison of the structures of functionary related proteins has shown that mutation may occur but necessary CH/pi interactions are conserved. A considerable part of the non-polar interactions, broadly ascribed in the past to the van der Waals interaction or the so-called hydrophobic effect, has been suggested to be attributed to a more specific attractive force, the CH/pi interaction.

Yan J, Roy S, Apolloni A, Lane A, Hancock JF
Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase.
J Biol Chem. 1998; 273: 24052-6
Display abstract
Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. We show here, however, that in vivo there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimicks that of Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized.

Erickson HP
Atomic structures of tubulin and FtsZ.
Trends Cell Biol. 1998; 8: 133-7
Display abstract
The recently published atomic structures of tubulin and FtsZ are a research milestone. The N-terminal GTP-binding domains of tubulin and FtsZ are virtually identical in structure, as expected from the substantial sequence identity. Sequence identity is absent from the C-terminal domains, but they also have virtually identical structures. A surprising finding is that the N-terminal GTP-binding domain is structurally homologous to that of Ras and other G proteins, despite the completely different GTP-binding sequence motifs. This article discusses these findings and the molecular mechanisms that can now be addressed with the atomic structures.

Watari Y et al.
Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector.
Gene. 1998; 224: 53-8
Display abstract
Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.

Iouzalen N, Camonis J, Moreau J
Identification and characterization in Xenopus of XsmgGDS, a RalB binding protein.
Biochem Biophys Res Commun. 1998; 250: 359-63
Display abstract
We have previously isolated a cDNA coding for a RalB protein from a Xenopus maternal library. By mutagenesis of RalB and microinjection of its mRNA in embryos we show that RalB is involved in Xenopus early development. We have used the yeast two-hybrid system to screen a Xenopus maternal cDNA library in order to isolate proteins interacting to RalB. We have identified a full-length cDNA encoding a protein homologous to mammalian smgGDS. The Xenopus gene shows 84.6% identity with the mammalian counterpart and contains one additional armadillo repeat. The XsmgGDS mRNA is expressed from oogenesis up to late embryogenesis at a higher level than that in adult tissues. Thus RalB is another protein that interacts with smgGDS which suggests that RalB may be activated by a Ras independent pathway.

Ramocki MB, White MA, Konieczny SF, Taparowsky EJ
A role for RalGDS and a novel Ras effector in the Ras-mediated inhibition of skeletal myogenesis.
J Biol Chem. 1998; 273: 17696-701
Display abstract
Oncogenic Ras inhibits the differentiation of skeletal muscle cells through the activation of multiple downstream signaling pathways, including a Raf-dependent, mitogen-activated or extracellular signal-regulated kinase kinase/mitogen-activated protein kinase (MEK/MAPK)-independent pathway. Here we report that a non-Raf binding Ras effector-loop variant (H-Ras G12V,E37G), which retains interaction with the Ral guanine nucleotide dissociation stimulator (RalGDS), inhibits the conversion of MyoD-expressing C3H10T1/2 mouse fibroblasts to skeletal muscle. We show that H-Ras G12V,E37G, RalGDS, and the membrane-localized RalGDS CAAX protein inhibit the activity of alpha-actin-Luc, a muscle-specific reporter gene containing a necessary E-box and serum response factor (SRF) binding site, while a RalGDS protein defective for Ras interaction has no effect on alpha-actin-Luc transcription. H-Ras G12V,E37G does not activate endogenous MAPK, but does increase SRF-dependent transcription. Interestingly, RalGDS, RalGDS CAAX, and RalA G23V inhibit H-Ras G12V, E37G-induced expression of an SRF-regulated reporter gene, demonstrating that signaling through RalGDS does not duplicate the action of H-Ras G12V,E37G in this system. As additional evidence for this, we show that H-Ras G12V,E37G inhibits the expression of troponin I-Luc, an SRF-independent muscle-specific reporter gene, whereas RalGDS and RalGDS CAAX do not. Although our studies show that signaling through RalGDS can interfere with the expression of reporter genes dependent on SRF activity (including alpha-actin-Luc), our studies also provide strong evidence that an additional signaling molecule(s) activated by H-Ras G12V,E37G is required to achieve the complete inhibition of the myogenic differentiation program.

Mao J, Yuan H, Xie W, Wu D
Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein alpha subunit Galpha13.
Proc Natl Acad Sci U S A. 1998; 95: 12973-6
Display abstract
Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein alpha subunits were characterized in transfection systems. Galphaq, Galpha12, and Galpha13, but not Galphai, activate SRF through RhoA. When Galphaq, alpha12, or alpha13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Galpha13, but not Galphaq or Galpha12, showed synergistic activation of SRF with GEF115. The synergy between Galpha13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Galpha13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Galpha13- and Galpha12-induced, but not GEF115 itself- or Galphaq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Galpha12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Galpha12 and Galpha13. Thus, the inhibition of Galpha12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Galpha13 and GEF115 indicates that GEF115 mediates Galpha13-induced activation of Rho and SRF.

Bos JL
All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral.
EMBO J. 1998; 17: 6776-82
Display abstract
Ras, Rap1 and Ral are related small GTPases. While the function of Ras in signal transduction is well established, it has been recognized only recently that Rap1 and Ral also are activated rapidly in response to a large variety of extracellular signals. Between the three GTPase an intriguing interconnectivity exists, in that guanine nucleotide exchange factors for Ral associate with the GTP-bound form of both Ras and Rap1. Furthermore, Rap1 is considered to function as an antagonist of Ras signalling by trapping Ras effectors in an inactive complex. Here, I summarize the recent developments in understanding the functional relationship between these three GTPase and argue that Rap1 functions in a signalling pathway distinct from Ras, while using similar or identical effectors.

Werner LA, Manseau LJ
A Drosophila gene with predicted rhoGEF, pleckstrin homology and SH3 domains is highly expressed in morphogenic tissues.
Gene. 1997; 187: 107-14
Display abstract
We have identified a Drosophila gene that has substantial sequence homology to a distinct class of proto-oncogenes that includes DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately downstream of the Rho/RacGEF. Rho/RacGEFs catalyze the dissociation of GDP from the Rho/Rac subfamily of Ras-like GTPases, thus activating the target Rho/Rac (Takai et al. (1995) Trends Biochem. Sci. 20, 227-231]. Members of the Rho/Rac subfamily regulate organization of the actin cytoskeleton, which controls the morphology, adhesion and motility of cells (Nobes et al. (1995) J. Cell Sci. 108, 225 233; Ridley and Hall (1992) Cell 70, 389-399; Ridley et al. (1992) Cell 70, 401-410]. Message from this gene is found throughout oogenesis and embryogenesis. Of particular interest, message is most abundant in furrows and folds of the embryo where cell shapes are changing and the cytoskeleton is likely to be undergoing reorganization.

Abdellatif M, Schneider MD
An effector-like function of Ras GTPase-activating protein predominates in cardiac muscle cells.
J Biol Chem. 1997; 272: 525-33
Display abstract
In contrast to familiar role for Ras in proliferation, we and others previously suggested that Ras also mediates hypertrophy, the increase in cell mass characteristic of post-natal ventricular muscle. We showed that activated (G12R) and dominant-negative (S17N) Ha-Ras regulate "constitutive" and growth factor-responsive genes equivalently, in both cardiac myocytes and non-cardiac, Mv1Lu cells. Here, we attempt to delineate pathways by which Ras exerts this global effect. The E63K mutation, which impairs binding of guanine nucleotide releasing factor to Ras, alleviated suppression by S17N, consistent with sequestration of exchange factors as the mechanism for inhibition. To compare potential Ras effector proteins, we first engineered G12R/D38N, to abolish binding of Raf and phosphatidylinositol-3-kinase and established that this site was indispensable for augmenting gene expression. To distinguish between inhibition of Ras by Ras GTPase-activating protein (GAP) versus a potential effector function of GAP, we tested the effector domain substitution P34R: this mutation, which abolishes GAP binding, enhanced Ras-dependent transcription in Mv1Lu cells, yet interfered with Ras-dependent expression in ventricular myocytes. To examine the dichotomous role of Ras-GAP predicted from these P34R results, we transfected both cell types with full-length GAP, the C-terminal catalytic domain (cGAP), or N-terminal Src homology domains (nGAP). In Mv1Lu cells, cGAP markedly inhibited both reporter genes, whereas GAP and nGAP had little effect. Antithetically, in ventricular myocytes, GAP and nGAP activated gene expression, whereas cGAP was ineffective. Thus, Ras activates gene expression through differing effectors contingent on cell type, and an effector-like function of GAP predominates in ventricular muscle.

Lawton DG, Gorman C, Lowe PN
Small G protein characterisation by isothermal titration calorimetry.
Biochem Soc Trans. 1997; 25: 510-510

Roy S, Lane A, Yan J, McPherson R, Hancock JF
Activity of plasma membrane-recruited Raf-1 is regulated by Ras via the Raf zinc finger.
J Biol Chem. 1997; 272: 20139-45
Display abstract
Ras recruits Raf to the plasma membrane for activation by a combination of tyrosine phosphorylation and other as yet undefined mechanism(s). We show here that the Raf zinc finger is not required for plasma membrane recruitment of Raf by Ras but is essential for full activation of Raf at the plasma membrane. Membrane targeting cannot compensate for the absence of the zinc finger. One facet of the zinc finger activation defect is revealed using a constitutively activated Raf mutant. Targeting Raf Y340D,Y341D to the plasma membrane increments activity, but full activation requires coexpression with activated Ras. This sensitivity to regulation by Ras at the plasma membrane is abrogated by mutations in the Raf zinc finger but is unaffected by mutation of the minimal Ras binding domain. These data show for the first time that Ras has two separate roles in Raf activation: recruitment of Raf to the plasma membrane through an interaction with the minimal Ras binding domain and activation of membrane-localized Raf via a mechanism that requires the Raf zinc finger.

Stang S, Bottorff D, Stone JC
Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.
Mol Cell Biol. 1997; 17: 3047-55
Display abstract
v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.

de Gunzburg J
[Isoprenylated proteins and cell proliferation: regulators and effectors of Ras proteins]
C R Seances Soc Biol Fil. 1997; 191: 195-210
Display abstract
Ras proteins play a central role in the control of cellular proliferation. They are 189 amino acid monomeric GTP-binding proteins that cycle between an inactive GDP-bound and the active GTP-bound state, and carry a slow intrinsic GTPase activity. Ras proteins are activated by growth promoting signals incoming from receptor tyrosine kinases via SH2 domain and SH3 domain containing adapter proteins and the Ras exchange factor Sos, as well as from serpentine receptors via the beta gamma subunits of heterotrimeric G proteins and the Ras exchange factor Ras-GRF (or Cdc25). Proteins that can stimulate the GTPase activity of Ras (GAPs) ensure that following mitogenic stimulations, they return to their inactive GDP-bound state; amongst these proteins are p120-GAP, neurofibomin (the product of the susceptibility gene to type I neurofibromatosis), as well as the inositol 1,3,4,5-tetrakisphosphate-dependent GAPIP4BF. Several effectors have been identified that mediate the biological effects of Ras. The serine/threonine kinase Raf-1, as well as the closely related protein B-Raf, elicit the ERK cascade of MAP kinases. Phosphatidylinositol-3-OH kinase is involved in the activation of the Rac/Rho family proteins that play a role in the control of actin polymerisation, as well as in growth control, RalGDS, RGL and Rlf, are responsible for the activation of the Ras-related protein Ral. Recent evidence, using effector domain mutants of Ras, demonstrates that these pathways cooperate to elicit the growth promoting effects of Ras proteins.

Ito Y et al.
Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein.
Biochemistry. 1997; 36: 9109-19
Display abstract
The backbone 1H, 13C, and 15N resonances of the c-Ha-Ras protein [a truncated version consisting of residues 1-171, Ras(1-171)] bound with GMPPNP (a slowly hydrolyzable analogue of GTP) were assigned and compared with those of the GDP-bound Ras(1-171). The backbone amide resonances of amino acid residues 10-13, 21, 31-39, 57-64, and 71 of Ras(1-171).GMPPNP, but not those of Ras(1-171).GDP, were extremely broadened, whereas other residues of Ras(1-171).GMPPNP exhibited amide resonances nearly as sharp as those of Ras(1-171). GDP. The residues exhibiting the extreme broadening, except for residues 21 and 71, are localized in three functional loop regions [loops L1, L2 (switch I), and L4 (switch II)], which are involved in hydrolysis of GTP and interactions with other proteins. From the temperature and magnetic field strength dependencies of the backbone amide resonance intensities, the extreme broadening was ascribed to the exchange at an intermediate rate on the NMR time scale. It was shown that the Ras(1-171) protein bound with GTP or GTPgammaS (another slowly hydrolyzable analogue of GTP) exhibits the same type of broadening. Therefore, it is a characteristic feature of the GTP-bound form of Ras that the L1, L2, and L4 loop regions, but not other regions, are in a rather slow interconversion between two or more stable conformers. This phenomenon, termed a "regional polysterism", of these loop regions may be related with their multifunctionality: the GTP-dependent interactions with several downstream target groups such as the Raf and RalGDS families and also with the GTPase activating protein (GAP) family. In fact, the binding of Ras(1-171).GMPPNP with the Ras-binding domain (residues 51-131) of c-Raf-1 was shown to eliminate the regional polysterism nearly completely. It was indicated, therefore, that each target/regulator selects its appropriate conformer among those presented by the "polysteric" binding interface of Ras. As the downstream target groups exhibit no apparent sequence homology to each other, it is possible that one target group prefers a conformer different from that preferred by another group. The involvement of loop L1 in the regional polysterism might suggest that the negative regulators, GAPs, bind to the polysteric binding interface (loops L2 and L4) of Ras and cooperatively select a conformer suitable for transition of the GTPase catalytic center, involving loops L1 and L4, into the highly active state.

Huff SY, Quilliam LA, Cox AD, Der CJ
R-Ras is regulated by activators and effectors distinct from those that control Ras function.
Oncogene. 1997; 14: 133-43
Display abstract
Like Ras, constitutively activated mutants of the Ras-related protein R-Ras cause tumorigenic transformation of NIH3T3 cells. However, since R-Ras causes a transformed phenotype distinct from that induced by Ras, it is likely that R-Ras controls signaling pathways and cellular processes distinct from those regulated by Ras. To address this possibility, we determined if R-Ras is regulated by activators and effectors distinct from those that regulate Ras function. We observed that Ras guanine nucleotide exchange factors failed to activate R-Ras in vivo, indicating that R-Ras is activated by distinct GEFs. Consistent with this, mutants of R-Ras with mutations analogous to the Ras(15A)/(17N) dominant negative proteins did not antagonize Ras GEF function and lacked the growth inhibitory activity seen with these mutant Ras proteins. Thus, R-Ras, but not Ras, is dispensable for the viability of NIH3T3 cells. Finally, whereas constitutively activated Ras can overcome the growth inhibitory action of the Ras(17N) dominant negative protein via Raf-dependent and -independent activities, transforming mutants of R-Ras failed to do so. This inability was consistent with our observation that Ras-, but not R-Ras-transformed, NIH3T3 cells possessed constitutively upregulated Raf kinase activities. Thus, R-Ras and Ras are regulators of distinct signaling pathways and cellular processes.

Clark GJ et al.
14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.
J Biol Chem. 1997; 272: 20990-3
Display abstract
Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

Katz ME, McCormick F
Signal transduction from multiple Ras effectors.
Curr Opin Genet Dev. 1997; 7: 75-9
Display abstract
Ras proteins activate a signaling cascade through direct binding of the serine/threonine kinase Raf. They also activate additional signaling pathways that are essential for full biological activity. Candidate effectors for these pathways include RalGDS and phosphatidyl inositol 3' kinase, as well as several other Ras binding proteins the biochemical and biological properties of which are poorly understood.

Camus C, Geymonat M, Garreau H, Baudet-Nessler S, Jacquet M
Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p.
Eur J Biochem. 1997; 247: 703-8
Display abstract
The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro. The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.

Ramocki MB, Johnson SE, White MA, Ashendel CL, Konieczny SF, Taparowsky EJ
Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.
Mol Cell Biol. 1997; 17: 3547-55
Display abstract
The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

Diaz JF, Wroblowski B, Schlitter J, Engelborghs Y
Calculation of pathways for the conformational transition between the GTP- and GDP-bound states of the Ha-ras-p21 protein: calculations with explicit solvent simulations and comparison with calculations in vacuum.
Proteins. 1997; 28: 434-51
Display abstract
The transitions between the water-equilibrated structures of the GTP and GDP forms of Ha-ras-p21 have been calculated by using the targeted molecular dynamics (TMD) method (Schlitter et al., Mol. Sim. 10:291-309, 1993) both in vacuo and with explicit solvent simulation. These constrained molecular dynamics calculations result in different pathways, depending on the nucleotide bound. Each pathway consists in a sequence of transitions affecting six segments of the protein, four of them forming a hydrophilic cleft around the nucleotide. The transitions are initiated by the removal or introduction of the gamma-phosphate of the nucleotide and proceed sequentially, crossing several low-energy transition states. The movements are transmitted either by direct interactions between the segments or through the nucleotide. The GTP to GDP pathway is initiated by the removal of the nucleotide gamma-phosphate. This gives some space to Gly12, Gly13, and Val14. Their movement is transmitted to the target recognition domain and the switch II region, forcing these segments to adopt another position. In a second step the target recognition domain and the switch II region undergo conformational transitions to reach an intermediate conformation. Finally, there is a relaxation of the target recognition domain to its final state that forces the switch II region to reach its target conformation. The calculated pathways allow the identification of many residues that play an important role in the conformational changes, explain the altered transformation properties of some, and suggest mutations to alter the pathway.

Luo Z, Diaz B, Marshall MS, Avruch J
An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ.
Mol Cell Biol. 1997; 17: 46-53
Display abstract
The function of the c-Raf-1 zinc finger domain in the activation of the Raf kinase was examined by the creation of variant zinc finger structures. Mutation of Raf Cys 165 and Cys 168 to Ser strongly inhibits the Ras-dependent activation of c-Raf-1 by epidermal growth factor (EGF). Deletion of the Raf zinc finger and replacement with a homologous zinc finger from protein kinase C gamma (PKC gamma) (to give gamma/Raf) also abrogates EGF-induced activation but enables a vigorous phorbol myristate acetate (PMA)-induced activation. PMA activation of gamma/Raf does not require endogenous Ras or PKCs and probably occurs through a PMA-induced recruitment of gamma/Raf to the plasma membrane. The impaired ability of EGF to activate the Raf zinc finger variants in situ is attributable, at least in part, to a major decrement in their binding to Ras-GTP; both Raf zinc finger variants exhibit decreased association with Ras (V12) in situ upon coexpression in COS cells, as well as diminished binding in vitro to immobilized, processed COS recombinant Ras(V12)-GTP. In contrast, Raf binding to unprocessed COS or prokaryotic recombinant Ras-GTP is unaffected by Raf zinc finger mutation. Thus, the Raf zinc finger contributes an important component to the overall binding to Ras-GTP in situ, through an interaction between the zinc finger and an epitope on Ras, distinct from the effector loop, that is present only on prenylated Ras.

Hu CD, Kariya Ki, Kotani G, Shirouzu M, Yokoyama S, Kataoka T
Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal region interferes with ras-dependent activation of Raf-1.
J Biol Chem. 1997; 272: 11702-5
Display abstract
Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.

Winkler DG, Johnson JC, Cooper JA, Vojtek AB
Identification and characterization of mutations in Ha-Ras that selectively decrease binding to cRaf-1.
J Biol Chem. 1997; 272: 24402-9
Display abstract
The oncoprotein Ras transforms cells by binding to one or more effector proteins. Effector proteins have been identified by their ability to bind to Ras in the GTP but not GDP form, and by their requirement for the Ras effector domain for binding. The best understood Ras effectors are serine/threonine kinases of the Raf family, but other candidate Ras effectors, including a Ral guanine nucleotide dissociation stimulator and phosphatidylinositol 3-kinase (PI3 kinase) have also been identified. To investigate the mechanism of binding of cRaf-1 to Ras, and to investigate the roles of other candidate Ras effectors in transformation, we have isolated and characterized mutants of activated Ras with decreased binding to cRaf-1 relative to other candidate effectors. Examination of these mutants indicates that surface-exposed residues of Ras outside the minimal effector domain interact differentially with cRaf-1 and other Ras-binding proteins, and that fibroblast transformation correlates with cRaf-1 binding and mitogen-activated protein (MAP) kinase activation. Furthermore, activation of PI3 kinase can occur in the absence of significant MAP kinase activation, suggesting that PI3 kinase activation is a primary effect of Ras.

Yee WM, Worley PF
Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals.
Mol Cell Biol. 1997; 17: 921-33
Display abstract
Rheb is a recently described member of the Ras family that was originally identified as an immediate-early gene in brain but is also widely expressed in other tissues. Here we demonstrate that Rheb interacts with and appears to regulate Raf-1 kinase, an essential component of the H-Ras signaling pathway. In direct contrast to H-Ras, however, the interaction of Rheb with Raf-1 is potentiated by growth factors in combination with agents that increase cyclic AMP (cAMP) levels. Protein kinase A-dependent phosphorylation of serine 43 within the regulatory domain of Raf-1 reciprocally potentiates its interaction with Rheb and decreases its interaction with H-Ras. A single amino acid in the G2 effector domain is critical for the differential properties of Rheb. Since Rheb is an immediate-early gene, our studies suggest that Rheb functions in concert with H-Ras to dynamically integrate cAMP and growth factor signaling.

Lander HM et al.
A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction.
J Biol Chem. 1997; 272: 4323-6
Display abstract
We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.

Chen JM, Monaco R, Manolatos S, Brandt-Rauf PW, Friedman FK, Pincus MR
Molecular dynamics on complexes of ras-p21 and its inhibitor protein, rap-1A, bound to the ras-binding domain of the raf-p74 protein: identification of effector domains in the raf protein.
J Protein Chem. 1997; 16: 619-29
Display abstract
We have computed the average structures for the ras-p21 protein and its strongly homologous inhibitor protein, rap-1A, bound to the ras-binding domain (RBD) of the raf protein, using molecular dynamics. Our purpose is to determine the differences in structure between these complexes that would result in no mitogenic activity of rap-1A-RBD but full activity of p21-RBD. We find that despite the similarities of the starting structures for both complexes, the average structures differ considerably, indicating that these two proteins do not interact in the same way with this vital target protein. p21 does not undergo major changes in conformation when bound to the RBD, while rap-1A undergoes significant changes in structure on binding to the RBD, especially in the critical region around residue 61. The p21 and rap-1A make substantially different contacts with the RBD. For example, the loop region from residues 55-71 of rap-1a makes extensive hydrogen-bond contacts with the RBD, while the same residues of p21 do not. Comparison of the structures of the RBD in both complexes reveals that it undergoes considerable changes in structure when its structure bond to p21 is compared with that bound to rap-1A. These changes in structure are due to displacements of regular structure (e.g., alpha-helices and beta-sheets) rather than to changes in the specific conformations of the segments themselves. Three regions of the RBD have been found to differ significantly from one another in the two complexes: the binding interface between the two proteins at residues 60 and 70, the region around residues 105-106, and 118-120. These regions may constitute effector domains of the RBD whose conformations determine whether or not mitogenic signal transduction will occur.

Chung D et al.
Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the ras-binding domain of the raf-p74 protein, identified from molecular dynamics calculations.
J Protein Chem. 1997; 16: 631-5
Display abstract
In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein-protein interaction interface, e.g., between residues 60 and 72, residues 97-110, and 111-121. Since the rap-1A-RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62-76, 97-110, and 111-121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p21. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97-110 peptide is the strongest inhibitor of oncogenic p21, while the 111-121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.

van den Berghe N, Cool RH, Horn G, Wittinghofer A
Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N).
Oncogene. 1997; 15: 845-50
Display abstract
A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli. It was purified and its interaction with GTP-binding proteins was investigated using fluorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM. Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.

Miller MJ et al.
RalGDS functions in Ras- and cAMP-mediated growth stimulation.
J Biol Chem. 1997; 272: 5600-5
Display abstract
Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.

Kishida S, Koyama S, Matsubara K, Kishida M, Matsuura Y, Kikuchi A
Colocalization of Ras and Ral on the membrane is required for Ras-dependent Ral activation through Ral GDP dissociation stimulator.
Oncogene. 1997; 15: 2899-907
Display abstract
Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslationally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells. The RalGDS action on Ral was enhanced by an active form of Ras but not a Ras mutant which was not post-translationally modified in the cells. The RalGDS activity was inhibited by acidic membrane phospholipids such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral was incorporated alone in the liposomes, RalGDS did not stimulate the dissociation of GDP from Ral. When Ral was incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-bound form of Ras did not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS required the Ras-binding domain of RalGDS. Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap did not enhance the RalGDS action in COS cells. Taken together with our previous observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells.

Cruise JL, Rafferty MP, Riehle MM
Cell-cycle regulated expression of Rap1 in regenerating liver.
Biochem Biophys Res Commun. 1997; 230: 578-81
Display abstract
Rap1 proteins are capable of competing with Ras p21 for binding to effectors, and of antagonizing some Ras-induced effects, but their participation in normal growth regulation has not been established. The level of Rap1 protein and the expression of the rap1A gene were examined by immunoblotting and Northern analysis during the regenerative growth response in rat liver following partial hepatectomy. Protein and mRNA were significantly down-regulated prior to and during the onset of DNA synthesis. The timing of this effect is consistent with a model in which expression of Rap1 is turned off or down to allow the initiation of proliferation.

Mello LV, van Aalten DM, Findlay JB
Comparison of ras-p21 bound to GDP and GTP: differences in protein and ligand dynamics.
Protein Eng. 1997; 10: 381-7
Display abstract
This paper documents the first essential dynamics analysis of ras protein ligands and of the protein itself, showing important features of their dynamic properties. Essential dynamics analysis of 300 ps of full solvent molecular dynamics simulations revealed differences in structure and dynamics between GDP- and GTP-bound forms of H-ras-p21. Regions in the protein which exhibited a structural shift correspond to the switch regions described previously. Differences in dynamics between H-ras-p21 GDP and H-ras-p21 GTP may be related to interactions of ras with GAP and its receptor and effector. Molecular dynamics of free GDP (in the absence of protein) were performed in water for 2 ns and analysed using essential dynamics. The conformations of GDP and GTP when bound to the protein were compared with free GDP, revealing that the ligands bind to the protein in an energetically unfavourable conformation. GDP and GTP molecules from various other protein crystal structures were also analysed. These ligands adopt similar conformations to those seen in H-ras-p21.

Okazaki M et al.
Synergistic activation of c-fos promoter activity by Raf and Ral GDP dissociation stimulator.
Oncogene. 1997; 14: 515-21
Display abstract
Ral, a member of small GTP-binding protein (G protein) superfamily, has been suggested to act downstream of Ras, since Ral GDP dissociation stimulator (RalGDS) has been found to be an effector protein of Ras. In this study, we examined the effects of RalGDS and Ral on gene expression using c-fos promoter linked to the luciferase reporter gene (c-fos-luciferase). RalGDS interacted with RasG12V/E37G (in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf in COS cells. RafCAAX is an active Raf kinase targeted to the plasma membranes by virtue of the addition of a C-terminal localization signal from K-Ras. Transfection of either RalGDS or RafCAAX into NIH3T3 cells slightly stimulated c-fos-luciferase expression and cotransfection of both proteins greatly enhanced the expression. RalGDS and an activated Rac (RacG12V) did not act synergistically to stimulate c-fos-luciferase expression. Transfection of an activated Ral (RalG23V) stimulated c-fos-luciferase expression. Furthermore, cotransfection of RalG23V and an activated Ras (RasG12V) enhanced RasG12V-dependent c-fos-luciferase expression. However, RalG23V did not synergize with RafCAAX, RacG12V or RalGDS to stimulate the expression. These results show that RalGDS and Ral regulate c-fos promoter activity and suggest that RalGDS may activate c-fos promoter synergistically with the signal from Raf by transmitting the signal to a target other than Ral.

Koyama S, Kishida S, Kikuchi A
[RalGDS mediates an intracellular signal-transduction system from a proto-oncogene product Ras]
Seikagaku. 1996; 68: 1646-50

Lee CH, Della NG, Chew CE, Zack DJ
Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins.
J Neurosci. 1996; 16: 6784-94
Display abstract
cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases are Mucor racemosus Ras2 and Ras3, to which they show approximately 48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

White MA, Vale T, Camonis JH, Schaefer E, Wigler MH
A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation.
J Biol Chem. 1996; 271: 16439-42
Display abstract
Oncogenic Ras transforms cells through the activation of multiple downstream pathways mediated by separate effector molecules, one of which is Raf. Here we report the identification of a second ras-binding protein that can induce cellular transformation in parallel with activation of the Raf/mitogen-activated protein kinase cascade. The Ral guanine nucleotide dissociation stimulator (RalGDS) was isolated from a screen for Ras-binding proteins that specifically interact with a Ras effector-loop mutant, ras(12V,37G), that uncouples Ras from activation of Raf1. RalGDS, like ras(12V, 37G), cooperates synergistically with mutationally activated Raf to induce foci of growth and morphologically transformed NIH 3T3 cells. RalGDS does not significantly enhance MAP kinase activation by activated Raf, suggesting that the cooperativity in focus formation is due to a distinct pathway acting downstream of Ras and parallel to Raf.

Chen JM, Grad R, Monaco R, Pincus MR
Prediction of the three-dimensional structure of the rap-1A protein from its homology to the ras-gene-encoded p21 protein.
J Protein Chem. 1996; 15: 11-5
Display abstract
rap-1A, an anti-oncogene-encoded protein, is a ras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32-47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A at ras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure of ras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 A from that of ras-p21. The effector binding domains from residues 32-47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115-126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.

Okazaki M, Kishida S, Murai H, Hinoi T, Kikuchi A
Ras-interacting domain of Ral GDP dissociation stimulator like (RGL) reverses v-Ras-induced transformation and Raf-1 activation in NIH3T3 cells.
Cancer Res. 1996; 56: 2387-92
Display abstract
Ral GDP dissociation stimulator (RalGDS) and RalGDS like (RGL) are putative effector proteins of Ras and contain the Ras-interacting domain (RID) at their C-terminal regions. v-Ras is known to activate c-fos promoter/enhancer and Raf-1 and to transform NIH3T3 cells. It is also known that v-Raf activates c-fos promoter/enhancer and transforms NIH3T3 cells. In this study, we examined the effect of RID on the phenotype of the cells transformed by v-Ras and v-Raf. Overexpression of RID greatly reduced cell growth in low serum, colony-forming activity in soft agar, c-fos promoter/enhancer activity, and Raf-1 activity of v-Ras-transformed cells. However, overexpression of RID did not affect the phenotype of v-Raf-transformed cells. These results clearly indicate that RID of RGL specifically binds to Ras in mammalian cells, that it blocks the signal from Ras to Raf-1, and that it reverses v-Ras-induced malignant phenotype. It has been reported that Ras-binding domains of Raf-1 and neurofibromatosis type 1 (NF1) reverse v-Ras-induced malignant phenotype. Since there is no homology in primary structures of RGL, Raf-1, and NF1, there may be a similarity of secondary or tertiary structure among RID of RGL and Ras-binding domains of Raf-1 and NF1, and the structure might be useful for developing a potential medicine for human cancers caused by Ras.

Peterson SN et al.
Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1.
J Biol Chem. 1996; 271: 29903-8
Display abstract
Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

Shen PS, Sanford JC, Samuelson J
Entamoeba histolytica: isoprenylation of p21ras and p21rap in vitro.
Exp Parasitol. 1996; 82: 65-8

Kuroda S, Ohtsuka T, Yamamori B, Fukui K, Shimizu K, Takai Y
Different effects of various phospholipids on Ki-Ras-, Ha-Ras-, and Rap1B-induced B-Raf activation.
J Biol Chem. 1996; 271: 14680-3
Display abstract
We have recently purified a Ki-Ras- and Ha-Ras-dependent extracellular signal-regulated kinase kinase from bovine brain and identified it as B-Raf protein kinase complexed with 14-3-3 proteins (Yamamori, B., Kuroda, S., Shimizu, K., Fukui, K., Ohtsuka, T., and Takai, Y. (1995) J. Biol. Chem. 270, 11723-11726). Moreover, we found that Rap1B as well as Ki-Ras and Ha-Ras stimulate the B-Raf activity. Since B-Raf contains a cysteine-rich domain originally found in protein kinase C as a domain responsible for interaction with phosphatidylserine (PS) and diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate, we have examined here the effect of these compounds on the Ki-Ras-, Ha-Ras-, and Rap1B-induced activation of bovine brain B-Raf. Bovine brain PS enhanced Ki-Ras-stimulated B-Raf activity. Phosphatidic acid was slightly active, but other phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol (PI), PI-4-monophosphate, PI-4,5-bisphosphate, and PI-3,4,5-trisphosphate, were inactive. However, none of the above phospholipids affected the Ha-Ras-stimulated B-Raf activity, whereas PI, PS, phosphatidylethanolamine, and phosphatidic acid inhibited the Rap1B-stimulated B-Raf activity. Phosphatidylcholine or PI-4-monophosphate did not show any effect on the Rap1B-stimulated B-Raf activity. Synthetic PS with two unsaturated fatty acids, such as 1,2-dioleoyl-PS or 1,2-dilinoleoyl-PS, showed the same effect toward the Ki-Ras- and Rap1B-stimulated B-Raf activities, but synthetic PS with two saturated fatty acids, such as 1, 2-distearoyl-PS, was inactive. 12-O-Tetradecanoylphorbol-13-acetate did not affect the stimulatory or inhibitory effect of PS on the Ki-Ras- and Rap1B-stimulated B-Raf activities, respectively. PS did not affect the Ki-Ras-, Ha-Ras-, or Rap1B-independent basal B-Raf activity or the mitogen-activated protein kinase kinase or extracellular signal-regulated kinase activity. These results indicate that various phospholipids differently affect Ki-Ras-, Ha-Ras, and Rap1B-induced B-Raf activation.

Stahl B, Chou JH, Li C, Sudhof TC, Jahn R
Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.
EMBO J. 1996; 15: 1799-809
Display abstract
GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.

Urano T, Emkey R, Feig LA
Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.
EMBO J. 1996; 15: 810-6
Display abstract
Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation.

Sung YJ, Hwang MC, Hwang YW
The dominant negative effects of H-Ras harboring a Gly to Ala mutation at position 60.
J Biol Chem. 1996; 271: 30537-43
Display abstract
v-H-Ras harboring the Gly-60 to Ala mutation (G60A) lacks the ability to induce germinal vesicle breakdown in Xenopus oocytes. Moreover, this mutant is capable of inhibiting the activity of v-H-Ras to induce oocyte germinal vesicle breakdown when co-injected. The duration and the extent of inhibition depends on the molar ratio of v-H-Ras(G60A) to v-H-Ras. The inhibition is not due to a general toxicity of v-H-Ras(G60A) to oocytes because oocytes injected with v-H-Ras(G60A) can be readily induced to mature by other mitogenic agents, such as insulin, insulin-like growth factor 1, insulin-like growth factor 2, and phosphatidylcholine-specific phospholipase C. The dominant negative effect of v-H-Ras(G60A) requires proper membrane attachment of v-H-Ras(G60A). By using a competition assay, it was concluded that the dominant negative phenotype of v-H-Ras(G60A) resulted from sequestering H-Ras downstream effector(s). Raf-1 was identified as one of the sequestered targets.

Kikuchi A, Williams LT
Regulation of interaction of ras p21 with RalGDS and Raf-1 by cyclic AMP-dependent protein kinase.
J Biol Chem. 1996; 271: 588-94
Display abstract
RalGDS is a GDP/GTP exchange protein for ral p24, a member of small GTP-binding protein superfamily. We have recently shown that RalGDS interacts directly with the GTP-bound active form of ras p21 through the effector loop of ras p21 in vitro, in insect cells and in the yeast two-hybrid system. These results suggest that RalGDS functions as an effector protein of ras p21. Here, we report that RalGDS interacts with ras p21 in mammalian cells in response to an extracellular signal. Epidermal growth factor (EGF) induced the interaction of c-ras p21 and RalGDS in COS cells expressing both proteins, but not in the cells expressing RalGDS and c-ras p21T35A, which is an effector loop mutant of ras p21. We also found that cyclic AMP-dependent protein kinase (protein kinase A) regulated the selectivity of ras p21-binding to either RalGDS or Raf-1. Protein kinase A phosphorylated RalGDS as well as (1-149)Raf (amino acid residues 1-149). Although the phosphorylated (1-149)Raf had a lower affinity for ras p21 than the unphosphorylated (1-149)Raf, both the phosphorylated and unphosphorylated RalGDS had the similar affinities for ras p21. The phosphorylation of RalGDS did not affect its activity to stimulate the GDP/GTP exchange of ral p24. Pretreatment of COS cells with forskolin further stimulated the interaction of ras p21 and RalGDS induced by EGF under the conditions that EGF-dependent Raf-1 activity was inhibited. These results indicate that ras p21 distinguishes between RalGDS and Raf-1 by their phosphorylation by protein kinase A.

Nassar N, Horn G, Herrmann C, Block C, Janknecht R, Wittinghofer A
Ras/Rap effector specificity determined by charge reversal.
Nat Struct Biol. 1996; 3: 723-9
Display abstract
Members of the Ras subfamily of small GTP-binding proteins have been shown to be promiscuous towards a variety of putative effector molecules such as the protein kinase c-Raf and the Ral-specific guanine nucleotide exchange factor (Ral-GEF). To address the question of specificity of interactions we have introduced the mutations E30D and K31E into Rap and show biochemically, by X-ray structure analysis and by transfection in vivo that the identical core effector region of Ras and Rap (residues 32-40) is responsible for molecular recognition, but that residues outside this region are responsible for the specificity of the interaction. The major determinant for the switch in specificity is the opposite charge of residue 31--Lys in Rap, Glu in Ras--which creates a favourable complementary interface for the Ras-Raf interaction.

McCormick F
Ras biology in atomic detail.
Nat Struct Biol. 1996; 3: 653-5

Hinoi T, Kishida S, Koyama S, Ikeda M, Matsuura Y, Kikuchi A
Post-translational modifications of Ras and Ral are important for the action of Ral GDP dissociation stimulator.
J Biol Chem. 1996; 271: 19710-6
Display abstract
Ral GDP dissociation stimulator (RalGDS) is a GDP/GTP exchange protein of Ral and a new effector protein of Ras. Therefore, there may be a new signaling pathway from Ras to Ral. In this paper, we examined the roles of the post-translational modifications of Ras and Ral on this new signal transduction pathway. The post-translationally modified form of Ras bound to RalGDS more effectively than the unmodified form. The modification of Ras was required to regulate the distribution of RalGDS between the cytosol and membrane fractions in COS cells. The post-translational modification of Ral enhanced the activities of RalGDS to stimulate the dissociation of GDP from and the binding of GTP to Ral. Furthermore, the modified form of Ral bound to Ral-binding protein 1 (RalBP1), a putative effector protein of Ral, more effectively than the unmodified form. Taken together with the observations that Ras and Ral are localized to the membranes, these results suggest that the post-translational modifications of Ras and Ral play a role for transmitting the signal effectively on the membranes in the signal transduction pathway of Ras/RalGDS/Ral/RalBP1.

Isomura M, Okui K, Fujiwara T, Shin S, Nakamura Y
Isolation and mapping of RAB2L, a human cDNA that encodes a protein homologous to RalGDS.
Cytogenet Cell Genet. 1996; 74: 263-5
Display abstract
We have isolated a novel human cDNA, designated RAB2L, that encodes a protein homologous to Ral guanine nucleotide dissociation stimulator (RalGDS). The predicted sequence of 122 amino acids revealed 39.5% identity with the C-terminal region of human RalGDS. As the C-terminal region of RalGDS is considered to interact with RAS, the product of this novel gene may also play some role in the ras-mediated signal transduction pathway. Northern analysis revealed that the gene RAB2L is expressed in all human tissues examined. We assigned this gene locus to chromosome band 6p21.3 by fluorescence in situ hybridization.

McCormick F, Wittinghofer A
Interactions between Ras proteins and their effectors.
Curr Opin Biotechnol. 1996; 7: 449-56

Akasaka K et al.
Differential structural requirements for interaction of Ras protein with its distinct downstream effectors.
J Biol Chem. 1996; 271: 5353-60
Display abstract
Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

Clark GJ et al.
Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.
Proc Natl Acad Sci U S A. 1996; 93: 1577-81
Display abstract
A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Wittinghofer A, Nassar N
How Ras-related proteins talk to their effectors.
Trends Biochem Sci. 1996; 21: 488-91
Display abstract
More and more effectors for the Ras-related protein superfamily are being discovered and it is emerging that these GTP-binding proteins interact with more than one effector to generate more than one cellular signal. Atomic details for the interaction of Rap/Ras with one of the effectors, the protein kinase c-Raf-1, have recently become available by X-ray structure analysis. The implications for the specificity of the signal transduction pathway, and how the GTP-dependent switch mechanism modulates the interaction with effectors will be discussed here, using Ras as a paradigm.

Mahajan MA, Park ST, Sun XH
Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins.
Oncogene. 1996; 12: 2343-50
Display abstract
TAL1 is a basic helix-loop-helix (bHLH) protein involved in hematopoietic development. In T cell acute lymphoblastic leukemic cells, TAL1 is aberrantly overexpressed and is thought to contribute to oncogenesis. To identify proteins that interact with TAL1 in mediating leukemogenesis, we used TAL1 as a bait in a two-hybrid interaction screen, and isolated a cDNA clone that encodes a unique GTP binding protein, DRG. The interaction between DRG and TAL1 was confirmed both in vitro and in vivo. DRG was also shown to bind in vitro to two TAL1-related proteins, TAL2 and Lyl1. Mutational analyses showed that the HLH domain of TAL1 was necessary and sufficient for its interaction with the C-terminus of DRG. Furthermore, while DRG and E47 compete to interact with TAL1, TAL1 binds to DRG and E47 in a mutually exclusive manner. In rat embryonic fibroblast transformation assays, DRG stimulated the cotransforming activity of c-myc and ras. Based on these results, DRG appears to be a potential target for TAL-like oncoproteins.

Rosa JL, Casaroli-Marano RP, Buckler AJ, Vilaro S, Barbacid M
p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins.
EMBO J. 1996; 15: 4262-73
Display abstract
We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta-repeat domains characteristic of the beta-subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid-rich regions, a putative leucine zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy-terminal RCC1-like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1-like motif located at the amino-terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R-Ras2/TC21. These observations suggest that p619 is a Brefeldin A-sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.

Schieffer B, Paxton WG, Chai Q, Marrero MB, Bernstein KE
Angiotensin II controls p21ras activity via pp60c-src.
J Biol Chem. 1996; 271: 10329-33
Display abstract
Angiotensin II is the major effector peptide of the renin-angiotensin system, and it exerts its physiologic functions via a G protein-coupled cell surface receptor called AT1. We found that in rat aortic smooth muscle cells, angiotensin II stimulated the formation of Ras-GTP, Ras-Raf-1 complex formation, and the tyrosine phosphorylation of two important Ras GTPase-activating proteins (GAPs), p120 Ras-GAP and p190 Rho-GAP. Electroporation of anti-pp60c-src antibody into cultured, adherent smooth muscle cells blocked the angiotensin II stimulation of Ras-GAP and Rho-GAP tyrosine phosphorylation. In contrast electroporation of antibodies against c-Yes or c-Fyn had no effect. Anti-pp60c-src antibody also blocked angiotensin II-stimulated Ras activation and Ras-Raf-1 complex formation. These data strongly suggest that a G protein-coupled receptor such as the AT1 receptor can activate the Ras protein cascade via the tyrosine kinase pp60c-src.

Chen JM, Brandt-Rauf PW, Pincus MR
Oncogenic amino acid substitutions in the inhibitory rap-1A protein cause it to adopt a ras-p21-like conformation as computed using molecular dynamics.
J Biomol Struct Dyn. 1996; 13: 925-33
Display abstract
rap-1A is a membrane-bound G-protein in the ras superfamily that, like the ras-p21 protein, is activated by binding GTP in place of GDP. When activated, however, this protein inhibits the action of ras-p21, which is to induce mitogenesis in cells A chimeric protein containing RAS-p21 residues 1-65 and rap-1A residues 66-184 becomes ras-p21-like in its activity. The critical changes in sequence that result in this transformation are G26N, 127H, E30D, K31E, and E45V. All of these substitutions occur in or around a critical effector domain of p21 that is involved in interacting with GTPase activating protein (GAP), raf-p74 protein and inositol-3-hydroxy kinase. Using molecular dynamics, we have computed the average low energy structures for each of the three proteins, ras-p21, rap-1A and mutant rap1A, called rap-M, that contains these critical amino acid substitutions. We find that rap-M more closely superimposes on ras-p21 (rms deviation 1.9 A) than on wild-type rap-1A (rms deviation 3.4 A). In particular, the amino terminal domains (residues 3-59) of both RAS-p21 and rap-M are superimposable while they deviate when the average structures of these two proteins are superimposed on that of wild-type rap-1A. We have identified Pro 34 as a critical residue which may determine if the protein transforms cells or inhibits cell transformation. In addition, we have found that ras-p21 and rap-M proteins are superimposable in the region 96-110 except at Asp 105. The 96-110 domain of ras-p21 has been found to be involved in the binding of this protein to the nuclear transcription protein, jun and its kinase, jun kinase, JNK. Both segments differ in structure from that of the rap-1A segment at Asp 108, implicating this residue as also being important in determining the activity of the protein. Overall, the oncogenic substitutions introduced into the rap-1A protein cause it to adopt a conformation that is very similar to that of ras-p21 rather than wild-type rap-1A.

Hiwasa T, Kasama M, Nakadai T, Sawada T, Sakiyama S
Loss of Raf-1-binding activity of v-Ha-Ras by the deletion of amino acid residues 64-72 and 143-151.
Cell Signal. 1996; 8: 393-6
Display abstract
In order to elucidate the molecular events in signal transduction, examination of the interaction between Ras and Raf-1 seems crucial. Many Raf-1 mutants have been investigated in terms of their binding activities to Ras, where only a few Ras mutants have been examined thus far. We have investigated the Raf-1-binding activities of v-Ha-Ras and 21 insertion/deletion mutants of this protein. The results show that the mutants have varying levels of Raf-1-binding activity that are related neither to their transforming activity nor to their guanine nucleotide-binding activity. Deletion in the effector domain of Ras did not completely abolish Raf-1-binding, whereas the deletion in amino acid residues 64-72 or 143-151 resulted in complete loss of Raf-1-binding activity.

Block C, Wittinghofer A
Switching to Rac and Rho.
Structure. 1995; 3: 1281-4
Display abstract
Could the recent elucidation of the structure of the Rap-Raf complex have been the first glimpse of a universal arrangement between GTPase switches and kinase cascades, as a number of recent reports show that Ras is not unique in its ability to start a signalling 'chain reaction'?

Bax B, Jhoti H
Protein-protein interactions. Putting the pieces together.
Curr Biol. 1995; 5: 1119-21
Display abstract
What do the recently determined crystal structures of 14-3-3 proteins and of a complex between part of the protein kinase Raf and the Ras-related protein Rap tell us about how 14-3-3 and Ras regulate the function of Raf?

Jacquet E, Baouz S, Parmeggiani A
Characterization of mammalian C-CDC25Mm exchange factor and kinetic properties of the exchange reaction intermediate p21.C-CDC25Mm.
Biochemistry. 1995; 34: 12347-54
Display abstract
This work characterizes several aspects of the interaction between H-ras p21 and the catalytic domain of the mammalian GDP/GTP exchange factor (GEF) CDC25Mm (C-CDC25Mm), isolated in pure form as recombinant protein from Escherichia coli. Formation of a complex with nucleotide-free H-ras p21 could be analyzed on native gel electrophoresis by combining C-CDC25Mm and p21.GDP, as the result of the fast separation of GDP from p21. Therefore, in order to obtain highly purified heterodimer in preparative amounts, p21.GDP and C-CDC25Mm were exposed to an electric field and the complex purified by anionic chromatography. The rate of the C-CDC25Mm-mediated nucleotide exchange on p21 depended on the nature of the bound nucleotide (6 times faster for p21.GDP than p21.GTP) but was uninfluenced by the nature of the free nucleotide acting as second substrate. No saturating concentration of p21.GDP could be determined by measuring the C-CDC25Mm-mediated increase in the nucleotide exchange rate with a nitrocellulose binding assay. On this basis the Km and the kcat of the reaction were calculated to be > 11 microM and > 0.8 s-1, respectively. The dramatic increase in the nucleotide exchange rate was found to be almost exclusively based on the strong stimulation of the "off-rate". The "on-rate" of the nucleotide on the p21.C-CDC25Mm complex was similar for GDP and GTP and was little increased vs that on p21 alone. The observation that the apparent affinity of both nucleotides for the p21.C-CDC25Mm complex was lower than for p21 alone stressed the great affinity of this complex, whose Kd was calculated to be approximately 3 pM. These results are discussed and compared with the properties of other GEFs, from which C-CDC25Mm differs for a number of specific properties.

Ratnam S, Kent C
Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines.
Arch Biochem Biophys. 1995; 323: 313-22
Display abstract
The effects of expression of the H-ras oncogene on phosphatidylcholine metabolism were examined in C3H10T1/2 and NIH3T3 cells expressing ras constitutively or under the control of inducible promoters. Cell lines expressing ras under the control of the mouse metallothionein promoter and the Escherichia coli lac operator/repressor system and an NIH3T3 cell line stably transfected with the ras oncogene were studied. Phosphocholine levels were elevated in the cells constitutively expressing ras and were increased 4-6 h upon induction in the inducible cell lines. Glycerophosphocholine, which is elevated five- to sixfold in constitutively transfected ras cells, did not increase at early times of induction, suggesting the absence of increased phosphatidylcholine degradation via a phospholipase A. Choline kinase activity increased within 4-6 h upon induction and correlated well with the increase in phosphocholine levels. This increase in phosphocholine levels could be prevented by the addition of hemicholinium-3, a competitive inhibitor of choline kinase. Expression of activated c-raf or v-raf also increased choline kinase activity, suggesting that the induction of choline kinase by ras is downstream of the ras/raf interaction. Long-term and short-term labeling experiments failed to detect evidence for increased phospholipase C activity. These results suggest that the increase in choline kinase activity observed in cells expressing ras is an early, integral part of ras transformation and is the main contributor to increased phosphocholine levels accompanying morphological changes.

Ponting CP
AF-6/cno: neither a kinesin nor a myosin, but a bit of both.
Trends Biochem Sci. 1995; 20: 265-6

Herman WH, Simonson MS
Nuclear signaling by endothelin-1. A Ras pathway for activation of the c-fos serum response element.
J Biol Chem. 1995; 270: 11654-61
Display abstract
Endothelin-1 (ET-1) regulates gene expression and growth of vascular cells by triggering signals that link its cognate, G protein-coupled receptor in the plasma membrane to transcriptional activation of immediate early genes in the nucleus. To define the nature of these signals, we asked whether Ras proteins contribute to activation of the c-fos serum response element (SRE) by ET-1 in mesangial cells, a microvascular cell from the renal glomerulus. ET-1 stimulated Ras by increasing Ras GTP loading. Addition of ET-1 or transfection with a plasmid expressing v-Ha-Ras stimulated SRE-dependent transcription. Activation of the c-fos SRE by ET-1 was blocked by a dominant negative Asn-17 c-Ha-Ras mutant. Expression of v-Ha-Ras reversed inhibition of ET-1-stimulated SRE transcriptional activity by Asn-17 c-Ha-Ras. ET-1 also stimulated kinase activity of c-Raf-1, a downstream effector in Ras signaling cascades. Activation of the c-fos SRE by transfection with a plasmid expressing constitutively activated delta Raf-1 was consistent with a role for Ras-Raf-1 in ET-1 signaling. Interestingly, Ras-dependent SRE activation in cells treated with ET-1 was blocked by point mutations in the SRE CArG DNA sequence, which binds the serum response factor, but not by mutations that inhibit binding of ternary complex factors (p62TCF) to the Ets DNA sequence of the SRE. Thus, Ras contributes to a nuclear signaling cascade linking ET-1 receptors to transcriptional activation through the CArG cis-element of the c-fos SRE.

Poullet P, Crechet JB, Bernardi A, Parmeggiani A
Properties of the catalytic domain of sdc25p, a yeast GDP/GTP exchange factor of Ras proteins. Complexation with wild-type Ras2p, [S24N]Ras2p and [R80D, N81D]Ras2p.
Eur J Biochem. 1995; 227: 537-44
Display abstract
The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc25p-C was stronger for Ras2p.GDP than Ras2p.GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p.GDP (GTP) complex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc25p-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc25p-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) showing the same specific activity as the 64-kDa Sdc25p-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc25p.

Indolfi C et al.
Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo.
Nat Med. 1995; 1: 541-5
Display abstract
Proliferation of smooth muscle cells of the arterial wall in response to local injury is an important aetiologic factor of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. Ras proteins are key transducers of mitogenic signals from membrane to nucleus in many cell types. We investigated the role of ras proteins in the vascular response to arterial injury by inactivating cellular ras of rats in which the common carotid artery was subjected to balloon injury. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury. Our results indicate a key role for ras in smooth muscle cell proliferation and show that the local delivery of transdominant negative mutants of ras in vivo might prevent some of the acute vascular injury caused by balloon injury.

Monaco R, Chen JM, Friedman FK, Brandt-Rauf P, Chung D, Pincus MR
Structural effects of the binding of GTP to the wild-type and oncogenic forms of the ras-gene-encoded p21 proteins.
J Protein Chem. 1995; 14: 721-9
Display abstract
Molecular dynamics calculations have been performed to determine the average structures of ras-gene-encoded p21 proteins bound to GTP, i.e., the normal (wild-type) protein and two oncogenic forms of this protein, the Val 12- and Leu 61-p21 proteins. We find that the average structures for all of these proteins exhibit low coordinate fluctuations (which are highest for the normal protein), indicating convergence to specific structures. From previous dynamics calculations of the average structures of these proteins bound to GDP, major regional differences were found among these proteins [Monaco et al. (1995), J. Protein Chem., in press]. We now find that the average structures of the oncogenic proteins are more similar to one another when the proteins are bound to GTP than when they are bound to GDP [Monaco et al. (1995), J. Protein Chem., in press]. However, they still differ in structure at specific amino acid residues rather than in whole regions, in contradistinction to the results found for the p21-GDP complexes. Two exceptions are the regions 25-32, in an alpha-helical region, and 97-110. The two oncogenic (Val 12- and Leu 61-) proteins have similar structures which differ significantly in the region of residues 97-110. This region has recently been identified as being critical in the interaction of p21 with kinase target proteins. The differences in structure between the oncogenic proteins suggest the existence of more than one oncogenic form of the p21 protein that can activate different signaling pathways.

Hilgenfeld R
Regulatory GTPases.
Curr Opin Struct Biol. 1995; 5: 810-7
Display abstract
The past year has witnessed a tremendous increase in our understanding of the structures and interactions of the GTPases. The highlights include crystal structures of G alpha subunits, as well as the first complex between a GTPase (Rap1A) and an effector molecule (c-Raf1 Ras-binding domain). In the field of elongation factors (EFs), three very important structures have been determined: EF-G, the ternary complex of EF-Tu.GTP with aminoacyl-tRNA, and the EF-Tu.EF-Ts complex.

Zhong JM, Chen-Hwang MC, Hwang YW
Switching nucleotide specificity of Ha-Ras p21 by a single amino acid substitution at aspartate 119.
J Biol Chem. 1995; 270: 10002-7
Display abstract
We examined c-Ha-Ras harboring an aspartate to asparagine substitution at position 119 (mutation D119N). The Asp-119 is part of the conserved NKXD motif shared by members of the regulatory GTPase family. This asparagine residue has been proposed to participate in direct bonding to the guanine ring and to determine the guanine-nucleotide binding specificity. The D119N mutation was found to alter nucleotide specificity of Ha-Ras from guanine to xanthine, an observation that directly supports the essential role of hydrogen bonding between the side chain of the aspartic acid residue and the guanine ring in nucleotide binding specificity. Besides nucleotide binding specificity, the D119N mutation has little or no effect on the interaction of Ha-Ras with SDC25C, SOS1, GAP, or Raf. Neither does it affect the hydrolysis of nucleotide triphosphate. Like xanthine-nucleotide-specific EF-Tu, xanthine-nucleotide-specific Ras and related proteins will be useful tools for elucidating cellular systems containing multiple regulatory GTPases.

Qiu RG, Chen J, Kirn D, McCormick F, Symons M
An essential role for Rac in Ras transformation.
Nature. 1995; 374: 457-9
Display abstract
The GTPase Rac1 is a key component in the reorganization of the actin cytoskeleton that is induced by growth factors or oncogenic Ras1. Here we investigate the role of Rac1 in cell transformation and show that Rat1 fibroblasts expressing activated Val-12 Rac1 (Rac1 with valine at residue 12) display all the hallmarks of malignant transformation. In a focus-forming assay in NIH3T3 fibroblasts to measure the efficiency of transformation, we found that dominant-negative Asn-17 Rac1 inhibited focus formation by oncogenic Ras, but not by RafCAAX, a Raf kinase targeted to the plasma membrane by virtue of the addition of a carboxyterminal localization signal from K-Ras. This indicates that Rac is essential for transformation by Ras. In addition, Val-12 Rac1 synergizes strongly with RafCAAX in focus-formation assays, indicating that oncogenic Ras drives both the Rac and MAP-kinase pathways, which cooperate to cause transformation.

Quilliam LA et al.
Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.
Proc Natl Acad Sci U S A. 1995; 92: 1272-6
Display abstract
Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.

Hu CD et al.
Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.
J Biol Chem. 1995; 270: 30274-7
Display abstract
The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

Moodie SA, Paris M, Villafranca E, Kirshmeier P, Willumsen BM, Wolfman A
Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets.
Oncogene. 1995; 11: 447-54
Display abstract
Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.

Niehof M, Radziwill G, Klauser S, Moelling K
A small peptide derived from the aminoterminus of c-Raf-1 inhibits c-Raf-1/Ras binding.
Biochem Biophys Res Commun. 1995; 206: 46-50
Display abstract
Various domains of the aminoterminal part of c-Raf-1 expressed as glutathione-S-transferase fusion proteins were analyzed for Ras binding. The binding site was localized at the aminoterminus outside of the cysteine-rich region. A single aminoacid exchange at aminoacid residue 89 (Arg89 to Leu) of c-Raf-1 inhibits binding. A small synthetic peptide corresponding to c-Raf-1 aminoacids 77 to 101 comprising Arg89 in a central position competes for Ras binding and thereby characterizes the relevant binding domain of Ras on c-Raf-1.

Herrmann C, Martin GA, Wittinghofer A
Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase.
J Biol Chem. 1995; 270: 2901-5
Display abstract
The Ras-binding domain (RBD) of human Raf-1 was purified from Escherichia coli, and its interaction with Ras was investigated. Its dissociation constant with p21ras.guanyl-5'-yl imidodiphosphate was found to be 18 nM, with a slight preference for H-ras over K- and N-ras. Oncogenic forms bind with slightly lower affinity. The affinity of RBD for effector region mutants or the GDP-bound form of p21ras is in the micromolar range, which means that 100-fold lower affinity is not sufficient for signal transduction. The rate of the GTPase of p21ras is not modified by RBD. Since P(i) release is found not to be rate limiting, the Ras-Raf signal of the cell may be terminated by the intrinsic GTPase of p21ras.

Ishihara H, Kimura M, Kuzumaki N, Ono K
Specific detection of the precursor of ras p21 with a mouse monoclonal anti-C-terminal peptide antibody, SARA-K1.
J Immunol Methods. 1995; 185: 217-23
Display abstract
In an attempt to clarify the post-translational modifications of ras oncogene product p21, we have established a mouse monoclonal antibody specific for the precursor of p21. The C-terminal peptide (156-188) of K(4A)-ras oncogene product p21 (p21K(4A), termed K(4A)-peptide, was used as the immunogen. In Western blotting, monoclonal antibodies were examined for their differential reactivity between two types of p21K(4A) expressed in Escherichia coli (esh-p21K(4A)) and mammalian cell (mam-p21K(4A)). One monoclonal antibody, designated SARA-K1, reacted selectively with esh-p21K(4A). The epitope for SARA-K1 was defined on tryptic peptide (177-184), containing Cys180, of the K(4A)-peptide. Pulse-chase experiments of mam-p21K(4A) synthesis at 24 degrees C revealed that SARA-K1 precipitated a 21 kDa protein within a 7 min chase but not after a 10 min chase, indicating that SARA-K1 recognizes the precursor of mam-p21K(4A). Furthermore, in Triton X-114 partitioning experiments using mammalian cells pre-treated with Mevalotin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, SARA-K1 precipitated [35S]methionine-labeled, [3H]mevalonic acid-unlabeled mam-p21K(4A) in the aqueous phase, but did not precipitate [3H]mevalonic acid-labeled mam-p21K(4A) in either aqueous or detergent phase. The data presented clearly show that the SARA-K1 specifically recognizes the primary translational product pro-p21K(4A).

Ikeda M, Koyama S, Okazaki M, Dohi K, Kikuchi A
rap1 p21 regulates the interaction of ras p21 with RGL, a new effector protein of ras p21.
FEBS Lett. 1995; 375: 37-40
Display abstract
We have recently found that ralGDS family members (RGL and ralGDS) are putative effector proteins of ras p21. rap1 p21 is a small GTP-binding protein which has the same amino acid sequence as the effector loop of ras p21. We examined the effect of rap1 p21 on the interaction of ras p21 with RGL. The GTP-bound form of rap1 p21 interacted with RGL as well as did ras p21. rap1 p21 inhibited the interaction of ras p21 with RGL. RGL was phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). Phosphorylation of RGL did not affect its binding to ras p21 and rap1 p21 under the conditions that phosphorylation of Raf-1 reduced its affinity for ras p21. These results demonstrate that rap1 p21 but not protein kinase A regulates the interaction of ras p21 with RGL and suggest that rap1 p21 and protein kinase A may cooperate to distinguish the signal or ras p21 to RGL from that to Raf-1.

Barnard D et al.
Identification of the sites of interaction between c-Raf-1 and Ras-GTP.
Oncogene. 1995; 10: 1283-90
Display abstract
Specific sites of protein-protein interaction were identified in the 51-149 region of c-Raf-1 using contact epitope scanning and site-directed mutagenesis. Nineteen overlapping peptides based upon the primary sequence of the Ras binding domain of c-Raf-1 were tested for the ability to competitively inhibit complex formation between Ras-GTP and the c-Raf-1 N-terminus. A peptide containing c-Raf-1 residues 91-105 as well as five overlapping peptides covering a region extending from residues 118 to 143 interfered with Ras association, defining these sites as potential contact surfaces with Ras. Alanine scanning mutagenesis was used as a second probe for sites of Ras interaction with the c-Raf-1 N-terminus. Raf residues 64-67 and 80-103 were demonstrated as important for association with Ras-GTP with residues 66, 67, 84, 87, 89 and 91 identified as the most critical individual points of contact with the Ras protein. Alanine substitution of residues between 118-143 suggested only one potentially weak site of interaction defined by residues 120-125. The combined results of both peptide and mutagenic analyses suggest that the primary site of c-Raf-1 interaction with Ras maps to Raf residues 80-103, with secondary interactions occurring with residues 66 and 67 and possibly 120-125. Contact epitope scanning of the Ras effector region found maximum inhibition of Ras/Raf association with a peptide corresponding to Ras amino acids 37-51. A model is proposed for the GTP-dependent association of Ras and Raf.

Surridge C
Picture story. Ras and Rap opposition.
Nat Struct Biol. 1995; 2: 536-536

Moodie SA, Paris MJ, Kolch W, Wolfman A
Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf.
Mol Cell Biol. 1994; 14: 7153-62
Display abstract
We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.

Stokoe D, Macdonald SG, Cadwallader K, Symons M, Hancock JF
Activation of Raf as a result of recruitment to the plasma membrane.
Science. 1994; 264: 1463-7
Display abstract
The small guanine nucleotide binding protein Ras participates in a growth promoting signal transduction pathway. The mechanism by which interaction of Ras with the protein kinase Raf leads to activation of Raf was studied. Raf was targeted to the plasma membrane by addition of the COOH-terminal localization signals of K-ras. This modified form of Raf (RafCAAX) was activated to the same extent as Raf coexpressed with oncogenic mutant Ras. Plasma membrane localization rather than farnesylation or the presence of the additional COOH-terminal sequence accounted for the activation of RafCAAX. The activation of RafCAAX was completely independent of Ras; it was neither potentiated by oncogenic mutant Ras nor abrogated by dominant negative Ras. Raf, once recruited to the plasma membrane, was not anchored there by Ras; most activated Raf in cells was associated with plasma membrane cytoskeletal elements, not the lipid bilayer. Thus, Ras functions in the activation of Raf by recruiting Raf to the plasma membrane where a separate, Ras-independent, activation of Raf occurs.

Irie K, Gotoh Y, Yashar BM, Errede B, Nishida E, Matsumoto K
Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase.
Science. 1994; 265: 1716-9
Display abstract
Intracellular signaling from receptor tyrosine kinases in mammalian cells results in activation of a signal cascade that includes the guanine nucleotide-binding protein Ras and the protein kinases Raf, MEK [mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase], and MAPK. MAPK activation that is dependent on the coupling of Ras and Raf was reconstituted in yeast. Yeast genes were isolated that, when overexpressed, enhanced the function of Raf. One of them is identical to BMH1, which encodes a protein similar to members of the mammalian 14-3-3 family. Bacterially synthesized mammalian 14-3-3 protein stimulated the activity of Raf prepared from yeast cells expressing c-Raf-1. Thus, the 14-3-3 protein may participate in or be required for activation of Raf.

Emerson SD, Waugh DS, Scheffler JE, Tsao KL, Prinzo KM, Fry DC
Chemical shift assignments and folding topology of the Ras-binding domain of human Raf-1 as determined by heteronuclear three-dimensional NMR spectroscopy.
Biochemistry. 1994; 33: 7745-52
Display abstract
Raf-1 is a 74-kDa serine-threonine kinase which serves as the immediate downstream target of Ras in the cell growth signal transduction pathway. Recent genetic and biochemical experiments have demonstrated that (1) Ras interacts directly with the amino-terminal domain of Raf and (2) residues 51-131 of the Raf sequence are sufficient to mediate this interaction [Vojtek, A. B., Hollenberg, S. M., & Cooper, J. A. (1993) Cell 74, 205-214]. We have expressed a corresponding segment of the human Raf sequence (Raf55-132) in Escherichia coli as a fusion with maltose binding protein. The fusion protein was purified by affinity chromatography and cleaved at a pre-engineered site with factor Xa protease to liberate the 78-residue fragment of Raf. Raf55-132 bound to Ras with high affinity in a competition assay with GAP. An unlabeled version of Raf55-132 was studied by 2D homonuclear NMR, and uniformly 15N- and 13C/15N-labeled versions of Raf55-132 were studied by 2D and 3D heteronuclear NMR. Nearly complete sequence-specific assignments were made for the backbone HN, H alpha, 15N, and 13C alpha resonances. NOEs were used to determine regions of secondary structure and the overall folding topology. Raf55-132 is an independently folded domain composed of a five-stranded beta-sheet, a three-turn alpha-helix, and possibly an additional one-turn helix. Its structure resembles that of ubiquitin, even though there is no more than 11% sequence homology between the two proteins.

Buhl AM, Avdi N, Worthen GS, Johnson GL
Mapping of the C5a receptor signal transduction network in human neutrophils.
Proc Natl Acad Sci U S A. 1994; 91: 9190-4
Display abstract
Human neutrophils respond to chemoattractants, resulting in their accumulation at an inflammatory site. Chemoattractants such as the C5a peptide, derived from the C5 complement factor, bind to inhibitory guanine nucleotide binding protein (Gi)-coupled seven membrane-spanning receptors expressed in neutrophils. C5a receptor activation results in the Gi-dependent activation of the mitogen-activated protein (MAP) kinase pathway in human neutrophils. C5a receptor ligation activates both B-Raf and Raf-1, with B-Raf activation overlapping but temporally distinct from that of Raf-1. B-Raf and Raf-1 both efficiently phosphorylate MAP kinase kinase (MEK-1). C5a also stimulates guanine nucleotide exchange and activation of Ras. Ras and Raf activation in response to C5a involves protein kinase C-dependent and -independent pathways. Activation of both Raf-1 and B-Raf was inhibited by protein kinase A stimulation, consistent with the inhibitory effects of increased cAMP levels on neutrophil function. The findings define a functional signal transduction pathway linking the neutrophil C5a chemoattractant receptor to the regulation of Ras, B-Raf, Raf-1, and MAP kinase.

Chuang E, Barnard D, Hettich L, Zhang XF, Avruch J, Marshall MS
Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.
Mol Cell Biol. 1994; 14: 5318-25
Display abstract
Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.

Hallberg B, Rayter SI, Downward J
Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
J Biol Chem. 1994; 269: 3913-6
Display abstract
It has recently been shown that Ras proteins interact directly with Raf serine/threonine kinases in vitro and in the yeast two-hybrid system, leading to speculation that Raf proteins function as effectors for Ras. Here it is demonstrated that the endogenous Raf-1 protein co-immunoprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-Raf complex is absolutely dependent on prior treatment of the cells with a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epidermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cellular Raf-1 can be found in association with Ras. The association is not competed by addition of exogenous GST-Raf to the cell lysates and is therefore unlikely to be due to Ras-Raf binding after cell lysis. Specific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Ras to become GTP-bound.

Moodie SA, Wolfman A
The 3Rs of life: Ras, Raf and growth regulation.
Trends Genet. 1994; 10: 44-8
Display abstract
Cellular Ras proteins are essential elements in normal signal transduction pathways while activated Ras proteins are prevalent in many different forms of human cancers. Here, we discuss the mechanism through which Ras proteins, either cellular or activated, transmit a proliferative signal by activating cytoplasmic serine/threonine kinases.

Vaillancourt RR, Harwood AE, Winitz S
Analysis of guanine nucleotides associated with protooncogene ras.
Methods Enzymol. 1994; 238: 255-8

Sturgill TW
More evidence for a Gi-regulated ras exchanger.
J Clin Invest. 1994; 94: 471-471

Leevers SJ, Paterson HF, Marshall CJ
Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane.
Nature. 1994; 369: 411-4
Display abstract
A conserved tyrosine kinase-activated signal transduction pathway has recently been identified that comprises the plasma membrane-bound small guanine-nucleotide-binding protein Ras and the protein kinases Raf, MAP-kinase kinase and MAP kinase. GTP-bound Ras interacts directly with the amino-terminal regulatory domain of Raf, but although Ras and Raf can be coimmunoprecipitated from ligand-stimulated cells, Ras-GTP does not stimulate the kinase activity of Raf in vitro. Furthermore, we have failed to detect Ras in preparations of active detergent-solubilized Raf, demonstrating that once it is activated, Raf does not require Ras. Whereas Raf is normally cytosolic, in cells expressing active Ras, Raf is associated with the plasma membrane. This led us to investigate whether Ras is required to localize Raf to the plasma membrane in order for Raf to become activated. We fused the membrane localization signal of K-Ras(4B) to the carboxy terminus of Raf. This protein is constitutively active and can be further activated by epidermal growth factor, independently of Ras. Our results indicate that Ras functions as a regulated, membrane-bound anchor for Raf, and that other signal(s) also contribute to Raf activation.

Spaargaren M, Bischoff JR
Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap.
Proc Natl Acad Sci U S A. 1994; 91: 12609-13
Display abstract
To identify proteins that bind to the Ras-related protein R-ras we performed a yeast two-hybrid cDNA library screen. Several clones were obtained encoding the C-terminal region of the guanine nucleotide dissociation stimulator for Ral (RalGDS). The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions. Using the two-hybrid system, we show that RalGDS-RBD interacts with H-ras, K-ras, and Rap, and with active but not with inactive point mutants of these Ras-like GTPases. Moreover, using purified proteins, we demonstrate the direct GTP-dependent interaction of the Ras-like GTPases with RalGDS-RBD and full-length RalGDS in vitro. Furthermore, we show that RalGDS-RBD and the Ras-binding domain of Raf-1 compete for binding to the Ras-like GTPases. These data indicate that RalGDS is a putative effector molecule for R-ras, H-ras, K-ras, and Rap.

Cook S, McCormick F
Ras blooms on sterile ground.
Nature. 1994; 369: 361-2

Peterson SN, Lapetina EG
Platelet activation and inhibition. Novel signal transduction mechanisms.
Ann N Y Acad Sci. 1994; 714: 53-63

Gulbins E et al.
Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.
Mol Cell Biol. 1994; 14: 4749-58
Display abstract
We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Prendergast GC, Gibbs JB
Ras regulatory interactions: novel targets for anti-cancer intervention?
Bioessays. 1994; 16: 187-91
Display abstract
Advances in the understanding of Ras oncoprotein function suggest novel points for anti-tumor intervention. First, upstream-acting guanine nucleotide exchange factors and SH2/SH3 domain-containing adaptor proteins that link Ras with growth factor receptor tyrosine kinases have recently been characterized. Second, work on downstream-acting Ras effector functions including the Ras GTPase-activating protein (p120GAP) and the Raf kinase has revealed direct biochemical interactions that are functionally required for oncogenic Ras signalling. We summarize progress in these areas and discuss the potential for novel applications to anti-cancer chemotherapy.

Park W, Mosteller RD, Broek D
Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras.
Mol Cell Biol. 1994; 14: 8117-22
Display abstract
Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF). Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid. We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae. Also, peptides corresponding to residues 1364 to 1383 of CDC25GEF inhibit interaction between GEFs and H-Ras. We propose that residues 1374 of CDC25GEF and 63 of H-Ras form an ion pair and that when this ion pair is reversed, functional interaction can still occur.

Ghosh S, Xie WQ, Quest AF, Mabrouk GM, Strum JC, Bell RM
The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras.
J Biol Chem. 1994; 269: 10000-7
Display abstract
Different domains of the serine/threonine kinase, raf-1, were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (molar basis), similar to analogous cysteine-rich domains of protein kinase C. GST-fusion proteins, containing the cysteine-rich domain of raf-1, bound to liposomes in a phosphatidylserine-dependent manner. In contrast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind phorbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bound to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusion protein (residues 1-130 of raf-1) was about 10-fold weaker, inferring that a 17-amino acid sequence represents a critical binding determinant in intact raf-1. These residues are adjacent to the amino-terminal end of, and partially extend into, the cysteine-rich domain (amino acids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of the cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.

Minato T, Wang J, Akasaka K, Okada T, Suzuki N, Kataoka T
Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras proteins.
J Biol Chem. 1994; 269: 20845-51
Display abstract
Ras proteins appear to have two distinct downstream effectors, adenylyl cyclase in Saccharomyces cerevisiae and a product of raf-1 protooncogene in higher organisms. We found that in vitro activation of adenylyl cyclase by yeast Ras2 and human H-Ras proteins is subject to competitive inhibition by its leucine-rich repeats domain and by the N-terminal regulatory domain of human Raf-1 protein. Kinetic analyses of the inhibition patterns enabled us to determine exact dissociation constants (Kd) of the two polypeptides for Ras2 and H-Ras. The leucine-rich repeats domain bound to the posttranslationally modified Ras2 with the Kd of approximately 13 nM, which was close to the value (7 nM) of the whole adenylyl cyclase. The Kd of Raf-1 for the modified H-Ras, 3.5 nM, was significantly lower than that for Ras2, 24 nM, whereas adenylyl cyclase bound preferentially to Ras2. Similar inhibition was also observed in vivo by suppression of RAS2Val-19-dependent heat shock sensitivity and of Ras-dependent cAMP response to glucose upon overexpression of Raf-1 in yeast. These results indicate that the leucine-rich repeats domain contains the Ras protein-binding site and that Raf-1 and adenylyl cyclase, sharing no structural homology with each other, bind to a similar, if not identical, region of Ras with comparable affinities.

Klinz FJ
GTP-blot analysis of small GTP-binding proteins. The C-terminus is involved in renaturation of blotted proteins.
Eur J Biochem. 1994; 225: 99-105
Display abstract
Recombinant c-Ha-ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [alpha-32P]GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c-Has-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-32P]GTP was decreased many-fold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process. Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [alpha-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [alpha-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [alpha-32P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras. Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.

Demo SD, Kikuchi A, Peters KG, MacNicol AM, Muslin AJ, Williams LT
Signaling molecules that mediate the actions of FGF.
Princess Takamatsu Symp. 1994; 24: 243-9
Display abstract
Signaling by fibroblast growth factor (FGF) is critical for cellular growth and differentiation. We have studied the role of FGF and its receptor in development and also the signaling pathways utilized by these molecules. Transgenic mice expressing a dominant negative FGF receptor specifically in lung tissue fail to develop lungs. These findings suggest that FGF signaling is important for airway branching and alveolar formation. In Xenopus embryos, FGF is involved in the development of posterior structures. Induction of mesoderm by FGF is mediated by the serine-threonine kinase RAF. The expression of a dominant negative RAF selectively blocks the mesoderm-inducing action of FGF. Expression of activated RAF accentuated the development of posterior structures Other intracellular molecules that are involved with FGF signaling include RAS, a low-molecular weight GTP-binding protein. We have identified a novel gene (RIP, for RAS-interacting protein) that contains a RAS-binding domain. This domain is found in at least one other protein, ralGDS. Both RIP and ralGDS bind to activated RAS suggesting that these proteins are likely candidates for mediators of RAS function. These findings help elucidate the role of FGF signaling in development.

Ruggieri R, Macdonald SG, Callow M, McCormick F
Raf-1 interferes with Ras and Rap1A effector functions in yeast.
Mol Biol Cell. 1994; 5: 173-81
Display abstract
Raf-1 is a serine/threonine kinase that acts downstream of Ras in mitogenic signal transduction pathways, but the mechanism by which Ras transmits signals to Raf-1 is not known. We have examined the interaction between Raf-1 and human H-ras in three different systems that utilize H-ras-induced phenotypes in Saccharomyces cerevisiae. In each system, the effects of H-ras depend on guanosine triphosphate and appear to be mediated through the H-ras effector binding region. H-ras effector function was blocked in each case by expression of the N-terminal regulatory domain of Raf-1. These inhibitory effects did not require the Raf-1 kinase domain. Raf-1 also blocked Rap1A effector function in S. cerevisiae. Raf-1, therefore, appears to interact with H-Ras and Rap1A in these in vivo systems with properties that suggest it is an immediate downstream effector.

Feig LA
The many roads that lead to Ras.
Science. 1993; 260: 767-8

Warne PH, Viciana PR, Downward J
Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.
Nature. 1993; 364: 352-5
Display abstract
The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

Zhang XF et al.
Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1.
Nature. 1993; 364: 308-13
Display abstract
In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.

Van Aelst L, Barr M, Marcus S, Polverino A, Wigler M
Complex formation between RAS and RAF and other protein kinases.
Proc Natl Acad Sci U S A. 1993; 90: 6213-7
Display abstract
We used a Saccharomyces cerevisiae genetic system to detect the physical interaction of RAS and RAF oncoproteins. We also observed interaction between RAS and byr2, a protein kinase implicated as a mediator of the Schizosaccharomyces pombe ras1 protein. Interaction with RAS required only the N-terminal domains of RAF or byr2 and was disrupted by mutations in either the guanine nucleotide-binding or effector-loop domains of RAS. We observed interaction between MEK (a kinase that phosphorylates mitogen-activated protein kinases) and the catalytic domain of RAF. RAS and MEK also interacted but only when RAF was overexpressed.

Vojtek AB, Hollenberg SM, Cooper JA
Mammalian Ras interacts directly with the serine/threonine kinase Raf.
Cell. 1993; 74: 205-14
Display abstract
We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library. Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region. We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.

Koide H, Satoh T, Nakafuku M, Kaziro Y
GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.
Proc Natl Acad Sci U S A. 1993; 90: 8683-6
Display abstract
Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells.

Albright CF, Giddings BW, Liu J, Vito M, Weinberg RA
Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase.
EMBO J. 1993; 12: 339-47
Display abstract
ras-related GTPases participate in signaling for a variety of cellular processes. The GTPases cycle between a GTP-bound active state and a GDP-bound inactive state. This cycling is partially controlled by guanine nucleotide dissociation stimulators (GDS, also known as exchange factors). We report on the molecular cloning of cDNAs encoding a new mammalian GDS protein, using sequences derived from the yeast ras GDS proteins as probes. The encoded protein stimulates the dissociation of guanine nucleotides from the ras-related ralA and ralB GTPases at a rate at least 30-fold faster than the intrinsic nucleotide dissociation rate. This new GDS, ralGDS, is at least 20-fold more active on the ralA and ralB GTPases than on any other GTPase tested, including other members of the ras family (H-ras, N-ras, K-ras, R-ras, rap1a and rap2), members of the rho family (rhoA, rhoB and CDC42-Hs) and members of the rab family (rab3a and ypt1). While the ralGDS protein is phosphorylated on serine residues, we find no evidence that phosphorylation affects the activity of insect cell-expressed ralGDS towards the ralA or ralB GTPase. The 3600 nucleotide ralGDS mRNA and the 115 kDa protein were found in all tissues and cell lines examined.

Mistou MY et al.
Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.
EMBO J. 1992; 11: 2391-7
Display abstract
The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.

Trueb J, Trueb B
Molecular cloning of a novel ras-like protein from chicken.
FEBS Lett. 1992; 306: 181-4
Display abstract
We have isolated a cDNA clone from a chicken DNA expression library which codes for a ras-like polypeptide of 216 amino acid residues. This polypeptide is closely related to the human protein TC4 and to the yeast protein Spil, two novel proteins that may be involved in the coordination of the cell cycle. In the amino-terminal region, the three polypeptides possess a P-loop motif characteristic of GTP-binding proteins. At the carboxy-terminal end, however, they lack the typical CAAX-box which is usually responsible for membrane anchorage of ras-like proteins. It is therefore likely that the three polypeptides define a new subclass of GTP-binding proteins within the ras-like superfamily.

Buss JE et al.
The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.
Mol Cell Biol. 1991; 11: 1523-30
Display abstract
Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

Kawata M et al.
The molecular heterogeneity of the smg-21/Krev-1/rap1 proteins, a GTP-binding protein having the same effector domain as ras p21s, in bovine aortic smooth muscle membranes.
Oncogene. 1991; 6: 841-8
Display abstract
smg p21 is a member of the ras p21/ras p21-like small GTP-binding protein (G protein) superfamily, having the same putative effector domain as ras p21s. In the preceding report, we showed that smg p21 was a major G protein in bovine aortic smooth muscle membranes. Recently, two different smg p21 cDNA clones, designated smg-21A and -B, were isolated from a bovine brain cDNA library. In the present studies, we resolved the bovine aortic smg p21 fraction into two distinct G protein fractions on hydroxyapatite column chromatography and purified them separately to near homogeneity (22K G1 and -2). Both 22K G1 and -2 were specifically recognized by an anti-smg p21 polyclonal antibody. 22K G1 and -2 were identified as smg p21B and -A, respectively, by peptide map and amino acid sequence analyses. Purified smg p21A and -B showed GDP/GTP-binding and GTPase activities similar to each other. The GTPase activities of smg p21A and -B were equally stimulated by smg p21 GTPase activating protein 1 and -2. Moreover, both smg p21A and -B were phosphorylated by cyclic AMP-dependent protein kinase with a stoichiometry of one phosphate/molecule of protein. These results indicate that smg p21A and -B coexist in bovine aortic smooth muscle membranes and suggest that smg p21A and -B may serve as intermediates for cyclic AMP actions.

Cosic I, Hearn MT
'Hot spot' amino acid distribution in Ha-ras oncogene product p21: relationship to guanine binding site.
J Mol Recognit. 1991; 4: 57-62
Display abstract
Although the rules which describe the atomic basis of structure-function relationships of proteins have yet to be deciphered, they are nevertheless coded within the framework of the amino acid and nucleotide sequence. The objectives of the present investigations were to document a composite, new approach for the evaluation of the structure-function dependencies of proteins based on the analysis of the informational content of the primary amino acid sequence as well as the topological and functional regions of a protein. This approach is validated with the example of the p21 Ha-ras oncogene family of proteins. Using this approach, amino acids crucial for p21 transforming activity have been identified and these amino acid residue assignments compared with experimental data.

Quilliam LA et al.
Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils.
J Immunol. 1991; 147: 1628-35
Display abstract
The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.

Tong LA, de Vos AM, Milburn MV, Kim SH
Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP.
J Mol Biol. 1991; 217: 503-16
Display abstract
The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.

Winegar DA, Ohmstede CA, Chu L, Reep B, Lapetina EG
Antisera specific for rap 1 proteins distinguish between processed and nonprocessed rap 1b.
J Biol Chem. 1991; 266: 4375-80
Display abstract
Polyclonal antisera were generated against synthetic peptides corresponding to distinct regions of the rap 1 protein sequences. A "rap 1-common" antiserum, prepared against an 18-amino acid segment of the rap 1a protein near the proposed GTP-binding region, reacted with both rap 1a and rap 1b recombinant proteins expressed in Escherichia coli and with two low molecular weight GTP-binding proteins of 22 and 24 kDa in unstimulated human platelets. An antiserum raised against a carboxyl-terminal peptide of rap 1b containing the putative site of post-translational processing reacted strongly with bacterial-expressed recombinant rap 1b and with a 24-kDa GTP-binding protein in platelets, but not with recombinant rap 1a or a 22-kDa GTP-binding protein. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this rap 1b immunoreactive protein coincided with that of bacterial-expressed rap 1b and not with the faster migrating form of rap 1b that incorporates radioactivity from [3H]mevalonic acid in the insect/baculovirus system. This suggests that our rap 1b-specific antiserum recognizes only one form of rap 1b, that which has not undergone carboxyl-terminal post-translational processing.

Casey PJ, Thissen JA, Moomaw JF
Enzymatic modification of proteins with a geranylgeranyl isoprenoid.
Proc Natl Acad Sci U S A. 1991; 88: 8631-5
Display abstract
The prenylation of several proteins involved in oncogenesis and signal transduction plays an essential role in regulating their biological activities. Two distinct isoprenoids are known to be involved in this modification, the 15-carbon farnesyl and 20-carbon geranylgeranyl groups. Thus far, identified farnesylated proteins contain methionine or serine at the COOH terminus, while those modified by geranylgeranyl end in leucine. This report describes the characterization of an enzyme activity that transfers the geranylgeranyl group to candidate proteins. The enzyme, termed a "protein geranylgeranyltransferase," exhibits a marked preference for substrate proteins that contain leucine at the COOH terminus. In fact, the enzyme will efficiently modify a normally farnesylated protein, Ha-ras, if its COOH-terminal amino acid is switched from serine to leucine. Additional studies characterize this enzyme and suggest that it is responsible for the geranylgeranyl modification of a number of GTP-binding proteins (or their subunits) that contain a consensus prenylation sequence ending in leucine.

Kitayama H, Matsuzaki T, Ikawa Y, Noda M
Genetic analysis of the Kirsten-ras-revertant 1 gene: potentiation of its tumor suppressor activity by specific point mutations.
Proc Natl Acad Sci U S A. 1990; 87: 4284-8
Display abstract
Kirsten-ras-revertant 1 (Krev-1) cDNA encodes a ras-related protein and exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when introduced into a v-K-ras-transformed mouse NIH 3T3 cell line, DT. Toward understanding the mechanism of action of Krev-1 protein, we constructed a series of point mutants of Krev-1 cDNA and tested their biological activities in DT cells and HT1080 human fibrosarcoma cells harboring the activated N-ras gene. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp17 and Asn116), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys181), and at the unique Thr61 all decreased the transformation suppressor activity. On the other hand, substitutions such as Gly12 to Val12 and Gln63 to Glu63 were found to significantly increase the transformation suppressor/tumor suppressor activity of Krev-1. These findings are consistent with the idea that Krev-1 protein is regulated like many other G proteins by the guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.

Plutner H, Schwaninger R, Pind S, Balch WE
Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway.
EMBO J. 1990; 9: 2375-83
Display abstract
Synthetic peptides of the putative effector domain of members of the ras-related rab gene family of small GTP-binding proteins were synthesized and found to be potent inhibitors of endoplasmic reticulum (ER) to Golgi and intra-Golgi transport in vitro. Inhibition of transport by one of the effector domain peptides was rapid (t1/2 of 30 s), and irreversible. Analysis of the temporal site of peptide inhibition indicated that a late step in transport was blocked, coincident with a Ca2(+)-dependent prefusion step. The results provide novel biochemical evidence for the role of members of the rab gene family in vesicular transport in mammalian cells, and implicate a role for a new downstream Rab effector protein (REP) regulating vesicle fusion.

Arabshahi L, Khan NN, Butler M, Noonan T, Brown NC, Wright GE
(Difluoromethylene)phosphates of guanine nucleosides as probes of DNA polymerases and G proteins.
Biochemistry. 1990; 29: 6820-6
Display abstract
5'-Polyphosphates of N2-(p-n-butylphenyl)-2'-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5'-[beta,gamma-(Difluoromethylene)triphosphates], including that of 2'-deoxyguanosine, were prepared by reaction of the corresponding 5'-phosphates, activated by 1,1'-carbonyldiimidazole, with difluoromethanediphosphonate. The 5'-[(difluoromethylene)diphosphate] of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5'-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with 1,1'-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5'-[(alpha,beta-difluoromethylene)triphosphate]. Products were characterized by 31P and 19F NMR spectroscopy. The phosphonates were tested for their ability to displace [3H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase alpha from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF2 group was in the beta,gamma position, but not when it was in the alpha,beta position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF2 group had no effect on inhibition of DNA polymerase alpha by N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphospate]. 2'-Deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.

Chen JM, Lee G, Murphy RB, Brandt-Rauf PW, Pincus MR
Comparisons between the three-dimensional structures of the chemotactic protein CheY and the normal Gly 12-p21 protein.
Int J Pept Protein Res. 1990; 36: 1-6
Display abstract
The three-dimensional structure of a chemotactic protein CheY from Salmonella typhimurium has recently been determined by X-ray crystallography. The structure of this small protein, containing 129 amino acid residues, shows a domain consisting of a central beta-pleated sheet surrounded on both sides by alpha-helices. We have examined the sequence and the arrangement of the structural domains of the CheY protein and have compared them with other nucleotide binding protein sequences and structures. We find that the CheY protein has significant sequence homology to the ras-gene encoded p21 protein. In addition, the structural domains of the two proteins are arranged in a fundamentally similar manner, including the phosphate-binding site (both proteins bind phosphate-containing ligands). The striking similarity in the arrangement of the structural domains of the two proteins suggests that both may serve similar functions as signal transducers.

Frech M, Schlichting I, Wittinghofer A, Chardin P
Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli.
J Biol Chem. 1990; 265: 6353-9
Display abstract
The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.

Brandt-Rauf PW, Carty RP, Chen JM, Lee G, Rackovsky S, Pincus MR
The structure of the carboxyl terminus of the p21 protein. Structural relationship to the nucleotide-binding/transforming regions of the protein.
J Protein Chem. 1990; 9: 137-42
Display abstract
The carboxyl-terminal region of the ras oncogene-encoded p21 protein is critical to the protein's function, since membrane binding through the C-terminus is necessary for its cellular activity. X-ray crystal structures for truncated p21 proteins are available, but none of these include the C-terminal region of the protein (from residues 172-189). Using conformational energy analysis, we determined the preferred three-dimensional structures for this C-terminal octadecapeptide of the H-ras oncogene p21 protein and generated these structures onto the crystal structure of the remainder of the protein. The results indicate that, like other membrane-associated proteins, the membrane-binding C-terminus of p21 assumes a helical hairpin conformation. In several low-energy orientations, the C-terminal structure is in close proximity to other critical locales of p21. These include the central transforming region (around Gln 61) and the amino terminal transforming region (around Gly 12), indicating that extracellular signals can be transduced through the C-terminal helical hairpin to the effector regions of the protein. This finding is consistent with the results of recent genetic experiments.

John J, Schlichting I, Schiltz E, Rosch P, Wittinghofer A
C-terminal truncation of p21H preserves crucial kinetic and structural properties.
J Biol Chem. 1989; 264: 13086-92
Display abstract
The human c-Ha-ras protooncogene product p21C was truncated at the C terminus by 23 amino acids. The resulting G-binding domain, p21 (1-166) = p21C', can be crystallized as a complex with the slowly hydrolyzing GTP analogues guanosin-5'-[beta,gamma-imido]triphosphate, guanosin-5'-[beta,gamma-methylene]triphosphate, and guanosin-5'-O-(3-thiotriphosphate). We show here that this protein has biochemical properties very similar to those of the intact protein. Activating mutations in position 12 (Gly12----Val; Gly12----Arg) have the same effect on the properties of the truncated protein as on intact protein. Nuclear magnetic resonance (NMR) measurements show no apparent effect of the C-terminal deletion on the solution structure of p21. This suggests that neither the structure of the G-binding domain nor any of its biochemical properties are markedly influenced by the truncation.

Garrett MD, Self AJ, van Oers C, Hall A
Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins.
J Biol Chem. 1989; 264: 10-3
Display abstract
The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.

Gautam N, Baetscher M, Aebersold R, Simon MI
A G protein gamma subunit shares homology with ras proteins.
Science. 1989; 244: 971-4
Display abstract
Guanine nucleotide binding proteins (G proteins) that transduce signals from cell surface receptors to effector molecules are made up of three subunits, alpha, beta, and gamma. A complementary DNA clone that encodes a 71-amino acid protein was isolated from bovine brain; this protein contains peptide sequences that were derived from the purified gamma subunit of Gi and Go. The primary sequence of this G protein gamma subunit (G gamma) has 55 percent homology to the gamma subunit of transducin (T gamma) and also has homology to functional domains of mammalian ras proteins. The probe for isolating the clone was generated with the use of the polymerase chain reaction (PCR). The extent of divergence between T gamma and G gamma, the isolation of homologous PCR-generated fragments, and the differences between the predicted amino acid sequence of G gamma and that derived from the gamma subunit of Gi and Go indicate that gamma subunits are encoded by a family of genes.

Kim S, Kikuchi A, Mizoguchi A, Takai Y
Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain.
Brain Res Mol Brain Res. 1989; 6: 167-76
Display abstract
We have purified to near homogeneity and characterized three small molecular weight (Mr) GTP-binding proteins, the c-Ki-ras protein (c-Ki-ras p21), the rho protein (rho p20) and a novel smg-25A protein (smg p25A), from bovine brain crude membranes. In the present studies, the intrasynaptosomal distribution of these 3 small Mr G protein has been investigated using bovine brain. ras p21 and rho p20 are found in the synaptosomal membrane fraction but not in the synaptosomal soluble fraction. In contrast, smg p25A is found in both the synaptosomal membrane and soluble fractions. These results indicate that the intrasynaptosomal distribution of these small Mr G proteins is different and suggest that they are involved in different neuronal functions.

Holbrook SR, Kim SH
Molecular model of the G protein alpha subunit based on the crystal structure of the HRAS protein.
Proc Natl Acad Sci U S A. 1989; 86: 1751-5
Display abstract
A structural model of guanine nucleotide-binding regulatory protein alpha subunits (G alpha subunits) is proposed based on the crystal structure of the catalytic domain of the human HRAS protein (p21ras). Because of low overall sequence similarity, structural and functional constraints were used to align the G alpha consensus sequence with that of p21ras. The resulting G alpha model specifies the spatial relationship among the guanine nucleotide-binding site, the binding site of the beta gamma subunit complex, likely regions of effector and receptor interaction, and sites of cholera and pertussis toxin modification. The locations in the model of the experimentally determined sites of proteolytic digestion, point mutation, monoclonal antibody binding, and toxin modification are consistent with and help explain the observed biological activity. Two important findings from our model are (i) the orientation of the G alpha model with respect to the membrane and (ii) the identification of the spatial proximity of the N- and C-terminal regions. Furthermore, by analogy to p21ras, the model assigns specific residues in G alpha required for binding the guanosine (G-box) and phosphates (PO4-box) and identifies residues potentially involved in the conformational switch mechanism (S-box). Specification of these critical regions in the G alpha model suggests guidelines for construction of mutants and chimeric proteins to experimentally test structural and functional hypotheses.

Aktories K, Braun U, Rosener S, Just I, Hall A
The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3.
Biochem Biophys Res Commun. 1989; 158: 209-13
Display abstract
The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.

Quinn MT, Parkos CA, Walker L, Orkin SH, Dinauer MC, Jesaitis AJ
Association of a Ras-related protein with cytochrome b of human neutrophils.
Nature. 1989; 342: 198-200
Display abstract
Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b with other cytosolic and membrane proteins. We have now co-isolated by conventional purification procedures a protein of relative molecular mass 22,000 with cytochrome b. This Ras-related protein is not a fragment of either of the subunits of cytochrome b, and its primary structure, as determined by the sequencing of its complementary DNA, is identical to that predicted from a recently cloned ras-related gene, rap1 (also termed Krev-1). Immunoaffinity purification on anti-cytochrome and anti-Ras immunoaffinity matrices indicates an association between cytochrome b and the Ras-related protein. The association of a Ras-related GTP-binding protein with cytochrome b of human neutrophils could indicate a role for such a protein in the transduction, regulation or structure of the superoxide generating system.

de Gunzburg J, Riehl R, Weinberg RA
Identification of a protein associated with p21ras by chemical crosslinking.
Proc Natl Acad Sci U S A. 1989; 86: 4007-11
Display abstract
The products of the ras oncogenes (p21ras) are ubiquitous membrane-associated proteins that bind guanine nucleotides and possess an intrinsic GTPase activity. Because of their functional homologies with regulatory guanine nucleotide-binding proteins, they are thought to be involved in the control of cellular proliferation as transducers of incoming growth signals. In an effort to identify proteins interacting with p21ras, we have used in vivo crosslinking techniques on Rat-1 fibroblasts and derived cell lines overexpressing p21ras and immunoprecipitation with polyclonal anti-p21ras antibodies. Under those conditions, using the homobifunctional crosslinker dithiobis(succinimidyl propionate), a protein of Mr 60,000 (p60) is found to be associated with p21ras, and this association is enhanced by the treatment of quiescent cells with serum. Upon sedimentation of detergent extracts from crosslinked cells on sucrose gradients, a p21-p60 complex could be demonstrated with a Mr of 200,000-300,000, p60 does not appear to be related to pp60src nor to the cytosolic GTPase activating protein that interacts with p21ras to enhance its GTPase activity. The amount of p60 seems to be limiting relative to p21ras in fibroblasts, since similar levels of p60 are immunoprecipitated from Rat-1 cells and transfectants overexpressing Ha-, Ki-, and N-ras p21s; the same protein is also found to associate with p21ras in numerous mammalian cell lines. The relevance of this component to the role of ras proteins in signal transduction is discussed.

Ueda T, Kikuchi A, Ohga N, Yamamoto J, Takai Y
GTPase activating proteins for the smg-21 GTP-binding protein having the same effector domain as the ras proteins in human platelets.
Biochem Biophys Res Commun. 1989; 159: 1411-9
Display abstract
Two proteins stimulating the GTPase activity of the smg-21 GTP-binding protein (smg p21) having the same effector domain as the ras proteins (ras p21s) are partially purified from the cytosol fraction of human platelets. These proteins, designated as smg p21 GTPase activating protein (GAP) 1 and 2, do not stimulate the GTPase activity of c-Ha-ras p21. The GAP activity for c-Ha-ras p21 is also detected in the cytosol fraction of human platelets. smg p21 GAP1 and 2 are separated from c-Ha-ras p21 GAP by column chromatographies. The activity of smg p21 GAP1 and 2 is killed by tryptic digestion or heat boiling. The Mr values of smg p21 GAP1 and 2 are similar and are estimated to be 2.5-3.5 x 10(5) by gel filtration analysis. These results indicate that there are two GAPs for smg p21 in addition to a GAP for c-Ha-ras p21 in human platelets.

Coburn J, Wyatt RT, Iglewski BH, Gill DM
Several GTP-binding proteins, including p21c-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzyme S.
J Biol Chem. 1989; 264: 9004-8
Display abstract
Pseudomonas aeruginosa exoenzyme S has appeared to be relatively indiscriminate in its choice of substrates, but in fact it ADP-ribosylates only a small subset of cellular proteins and exhibits a marked preference for several different membrane-associated proteins of apparent Mr = 23,000-25,000, at least some of which appear to bind GTP. One of these is the p21 product of the proto-oncogene c-H-ras, which can be labeled to completion. ADP-ribosylation does not alter the interaction of p21c-H-ras with guanyl nucleotides, but does cause a shift in electrophoretic mobility that implies a large conformational change. Exoenzyme S modifies all of its substrates at arginine residues.

Didsbury J, Weber RF, Bokoch GM, Evans T, Snyderman R
rac, a novel ras-related family of proteins that are botulinum toxin substrates.
J Biol Chem. 1989; 264: 16378-82
Display abstract
A new family of ras-related proteins, designated rac (ras-related C3 botulinum toxin substrate) has been identified. rac1 and rac2 cDNA clones were isolated from a differentiated HL-60 library and encode proteins that are 92% homologous and share 58% and 26-30% amino acid homology with human rhos and ras, respectively. Nucleotide sequence analysis predicts both rac1 and rac2 proteins to contain 192 amino acids with molecular masses of 21,450 and 21,429 daltons, respectively. rac1 and rac2 possess four of the five conserved functional domains in ras associated with binding and hydrolysis of guanine nucleotides. They also contain the COOH-terminal consensus sequence Cys-X-X-X-COOH which localizes ras to the inner plasma membrane and the residues Gly12 and Ala59, at which sites mutations elicit transforming potential to ras. The rac transcripts, particularly rac2, display relative myeloid tissue selectivity. Both rac1 transcripts (2.4 and 1.1 kilobases (kb] increase when HL-60 cells differentiate to neutrophil-like morphology. In contrast, differentiation of U937 cells to monocyte-like morphology causes no change in the 2.4-kb mRNA and a decrease in the 1.1-kb mRNA species. rac2 mRNA (1.45 kb) increases 7-9-fold and 3-fold upon differentiation of HL-60 and U937 cells, respectively. Neither rac mRNAs are present in a Jurkat T cell line, and unlike rac1, rac2 mRNA is absent in human brain and liver tissue. Transfection experiments permitted the demonstration that rac1 and rac2 are substrates for ADP-ribosylation by the C3 component of botulinum toxin. The data suggest that racs are plasma membrane-associated GTP-binding proteins which could regulate secretory processes, particularly in myeloid cells.

Pizon V, Chardin P, Lerosey I, Olofsson B, Tavitian A
Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region.
Oncogene. 1988; 3: 201-4
Display abstract
We have characterized two new ras-related genes rap1 and rap2 from a human cDNA library, by hybridization with the Drosophila Dras3 gene at low stringency conditions. The rap1 and rap2 genes encode proteins of 184 and 183 amino acid respectively with molecular weights of 20.9 kd and 20.7 kd. These proteins are 53% and 46% identical to the human K-ras protein and share several properties with the classical ras proteins. The C-terminal cysteine involved in the membrane anchoring as well as the GTP binding regions of the p21 ras proteins are present in the rap proteins suggesting that these proteins could bind GTP/GDP and have a membrane localization. The most striking difference between the rap and ras proteins resides in their 61st amino acid. As in the Drosophila Dras3 protein, both rap proteins have a threonine instead of the glutamine found at position 61 of the classical ras proteins. Furthermore the putative effector domain of the ras proteins is strictly conserved in the rap1 protein whereas only one amino acid difference is found in the rap2 protein. This suggests that the rap proteins might interact with the same effector as the ras proteins.

Bokoch GM, Parkos CA, Mumby SM
Purification and characterization of the 22,000-dalton GTP-binding protein substrate for ADP-ribosylation by botulinum toxin, G22K.
J Biol Chem. 1988; 263: 16744-9
Display abstract
GTP-binding proteins were purified from human neutrophils, including a 40,000-Da pertussis toxin substrate (Gn) and 22,000-, 24,000-, and 26,000-Da proteins, termed G22K, G24K, and G26K, respectively. The latter proteins were shown to be immunologically unrelated to Gn. G22K cross-reacted with anti-ras monoclonal antibody 142-24EO5, but not with monoclonal antibody Y13-259. A single 22,000-Da substrate for botulinum toxin-catalyzed ADP-ribosylation present in neutrophil membranes co-migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with G22K. In the presence of a cytosolic factor, G22K could serve as a specific botulinum toxin substrate. The 22,000-Da botulinum toxin substrate in neutrophil membranes could be immunoprecipitated by antibody 142-24EO5, but not by antibody Y13-259. G22K appears to be a unique GTP-binding protein which serves as a substrate for ADP-ribosylation by a component of botulinum toxin and which may be involved in exocytotic secretion or cellular differentiation.

Bokoch GM, Parkos CA
Identification of novel GTP-binding proteins in the human neutrophil.
FEBS Lett. 1988; 227: 66-70
Display abstract
We describe here the existence of previously undescribed GTP-binding proteins within the human neutrophil. These proteins specifically bind guanine nucleotides under conditions in which the previously characterized G-proteins are unable to bind. We have partially purified these proteins and present both functional and immunologic data which indicate that they are unrelated to Gn, the major neutrophil pertussis toxin substrate. An additional protein, of apparent molecular mass 22 kDa, may be related to the ras G-protein family. Analysis of the structural and functional characteristics of these novel proteins will promote a better understanding of the process of neutrophil activation.

Quilliam LA, Brown JH, Buss JE
A 22 kDa ras-related G-protein is the substrate for an ADP-ribosyltransferase from Clostridium botulinum.
FEBS Lett. 1988; 238: 22-6
Display abstract
A ribosyltransferase from C. botulinum type D ADP-ribosylated a protein of 22 kDa (p22) in human astrocytoma (1321N1) cells. ADP-ribosylation of membrane-bound p22 was potentiated by 2 mM MgCl2 or guanine nucleotides but was much reduced in the presence of 10 mM Mg2+ plus GTP gamma S. p22 was immunoprecipitated by a monoclonal antibody (142-24E05) raised against a peptide sequence common to the ras gene family but not by other ras or G-protein antibodies. p22 was also ADP-ribosylated in Drosophila but was not detected in Dictyostelium. These data suggest that the 22 kDa botulinum toxin substrate is a GTP-binding protein and a member of the ras protein family.

Saikumar P, Ulsh LS, Clanton DJ, Huang KP, Shih TY
Novel phosphorylation of c-ras p21 by protein kinases.
Oncogene Res. 1988; 3: 213-22
Display abstract
Novel p21 phosphorylation was found in cells expressing high levels of this product of c-H- and c-K-ras genes. Phorbol 12,13-dibutyrate, a protein kinase C (PKC) activator, and permeable c-AMP derivatives, which activate protein kinase A (PKA), stimulated phosphorylation of K-ras(4B) p21 in 416B cells 3 to 5 fold. By tryptic peptide mapping, it was found that both PKC and PKA phosphorylated in vitro the K-ras p21 at the same site as was found in p21 from cells labeled with [32P]orthophosphate in vivo. A common site of H-ras p21 was also phosphorylated by both PKC and PKA, although phosphopeptides of H-ras p21 were distinct from those of K-ras p21. The construction of a mutant by site-directed mutagenesis allowed the identification of serine-177 as the phosphorylation site of H-ras p21. This novel phosphorylation site lies in the hypervariable region, which links the globular catalytic domain of p21 to the membrane-anchoring site at the C-terminus, a location suggesting that this phosphorylation plays a role in modulating transmembrane signaling.

Kawata M, Matsui Y, Kondo J, Hishida T, Teranishi Y, Takai Y
A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization.
J Biol Chem. 1988; 263: 18965-71
Display abstract
In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.

Longo PA, Broido MS, Chen J, Kung HF, Pincus MR
The structure of the amino terminal transforming segment of the p21 protein, Tyr4-Thr20 (with Asp12), by two-dimensional NMR.
Biochem Biophys Res Commun. 1988; 157: 776-82
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The structure of a peptide from the transforming region (residues 4-20) of the p21 protein has been determined using two-dimensional NMR. In the normal protein, this segment contains a Gly residue at the critical 12 position; any substitution, other than Pro, at this position results in a transforming protein. Previously performed energy calculations indicated that this peptide segment is a structured one. In this study we find that the Asp12 containing peptide has a surprisingly well-defined structure in solution which has more similarity to the GDP-binding loop region in EF-tu than to that in p21.

Miura K, Kamiya H, Kubota S, Ikehara M, Nishimura S, Ohtsuka E
Conversion of the guanine nucleotide binding sites of ras protein resulting in the reduction of base specificity.
Protein Eng. 1988; 2: 227-31
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A gene coding for the novel ras protein, p21X, in which the domains of guanine binding and phosphate binding were exchanged, was constructed and expressed in Escherichia coli. The gene product, p21X, showed GTP binding activity, but no GPTase activity. In addition, p21X revealed binding activity toward ATP and CTP. In a competitive binding assay, [3H]GTP binding to p21X was inhibited in the presence of ATP, CTP and UTP, ITP as well as GDP, GTP and dGTP.

Lalaiants IE
[Possible mechanisms of disorders of GTPase activity with substitutions at the 12th and 61st codons of the p21 H-ras protein]
Vopr Onkol. 1987; 33: 9-15

Hiwasa T, Yokoyama S, Ha JM, Noguchi S, Sakiyama S
c-Ha-ras gene products are potent inhibitors of cathepsins B and L.
FEBS Lett. 1987; 211: 23-6
Display abstract
c-Ha-ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c-Ha-ras proteins and thiol proteinase inhibitors has been found.

Ishihara H, Nakagawa H, Ono K, Fukuda A
Antibodies against synthetic carboxy-terminal peptides distinguish H-ras and K-ras oncogene products p21.
J Immunol Methods. 1987; 103: 131-9
Display abstract
Synthetic peptides corresponding to the carboxy-terminal region of H-ras, K-ras, and N-ras oncogene product p21 proteins are used to obtain antibodies specific to each ras oncogene product. The synthetic peptides of 32 amino acids are immunogenic in rabbits without being coupled to carriers. Specific antibodies are purified by absorption of the antisera with the other peptides coupled to CH-Sepharose 4B, and antibodies reacting with all three peptides are obtained by affinity chromatography. These findings imply that antibodies specific to each peptide recognize the variable carboxy-terminal region while antibodies reacting with all three peptides recognize the constant region of the carboxy-terminal amino acid sequence of p21 proteins. The affinity-purified antibodies against H-ras and K-ras peptides are shown to react specifically with c-H-ras and v-K-ras p21 proteins expressed in E. coli and eukaryotic cells, respectively. These antibodies may be useful tools to study the functional roles of p21 carboxy-terminal domain and to detect differential expression of the family of ras oncogenes in cancerous tissues. The affinity-purified anti-N-ras peptide antibody, however, fails to react with N-ras p21 in spite of its positive reactivity with the N-ras peptide.

Doniger J
Differential conservation of non-coding regions within human and guinea pig N-ras genes.
Oncogene. 1987; 1: 331-4

Hattori S et al.
Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.
Mol Cell Biol. 1987; 7: 1999-2002
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The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

Clanton DJ, Hattori S, Shih TY
Mutations of the ras gene product p21 that abolish guanine nucleotide binding.
Proc Natl Acad Sci U S A. 1986; 83: 5076-80
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We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein. Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21. These activities are retained by proteins with a mutation at position 117 or 118. Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities. Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay. By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated. Cells transfected with 116K or 116Y mutant DNA are contact inhibited. In contrast to competent clones, the defective mutants have no detectable phosphorylated p21. The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function.

Shih TY et al.
Structure and function of p21 ras proteins.
Gene Amplif Anal. 1986; 4: 53-72
Display abstract
Cancer is a malfunction of cellular growth control. The discovery of oncogenes, first in transforming retroviruses, and later in human and animal tumors, may have uncovered the key to understanding one of the most elusive subjects of basic cell biology, namely, the controlling mechanisms of cell growth. The ras gene family encodes a group of closely related 21,000 dalton (p21) proteins with special affinity for guanine nucleotides. Other cellular proteins with similar biochemical properties, collectively known as G-proteins, include the regulatory G proteins of adenylate cyclase, the alpha subunit of transducin of retina rod outer segments, the recently identified rho gene proteins, and perhaps also the elongation factors, EF-Tu and EF-G, of the protein synthesis system. These G-proteins have roles in cellular signal transduction; by analogy p21 may have a similar cellular function in mediating the flow of growth control signals. Recent progress in the cloning and sequencing of these genes, overproduction of gene products in E. coli, protein engineering, detailed biochemical characterization, and the molecular structure determined by high resolution X-ray crystallography, have helped to elucidate in great detail the structure and function of p21 ras proteins. p21 appears to have a small membrane binding domain at the C-terminus, which contains a palmitylation site at cysteine-186, four amino acid residues from the end. Separated by a variable "hinge" region, most of the rest of ras amino acid sequences are highly conserved in nature. Four regions of extensive sequence homology among G-proteins constitute the GTP/GDP binding domain. In the crystal structure of EF-Tu, four peptide loops connecting beta sheets and alpha helices form the pocket for binding GDP. Studies using site-directed mutagenesis and immnochemical probes, indicate that the basic structure of the GDP binding site is conserved between p21 and EF-Tu. Furthermore, these studies also conclude that GTP binding is crucial for p21 ras cellular function. Although the precise target molecules for p21 are still unknown, the finding of the on/off switch function for ras genes have provided a better understanding of the mechanism of proto-oncogene activation, and may also provide further impetus to explore means of cancer intervention by interfering with the switch function.

Backer JM, Weinstein IB
p21 ras proteins and guanine nucleotides modulate the phosphorylation of 36- and 17-kilodalton mitochondria-associated proteins.
Proc Natl Acad Sci U S A. 1986; 83: 6357-61
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We have found that, when isolated rat liver mitochondria are incubated with [gamma-32P]ATP, there is phosphorylation of 36- and 17-kDa proteins. These proteins together with their protein kinase(s) are released as a complex by incubation of the isolated rat liver mitochondria at 20 degrees C for 30 min with 10 mM glucose 6-phosphate, 0.5 mM inositol phosphate, or 0.01 mM inositol triphosphate. Phosphorylation of the 36- and 17-kDa proteins in this soluble protein fraction is modulated by p21 proteins encoded by ras oncogenes and synthesized in Escherichia coli via recombinant DNA methods. A normal p21 ras protein stimulates phosphorylation of the 36-kDa protein and inhibits phosphorylation of the 17-kDa protein, whereas two transforming p21 ras proteins inhibit phosphorylation of both the 36- and 17-kDa proteins. Although GDP and 5'-guanylyl imidodiphosphate also influence the phosphorylation of these proteins, we present evidence that the effects of p21 ras protein are not simply due to their bound GDP. This novel system may be useful for further studies on the biochemical functions of the p21 ras proteins.

Der CJ, Pan BT, Cooper GM
rasH mutants deficient in GTP binding.
Mol Cell Biol. 1986; 6: 3291-4
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Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.

Pincus MR, Brandt-Rauf PW
Protein structure and cancer.
Cancer Invest. 1986; 4: 185-95

Srivastava SK, Lacal JC, Reynolds SH, Aaronson SA
Antibody of predetermined specificity to a carboxy-terminal region of H-ras gene products inhibits their guanine nucleotide-binding function.
Mol Cell Biol. 1985; 5: 3316-9
Display abstract
The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecule specifically recognized H-ras-encoded p21 proteins. This antibody was also shown to strikingly and specifically inhibit the guanine nucleotide-binding function of the p21 protein. The inability of p21 protein to bind guanine nucleotides was associated with a lack of autophosphorylation or GTPase activities. These studies suggest that a region toward its carboxy terminus is directly or indirectly involved in the guanine nucleotide-binding function of the p21 molecule.

LeBien TW, Kersey JH
No evidence for structural or functional identity between p24/CD9 and p21/ras.
Leuk Res. 1985; 9: 1565-70
Display abstract
Experiments were undertaken to test the hypothesis that the p24/CD9 cell surface molecule (originally detected on acute lymphoblastic leukemia cells) and the p21 protein encoded by the ras gene family are related. Immunoprecipitation and guanine nucleotide binding assays conducted with BA-2 and anti-p21/ras monoclonal antibodies showed no evidence that p24/CD9 is structurally or functionally related to p21/ras.

Schejter ED, Shilo BZ
Characterization of functional domains of p21 ras by use of chimeric genes.
EMBO J. 1985; 4: 407-12
Display abstract
Comparison of the predicted amino acid sequences of different members of the ras family in vertebrates has shown that the N-terminal 120 residues are highly conserved while the C terminus is variable. To test the possible role of the variable residues in cell transformation, chimeras were constructed containing the N-terminal 111 amino acids of the human Ha-ras EJ oncogene and the C terminus of two Drosophila ras genes. We show that one of these constructs which has only 20 conserved residues between positions 121 and 189, can transform rat-1 cells, and the transformed cells are capable of inducing lethal tumors in rats. The second construct containing the C terminus of another Drosophila ras gene exhibits a transforming capacity as well, but only after linkage to a viral transcriptional promoter. These results show that the majority of residues within the C terminus can be replaced without abolishing the transforming potential of p21 ras.

Gibbs JB, Ellis RW, Scolnick EM
Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.
Proc Natl Acad Sci U S A. 1984; 81: 2674-8
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The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.

Hurley JB, Simon MI, Teplow DB, Robishaw JD, Gilman AG
Homologies between signal transducing G proteins and ras gene products.
Science. 1984; 226: 860-2
Display abstract
The guanosine triphosphate-binding proteins (G proteins) found in a variety of tissues transduce signals generated by ligand binding to cell surface receptors into changes in intracellular metabolism. Amino acid sequences of peptides prepared by partial proteolysis of the alpha subunit of a bovine brain G protein and the alpha subunit of rod outer-segment transducin were determined. The two proteins show regions of sequence identity as well as regions of diversity. A portion of the amino-terminal peptide sequence of each protein is highly homologous with the corresponding region in the ras protein (a protooncogene product). These similarities suggest that G proteins and ras proteins may have analogous functions.

Rasheed S, Norman GL, Heidecker G
Nucleotide sequence of the Rasheed rat sarcoma virus oncogene: new mutations.
Science. 1983; 221: 155-7
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The nucleotide sequence of the oncogene of the Rasheed strain of rat sarcoma virus was determined. The oncogene (Ra-v-ras) encodes a 29,000-dalton (p29) transforming protein. This protein is distinct from the immunologically related 21,000-dalton protein (p21) of the Harvey murine sarcoma virus in its amino terminus and in having additional mutations in its carboxyl terminus. Although the functional significance of these changes is unknown, they appear to occur only in rat sarcoma virus.