SMART MODE:

NORMAL GENOMIC

• Aznar S, Lacal JC
• Searching new targets for anticancer drug design: the families of Ras and Rho GTPases and their effectors.
• Prog Nucleic Acid Res Mol Biol. 2001; 67: 193-234
• Display abstract

• The Ras superfamily of low-molecular-weight GTPases are proteins that, in response to diverse stimuli, control key cellular processes such as cell growth and development, apoptosis, lipid metabolism, cytoarchitecture, membrane trafficking, and transcriptional regulation. More than 100 genes of this superfamily grouped in six subfamilies have been described so far, pointing to the complexities and specificities of their cellular functions. Dysregulation of members of at least two of these families (the Ras and the Rho families) is involved in the events that lead to the uncontrolled proliferation and invasiveness of human tumors. In recent years, the cloning and characterization of downstream effectors for Ras and Rho proteins have given crucial clues to the specific pathways that lead to aberrant cellular growth and ultimately to tumorigenesis. A direct link between the functions of some of these effectors with the appearance of transformed cells and their ability to proliferate and invade surrounding tissues has been made. Accordingly, drugs that specifically alter their functions display antineoplasic properties, and some of these drugs are already under clinical trials. In this review, we survey the progress made in understanding the underlying molecular connections between carcinogenesis and the specific cellular functions elicited by some of these effectors. We also discuss new drugs with antineoplastic or antimetastatic activity that are targeted to specific effectors for Ras or Rho proteins.

• Sayed A et al.
• ATPase and GTPase activities copurifying with GTP-binding proteins in E. coli.
• J Mol Microbiol Biotechnol. 2000; 2: 261-3
• Display abstract

• Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway. We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions. Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations. Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations. From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions. Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.

• Bischoff F, Molendijk A, Rajendrakumar CS, Palme K
• GTP-binding proteins in plants.
• Cell Mol Life Sci. 1999; 55: 233-56
• Display abstract

• GTP-binding proteins are found in all organisms. They are important switches that cycle between an active and an inactive state, ensuring vectorial flow of information on the expense of guanosine triphosphate (GTP). In this review, we discuss current progress in the molecular characterization and functional analysis of plant genes encoding heterotrimeric and small GTPases. An up-to-date list including all cloned plant GTPase genes is given and a systematic classification is proposed.

• Jiang Y, Ma W, Wan Y, Kozasa T, Hattori S, Huang XY
• The G protein G alpha12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain.
• Nature. 1998; 395: 808-13
• Display abstract

• Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are signal transducers that relay messages from many receptors on the cell surface to modulate various cellular processes. The direct downstream effectors of G proteins consist of the signalling molecules that are activated by their physical interactions with a G alpha or Gbetagamma subunit. Effectors that interact directly with G alpha12 G proteins have yet to be identified. Here we show that G alpha12 binds directly to, and stimulates the activity of, Bruton's tyrosine kinase (Btk) and a Ras GTPase-activating protein, Gap1m, in vitro and in vivo. G alpha12 interacts with a conserved domain, composed of the pleckstrin-homology domain and the adjacent Btk motif, that is present in both Btk and Gap1m. Our results are, to our knowledge, the first to identify direct effectors for G alpha12 and to show that there is a direct link between heterotrimeric and monomeric G proteins.

• Gawler DJ
• Points of convergence between Ca2+ and Ras signalling pathways.
• Biochim Biophys Acta. 1998; 1448: 171-82
• Display abstract

• p21 Ras proteins play a critical role in the regulation of cellular growth and differentiation. In addition, Ras and proteins which regulate Ras activity have been implicated in long-term memory consolidation and long-term potentiation processes. Over the last few years, much evidence has emerged which indicates that changes in cytoplasmic Ca2+ levels can regulate Ras protein activity and subsequent biological function. Also, Ras proteins themselves can modulate intracellular Ca2+ levels by regulating both Ca2+ release and Ca2+ influx processes. Here we examine the signalling components which regulate Ras activity and, in particular, consider points of convergence between intracellular Ca2+ and p21 Ras signalling processes. In addition, we consider the possible biological consequences resulting from the integration of these signalling pathways and highlight the importance of our understanding protein protein interactions. Finally, we discuss the possibility of protein-protein interactions mediated via Ca2+-responsive structural domains, such as the C2 and IQ domains, playing important roles in Ca2+-dependent Ras functions yet to be established.

• Iiri T, Bourne HR
• G proteins propel surprise.
• Nat Genet. 1998; 18: 8-10

• Richardson CJ, Jones S, Litt RJ, Segev N
• GTP hydrolysis is not important for Ypt1 GTPase function in vesicular transport.
• Mol Cell Biol. 1998; 18: 827-38
• Display abstract

• GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37 degrees C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

• Overmeyer JH, Wilson AL, Erdman RA, Maltese WA
• The putative "switch 2" domain of the Ras-related GTPase, Rab1B, plays an essential role in the interaction with Rab escort protein.
• Mol Biol Cell. 1998; 9: 223-35
• Display abstract

• Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the alpha2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative alpha2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the alpha3 and alpha4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B alpha2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B alpha2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the alpha2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

• Olson MF, Paterson HF, Marshall CJ
• Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1.
• Nature. 1998; 394: 295-9
• Display abstract

• Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation, and the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signalling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signalling pathways to varying degrees. Other signalling pathways could also influence the consequences of Ras signalling. Here we show that when signalling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Cells that lack p21Waf1/Cip1 do not require Rho signalling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signalling is the suppression of p21Waf1/Cip1 induction.

• Lacal JC
• Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid-dependent signaling.
• FEBS Lett. 1997; 410: 73-7
• Display abstract

• Small GTPases are molecular switches that control signaling pathways critical for diverse cellular functions. Recent evidence indicates that multiple effector molecules can be activated by small GTPases. As a result, complex biological processes such as cell proliferation and apoptosis are turned on. Thus, rather than a single linear pathway from the membrane to the nucleus, the integration of complementary signals is required for these events to occur. In fact, the coordinated activation of small GTPases may constitute some of the critical modulators of those signals triggering either proliferation or cell death. In addition to the activation of specific kinases cascades, phospholipid-derived messengers are candidates to compose some of the most critical elements associated to regulation of signaling cascades capable of discerning among life and death. Both proliferation and apoptosis needs competence and progression signals. Phospholipase D and sphingomyelinase may be important players in this decision-maker step.

• Wang KL, Khan MT, Roufogalis BD
• Identification and characterization of a calmodulin-binding domain in Ral-A, a Ras-related GTP-binding protein purified from human erythrocyte membrane.
• J Biol Chem. 1997; 272: 16002-9
• Display abstract

• A 28-kDa protein (p28) has been purified from Triton X-100 extracts of human erythrocyte plasma membrane by calmodulin affinity chromatography. Based on internal peptide sequencing and its protein amino acid composition, this protein has been shown to be highly related, if not identical to, Ral-A, a Ras-related GTP-binding protein. This protein assignment is consistent with the findings that p28 binds [32P]GTP specifically and has low GTPase activity. In this study we describe the identification and characterization of a calmodulin-binding domain in Ral-A. The Ca2+-dependent interaction of p28 with calmodulin was first detected by a calmodulin affinity column. Gel overlay experiments of both p28 and recombinant Ral-A with biotinylated calmodulin provided strong evidence that Ral-A is a calmodulin-binding protein. A peptide of 18 residues (P18) with the sequence SKEKNGKKKRKSLAKRIR has been identified as a putative calmodulin-binding domain in Ral-A, because it comprises a basic/hydrophobic composition with the propensity to form an amphiphilic helix. P18 was synthesized, and its interaction with calmodulin by gel overlay was shown to be Ca2+-dependent. Circular dichroism analysis demonstrated that this interaction results in less alpha-helical content upon calmodulin complex formation. These results indicate that Ral-A is a calmodulin-binding protein, raising the possibility that it may be associated with Ca2+-dependent intracellular signaling pathways.

• Chisholm RL
• Cytokinesis: a regulatory role for Ras-related proteins?
• Curr Biol. 1997; 7: 64850-64850

• Kadono-Okuda K, Andres DA
• An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay.
• Anal Biochem. 1997; 254: 187-91
• Display abstract

• We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [alpha-32P]GTP. The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E. coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-like small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This results in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particularly useful for the cloning of novel Ras-related GTP-binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

• Schweins T, Scheffzek K, Assheuer R, Wittinghofer A
• The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+ versus Mg2+.
• J Mol Biol. 1997; 266: 847-56
• Display abstract

• GTP and ATP hydrolysing proteins have an absolute requirement for a divalent cation, which is usually Mg2+, as a cofactor in the enzymatic reaction. Other phosphoryl transfer enzymes employ more than one divalent ion for the enzymatic reaction. It is shown here for p21ras, a well studied example of GTP hydrolysing proteins, that the GTP-hydrolysis rate is significantly faster if Mg2+ is replaced by Mn2+, both in the presence or absence of its GTPase-activating protein Ras-GAP. This effect is not due to a different stoichiometry of metal ion binding, since one metal ion is sufficient for full enzymatic activity. To determine the role of the metal ion, the crystal structure of p21(G12P). GppCp complexed with Mn2+ was determined and shown to be very similar to the corresponding p21(G12P). GppCp.Mg2+ structure. Especially the coordination sphere around the metal ions is very similar, and no second metal ion binding site could be detected, consistent with the assumption that one metal ion is sufficient for GTP hydrolysis. In order to explain the biochemical differences, we analysed the GTPase reaction mechanism with a linear free energy relationships approach. The result suggests that the reaction mechanism is not changed with Mn2+ but that the transition metal ion Mn2+ shifts the pKa of the gamma-phosphate by almost half a unit and increases the reaction rate due to an increase in the basicity of GTP acting as the general base. This suggests that the intrinsic GTPase reaction could be an attractive target for anti-cancer drug design. By using Rap1A and Ran, we show that the acceleration of the GTPase by Mn2+ appears to be a general phenomenon of GTP-binding proteins.

• Nisimoto Y, Freeman JL, Motalebi SA, Hirshberg M, Lambeth JD
• Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase.
• J Biol Chem. 1997; 272: 18834-41
• Display abstract

• Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with Kd values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558.

• Basi NS, Rebois RV
• Rate zonal sedimentation of proteins in one hour or less.
• Anal Biochem. 1997; 251: 103-9
• Display abstract

• Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 microliters) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of S20,w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

• Louis SA, Weeks G, Spiegelman GB
• Rap1 overexpression reveals that activated RasD induces separable defects during Dictyostelium development.
• Dev Biol. 1997; 190: 273-83
• Display abstract

• One of the Dictyostelium ras genes, rasD, is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymond et al., 1986, Nature, 323, 340-343). The ability of the Dictyostelium rap1 gene to suppress this abnormal developmental phenotype was investigated. The rap1 gene and G12V activated and G10V negative mutant forms of the rap1 gene were independently linked to the rasD promoter and each construct used to transform M1, a Dictyostelium cell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). The rap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louis et al., 1997, Mol. Biol. Cell, 8, 303-312). During the development of ME cells, ecmA mRNA levels were restored to those seen for Ax3, and tagB mRNA levels were also markedly reduced, although not to Ax3 levels. cotC expression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression of car1 mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed by rap1.

• McCormick F
• The superfamily of Ras-related GTPases.
• Jpn J Cancer Res. 1997; 88: 0-0

• Yanachkov I, Pan JY, Wessling-Resnick M, Wright GE
• Synthesis and effect of nonhydrolyzable xanthosine triphosphate derivatives on prenylation of Rab5D136N.
• Mol Pharmacol. 1997; 51: 47-51
• Display abstract

• A novel and convenient method for nucleoside triphosphate synthesis was applied to the preparation of potentially nonhydrolyzable xanthosine triphosphate derivatives. The N-methylimidazolide of xanthosine 5'-monophosphate reacted rapidly with methylenediphosphonic acid and imidodiphosphonic acid to give xanthosine 5'-(beta, gamma-methylene)triphosphate and xanthosine 5'-(beta, gamma-imido)triphosphate, respectively, in good yields. Both compounds inhibited the xanthosine-diphosphate-dependent prenylation of a mutant of Rab5, Rab5D136N, the nucleotide specificity of which had been converted from GTP to xanthosine triphosphate. The results indicate that xanthosine 5'-(beta, gamma-methylene)triphosphate and xanthosine 5'-(beta, gamma-imido)triphosphate bound to the mutant protein with similar affinities and were not hydrolyzed under the assay conditions. These novel derivatives may be useful tools for the study of the role of individual GTPases mutated to xanthosine triphosphate specificity in the background of other GTP-binding proteins.

• Shpakov AO
• [Structural elements of molecules of GTP-binding proteins and effectors mediating coupling between them]
• Ukr Biokhim Zh. 1997; 69: 3-20
• Display abstract

• Data available in literature on molecular mechanisms of the functional coupling of heterotrimeric G-proteins and small GTP-binding proteins (Ras-type) to effectors are analyzed. The segments of G-protein alpha-subunits which participate in formation of the effector-binding site are discussed. The induction of interaction between activated alpha-subunit and effector, following change of alpha-subunit nucleotide-binding site conformation as a result of GDP/GTP exchange is shown. The analysis of effector molecular determinants responsible for coupling to G-proteins are analyzed. The determinants are preferentially of the polycationic nature.

• Pennisi E
• Superoxides relay Ras protein's oncogenic message.
• Science. 1997; 275: 1567-8

• Klein NP, Schneider RJ
• Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras.
• Mol Cell Biol. 1997; 17: 6427-36
• Display abstract

• The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.

• Beckner SK
• G-protein activation by chemokines.
• Methods Enzymol. 1997; 288: 309-26

• Iida H, Tanaka S, Shibata Y
• Small GTP-binding protein, Rab6, is associated with secretory granules in atrial myocytes.
• Am J Physiol. 1997; 272: 1594601-1594601
• Display abstract

• Rab proteins, a subfamily of small GTP-binding proteins, have been shown to play key roles in regulation of vesicular traffic in eukaryotic cells. In this study, we have intended to identify, the atrial granule-associated Rab proteins that seem to be required for formation or intracellular transport of the granules. Atrial granules contained at least four small GTP-binding proteins, and we have demonstrated by biochemical analysis that one of the small GTP-binding proteins associated with the atrial granules is a Rab6 protein (Rab6p). Rab6p was also detected in highly purified zymogen granules of pancreatic exocrine gland. Immunogold electron microscopy performed on ultrathin cryosections of rat auricle revealed that Rab6p was associated with the atrial granule membranes. Association of Rab6p with the atrial granule membranes was also confirmed by immunodiffusion electron microscopy in agarose-embedded atrial granules. These data indicate that Rab6p is associated with the atrial granules and that it might function in the intracellular traffic of the secretory granules in the atrial myocytes.

• Hihara T, Tanaka K, Takai Y
• [Functions and modes of action of Ras and Rho small GTP-binding proteins]
• Tanpakushitsu Kakusan Koso. 1997; 42: 1501-8

• Neu M, Brachvogel V, Oschkinat H, Zerial M, Metcalf P
• Rab7: NMR and kinetics analysis of intact and C-terminal truncated constructs.
• Proteins. 1997; 27: 204-9
• Display abstract

• Rab proteins are a family of approximately 25kD ras-related GTPases which are associated with distinct intracellular membranes where they control vesicle traffic between intracellular compartments. The late-endosomal rab protein rab7(1-207), (lacking only the C-terminal lipids of the native molecule) and three C-terminal truncated constructs rab7(1-202), rab7(1-197) and rab7(1-182) were purified using an E. coli expression system. The C-terminal tail region of rab proteins is of special interest because it is thought to target rab proteins to particular intracellular membranes. A comparison of TOCSY-NMR spectra from intact rab7(1-207) and the tail-less construct rab7(1-182) suggested that much of the C-terminal tail is flexible in solution. The GTP hydrolysis, and GDP association and dissociation rates for all the truncated and intact constructs were similar, showing that the tail region of rab7(1-207) has little influence on the hydrolysis and exchange rates of the nucleotide.

• Bauer PH, Benjamin TL
• A novel 39-kilodalton membrane protein binds GTP in polyomavirus-transformed cells.
• J Virol. 1997; 71: 4128-32
• Display abstract

• To investigate the possible involvement of GTP-binding proteins in transformation by the DNA tumor virus polyomavirus, the GTP-binding activities of Ras-like proteins and G protein alpha subunit proteins were examined in polyomavirus-transformed cells. No differences in the degrees or patterns of expression of Ras-like proteins were observed. However, a 39-kDa protein specifically bound GTP in membranes from polyomavirus-transformed cells. This protein was not seen in nontransformed or lytically infected cells or in phenotypically normal revertants of polyomavirus-transformed cells. It reappeared, however, in spontaneous retransformants derived from the revertants. The 39-kDa protein was not found stably associated with polyomavirus T antigens, nor was it phosphorylated on tyrosine. The 39-kDa protein was not recognized by an antiserum specific for members of the Gi alpha subfamily of G proteins or by antisera against all other known GTP-binding proteins of similar molecular mass. These results suggest that this novel 39-kDa GTP-binding membrane protein is observed as part of a long-term response that accompanies stable transformation by the virus.

• Chen HK, Yeh NH
• The nucleolar phosphoprotein P130 is a GTPase/ATPase with intrinsic property to form large complexes triggered by F- and Mg2+.
• Biochem Biophys Res Commun. 1997; 230: 370-5
• Display abstract

• We have previously identified a human nucleolar phosphoprotein p130 whose alterations during mitosis are correlated well with the nucleolar disassembly and reassembly. Further studies found that p130 in the cell lysates or after being purified by immunoprecipitation was able to form large complexes triggered by F- and Mg2+. These sodium dodecyl sulfate-insoluble p130 molecules were readily dissociated by adding EDTA to the complexes. It is known that F- and Mg2+ act on many GTPases and ATPases through the induction of a conformational transition mimicking the nucleoside triphosphate-bound state. These initial observations led us to discover that p130 functions as a GTP/ATP binding protein with intrinsic GTPase/ATPase activities. The rate of GTP hydrolysis by purified p130 under our experimental conditions was 0.8 mol/min/mol of p130. These results imply that p130, a novel nucleolar GTPase/ATPase, may switch its conformation in a nucleotide-dependent manner.

• Hofmann F, Rex G, Aktories K, Just I
• The ras-related protein Ral is monoglucosylated by Clostridium sordellii lethal toxin.
• Biochem Biophys Res Commun. 1996; 227: 77-81
• Display abstract

• Clostridium sordellii lethal toxin (LT), a cytotoxin which causes preferential destruction of the actin cytoskeleton, has been recently identified as glucosyltransferase to modify the low molecular mass GTPases Rac, Ras and Rap. We report here on LT produced by C. sordellii strain 6018 which glucosylates in addition to Rac, Ras and Rap the Ral protein. LT from strain VPI9048 however does not glucosylate Ral. Besides recombinant Ral, cellular Ral is also substrate. In the GDP-bound form, Ral is a superior substrate to the GTP form. Acceptor amino acid for glucose is threonine-46 which is equivalent to threonine-35 in H-Ras located in the effector region. The Ral-glucosylating toxin is a novel isoform of Ras-modifying clostridial cytotoxins.

• Marshall CJ
• Ras effectors.
• Curr Opin Cell Biol. 1996; 8: 197-204
• Display abstract

• The search for proteins which interact with the active GTP-bound form of Ras in order to transmit signals for proliferation, differentiation and oncogenesis has been a long one. Now there are several strong candidates for Ras effectors that include protein kinases, lipid kinases and guanine nucleotide exchange factors. Structural information on how one Ras-binding domain in an effector interacts with Ras.GTP has recently been obtained. Recent data show that transformation by Ras oncoproteins requires the activation of several signal transduction pathways, including those which transmit signals via members of the Rho family of GTPases.

• Fischer R, Wei Y, Anagli J, Berchtold MW
• Calmodulin binds to and inhibits GTP binding of the ras-like GTPase Kir/Gem.
• J Biol Chem. 1996; 271: 25067-70
• Display abstract

• Recently, a new subfamily of Ras-related GTP-binding proteins consisting of Rad (Ras associated with diabetes), Gem (immediate early gene expressed in mitogen-stimulated T-cells), and Kir (tyrosine kinase-inducible Ras-like) was discovered. The C terminus of these proteins contains an extension of approximately 30 amino acids not present in other members of the Ras family and which exhibits all the hallmarks typical for calmodulin (CaM)-binding domains. A peptide corresponding to the putative CaM-binding domain of the Kir/Gem protein was synthesized, and its affinity for CaM was determined by fluorescence spectrometry. Titration of dansyl-CaM with the Kir/Gem peptide gave an affinity constant of 1 nM. Furthermore, a single point mutation of the peptide, W269G, abolished this high affinity interaction. Gel-shift analysis showed that the complex formation between CaM and the Kir/Gem peptide is strictly calcium-dependent. We also demonstrate with a newly developed [32P]CaM overlay technique that full-length Kir/Gem and Rad proteins bind CaM in a Ca2+-dependent fashion. The binding of CaM to glutathione S-transferase-Kir and GST-Gem inhibited the binding of GTP to Kir/Gem significantly. These results suggest the existence of a direct link between Ca2+/CaM and growth factor signal transduction pathways at the level of small Ras-like GTPases.

• Sacchi GA, Pirovano L, Lucchini G, Cocucci S
• A low-molecular-mass GTP-binding protein in the cytosol of germinated wheat embryos.
• Eur J Biochem. 1996; 241: 286-90
• Display abstract

• A low-molecular-mass protein able to bind GTP in both native and SDS-denaturating conditions was detected in the cytosol of embryos from wheat (Triticum aestivum L.) seeds germinated for 40 h. The protein fulfilled most of the distinguishing criteria common to eukaryotic small GTP-binding proteins. It retained the ability to bind GTP after SDS/PAGE and nitrocellulose blotting. The protein eluted from Sephadex G-200 gel filtration with a Ve/Vo value corresponding to a molecular mass of 18 kDa, whereas on SDS/PAGE the molecular mass was 20 kDa. The native protein, which showed an intrinsic GTPase activity highly sensitive to NaF, bound the guanine nucleotide with high specificity and with a relatively high affinity (Kd approximately 85 nM). The GTP-binding protein was not detectable in other subcellular fractions; in the microsomal fraction, two other peptides of low molecular mass (23.5 and 21.5 kDa) with GTP-binding activity were detected. These results indicate that in the cytosolic fraction of germinating wheat embryos there is a 20-kDa protein which is biochemically similar to the known small GTP-binding proteins that currently have been detected almost exclusively in the membrane fraction of plant material.

• Bokoch GM
• Interplay between Ras-related and heterotrimeric GTP binding proteins: lifestyles of the BIG and little.
• FASEB J. 1996; 10: 1290-5
• Display abstract

• Directed interactions of heterotrimeric G-proteins coupled to receptors responding to extracellular signals and small Ras-related GTPases that control a variety of intracellular processes are being elucidated by recent work. We explore two of the most well-characterized pathways that couple big G-proteins to their smaller relatives.

• von Eichel-Streiber C, Boquet P, Sauerborn M, Thelestam M
• Large clostridial cytotoxins--a family of glycosyltransferases modifying small GTP-binding proteins.
• Trends Microbiol. 1996; 4: 375-82
• Display abstract

• Some Clostridium species produce ABX-type protein cytotoxins of high molecular weight. These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily. These characteristics render the LCTs valuable tools for developmental and cell biologists.

• Zhang FL, Casey PJ
• Protein prenylation: molecular mechanisms and functional consequences.
• Annu Rev Biochem. 1996; 65: 241-69
• Display abstract

• Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. Known prenylated proteins include fungal mating factors, nuclear lamins, Ras and Ras-related GTP-binding proteins (G proteins), the subunits of trimeric G proteins, protein kinases, and at least one viral protein. Prenylation promotes membrane interactions of most of these proteins, which is not surprising given the hydrophobicity of the lipids involved. In addition, however, prenylation appears to play a major role in several protein-protein interactions involving these species. The emphasis in this review is on the enzymology of prenyl protein processing and the functional significance of prenylation in cellular events. Several other recent reviews provide more detailed coverage of aspects of prenylation that receive limited attention here owing to length restrictions (1-4).

• Homburger V, Lagriffoul A, Bockaert J
• [Large and small G proteins in vesicular transport]
• Ann Endocrinol (Paris). 1996; 57: 83-90
• Display abstract

• The movement of proteins between compartments of the exocytic and endocytic pathways of eukaryotic cells is mediated by carrier vesicles. They bud from a donor compartment and are targetted to and fuse with the acceptor compartment. GTPases, proteins which bind and hydrolyze GTP, play key roles in the regulation of this vesicular protein transport. Heterotrimeric and small GTPases are involved in this vesicular traffic.

• Self AJ, Hall A
• Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates.
• Methods Enzymol. 1995; 256: 67-76

• Bischoff FR, Klebe C, Kretschmer J, Wittinghofer A, Ponstingl H
• RanGAP1 induces GTPase activity of nuclear Ras-related Ran.
• Proc Natl Acad Sci U S A. 1994; 91: 2587-91
• Display abstract

• The nuclear Ras-related protein Ran binds guanine nucleotide and is involved in cell cycle regulation. Models of the signal pathway predict Ran to be active as Ran.GTP at the initiation of S phase upon activation by the nucleotide exchange factor RCC1 and to be inactivated for the onset of mitosis by hydrolysis of bound GTP. Here a nuclear homodimeric 65-kDa protein, RanGAP1, is described, which we believe to be the immediate antagonist of RCC1. It was purified from HeLa cell lysates and induces GTPase activity of Ran, but not Ras, by more than 3 orders of magnitude. The Ran mutant Q69L, modeled after RasQ61L, which is unable to hydrolyze bound GTP, is insensitive to RanGAP1.

• Wagner AC, Williams JA
• Low molecular weight GTP-binding proteins: molecular switches regulating diverse cellular functions.
• Am J Physiol. 1994; 266: 114-114
• Display abstract

• Low molecular weight GTP-binding proteins (LMWG proteins) form a family of proteins that shows homology with Ras, are 18-30 kDa in mass, and bind and hydrolyze GTP. They act as molecular switches, being active when binding GTP. Their activity is regulated by other proteins that influence the dissociation of GDP and the rate of GTP hydrolysis. Roles are emerging for these proteins in regulation of membrane fusion and cytoskeletal organization and growth. In the gastrointestinal tract, the best studied physiological processes that may be regulated by LMWG proteins are digestive enzyme and gastric acid secretion.

• Bokoch GM, Knaus UG
• Ras-related GTP-binding proteins and leukocyte signal transduction.
• Curr Opin Hematol. 1994; 1: 53-60
• Display abstract

• Many aspects of leukocyte function are regulated by both heterotrimeric and Ras-related GTP-binding proteins, but there is little definite information about their roles in the specialized processes utilized by leukocytes for cell killing. Recent progress in understanding the regulation of the phagocyte NADPH oxidase by the Rac GTP-binding proteins provides a basis for defining the operational characteristics of one such phagocyte system. It is clear from various studies that the activity of the NADPH oxidase can be modulated through the regulation of the GTP-GDP state of Rac. Proteins exist in leukocytes able to modify GTP-binding protein function in this manner, and their activity may be regulated by signals generated on phagocyte stimulation. Proteins of the Ras superfamily are likely to be involved in a variety of normal phagocyte functions through their ability to modulate the assembly of actin filaments, direct vesicle trafficking and fusion, and so forth.

• Schweins T, Wittinghofer A
• GTP-binding proteins. Structures, interactions and relationships.
• Curr Biol. 1994; 4: 547-50
• Display abstract

• Recently available crystal structures show that some, though not all, GTP-binding proteins have a common 'G-domain' topology, variations on which confer distinct functional properties.

• Maguire J, Santoro T, Jensen P, Siebenlist U, Yewdell J, Kelly K
• Gem: an induced, immediate early protein belonging to the Ras family.
• Science. 1994; 265: 241-4
• Display abstract

• A gene encoding a 35-kilodalton guanosine triphosphate (GTP)-binding protein, Gem, was cloned from mitogen-induced human peripheral blood T cells. Gem and Rad, the product of a gene overexpressed in skeletal muscle in individuals with Type II diabetes, constitute a new family of Ras-related GTP-binding proteins. The distinct structural features of this family include the G3 GTP-binding motif, extensive amino- and carboxyl-terminal extensions beyond the Ras-related domain, and a motif that determines membrane association. Gem was transiently expressed in human peripheral blood T cells in response to mitogenic stimulation; the protein was phosphorylated on tyrosine residues and localized to the cytosolic face of the plasma membrane. Deregulated Gem expression prevented proliferation of normal and transformed 3T3 cells. These results suggest that Gem is a regulatory protein, possibly participating in receptor-mediated signal transduction at the plasma membrane.

• Pasheva E, Janoueix-Lerosey I, Tavitian A, de Gunzburg J
• Characterization of the Ras-related RAP2A protein expressed in the baculovirus-insect cell system: processing of the protein in insect cells and comparison with the bacterially produced unprocessed form.
• Biochem Biophys Res Commun. 1994; 198: 973-82
• Display abstract

• The Ras-related protein Rap2A is a 21kD GTP-binding protein that exhibits 46% identity with Ras proteins and is similarly post-translationally modified by farnesyl and palmitate groups. Using a recombinant baculovirus, we expressed Rap2A in Sf9 insect cells. The protein is initially synthesized as a soluble hydrophilic precursor, that is post-translationally processed to a hydrophobic membrane-bound form (Rap2Am) that contains both isoprenoid and palmitate groups. The processed form of the protein was purified from the membranes of infected Sf9 cells, and its biochemical properties were compared with those of the unprocessed form produced in recombinant bacteria (Rap2Ab). Both proteins exhibited similar kinetics of GDP dissociation and GTP binding and displayed a weak intrinsic GTPase activity that was stimulated to the same extent by a factor present in bovine brain cytosol. We conclude that Rap2A is correctly processed in insect cells and that maturation does not alter its biochemical properties.

• Burton JL, Burns ME, Gatti E, Augustine GJ, De Camilli P
• Specific interactions of Mss4 with members of the Rab GTPase subfamily.
• EMBO J. 1994; 13: 5547-58
• Display abstract

• Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4-GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co-precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.

• Glomset JA, Farnsworth CC
• Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes.
• Annu Rev Cell Biol. 1994; 10: 181-205

• Yang C, Mollat P, Chaffotte A, McCaffrey M, Cabanie L, Goud B
• Comparison of the biochemical properties of unprocessed and processed forms of the small GTP-binding protein, rab6p.
• Eur J Biochem. 1993; 217: 1027-37
• Display abstract

• The rab6 protein (rab6p) belongs to a large family of ras-like low-molecular-mass GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. When expressed in the baculovirus/insect cell system, two major forms of rab6p are obtained; a 24-kDa cytosolic unprocessed form and a 23-kDa membrane-bound form which represents the processed lipid-modified protein. Here, we have purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar binding-rate constants (kon) for GDP and GTP (1-1.9 microM-1 min-1). However, significant differences exist in the dissociation constants of bound guanine nucleotides. Processed rab6p in low and high magnesium solutions displays similar koff values for GTP and GDP. However, unprocessed rab6p has a koff value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase activities also differ; unprocessed rab6p has an almost undetectable GTPase activity, whereas that of processed rab6p is in the same range as that reported for other ras and ras-like GTP-binding proteins (0.012 +/- 0.002 min-1). These results suggest that post-translational modifications of rab6p might induce subtle changes in the three-dimensional structure of the protein which affect the guanine-nucleotide-binding/hydrolysis activity.

• Hall A, Paterson HF, Adamson P, Ridley AJ
• Cellular responses regulated by rho-related small GTP-binding proteins.
• Philos Trans R Soc Lond B Biol Sci. 1993; 340: 267-71
• Display abstract

• Rho-related proteins are members of the ras superfamily of small GTP-binding proteins. Their function in fibroblasts has been analysed using microinjection of living cells. Rho appears to link plasma membrane receptors to the assembly of focal adhesions and actin stress fibres. The closely related protein rac, on the other hand, links receptors to the polymerization of actin at the plasma membrane to form membrane ruffles and pinocytotic vesicles. In phagocytic cells, rac has been shown to be required for activation of a membrane-bound NADPH oxidase in response to receptor activation. These systems provide the basis for a working model for the mechanism of action of the rho family of small GTPases.

• Ahmed S, Lee J, Kozma R, Best A, Monfries C, Lim L
• A novel functional target for tumor-promoting phorbol esters and lysophosphatidic acid. The p21rac-GTPase activating protein n-chimaerin.
• J Biol Chem. 1993; 268: 10709-12
• Display abstract

• Phorbol esters are potent tumor promoters widely used for investigating mechanisms of cell transformation with protein kinase C (PKC) generally considered as being their only protein target. Lysophosphatidic acid (LPA) can act as a mitogen, affecting cell shape and the actin cytoskeleton. There is no identified functional target for LPA. We have isolated a cDNA encoding a protein n-chimaerin that is a high affinity phorbol ester receptor and a p21rac-GTPase activating protein (rac-GAP). p21rac is a member of the ras superfamily of small molecular weight GTP-binding proteins, which stimulates actin microfilament formation in Swiss 3T3 cells and superoxide production by the neutrophil oxidase. We now show that the rac-GAP activity of n-chimaerin is stimulated by phosphatidylserine (PS) and phosphatidic acid (PA) and that phorbol esters can synergize with PS and PA. LPA, in contrast, was found to inhibit n-chimaerin. The phospholipid/phorbol ester modulation of the rac-GAP activity requires the PKC-like cysteine-rich domain of n-chimaerin. Thus, n-chimaerin is a novel functional target (distinct from PKC) for both phorbol esters and LPA. These data suggest that the physiological role of n-chimaerin is to link events initiating at the cell surface/membrane with p21rac effector pathways.

• Bokoch GM, Der CJ
• Emerging concepts in the Ras superfamily of GTP-binding proteins.
• FASEB J. 1993; 7: 750-9
• Display abstract

• The Ras superfamily of GTP-binding proteins (> 50 members) regulates a diverse spectrum of intracellular processes. These include cellular proliferation and differentiation, intracellular vesicular trafficking, cytoskeletal control, NADPH oxidase function, as well as others. In this review, we describe recent progress and emerging themes in the action and regulation of these important cellular regulatory molecules. Structural studies have provided insight into the function of low molecular weight GTP-binding proteins (LMWGs) as molecular switches, and are defining modes of interaction with associated regulatory molecules. Details of the enzymatic processes involved in the posttranslational processing of LMWGs, and how this processing is important for protein function, are being elucidated. A variety of GTPase activating proteins, GDP/GTP dissociation stimulators, and GDP dissociation inhibitors have been identified, and their ability to determine the activity of LMWG-regulated systems is being worked out. The discovery of multifunctional regulatory molecules has indicated that substantial "crosstalk" between various LMWG may occur. The continuing emergence of additional cellular functions that are regulated by LMWGs, and particularly the recent availability of in vitro analytical systems for studies of the mechanism (or mechanisms) of action of LMWGs, is resulting in a wealth of information about the regulation and integration of cellular signaling, form, and function.

• Grunicke HH, Maly K
• Role of GTPases and GTPase regulatory proteins in oncogenesis.
• Crit Rev Oncog. 1993; 4: 389-402
• Display abstract

• The GTPases comprise a superfamily of GTP-binding proteins with intrinsic GTPase activity. Some members of this family representing either heterotrimeric or small G-proteins are involved in the transmission of mitogenic signals. Mutations that lead to constitutively activated G-proteins have been shown to contribute to malignant transformation. These genes represent, therefore, putative oncogenes. Examples are the gsp and gip2 oncogenes, encoding GTPase deficient alpha-subnits of Gs or Gi-2 proteins. Representatives from the family of small G-proteins are the products of the Harvey-, Kirsten- or N-ras oncogenes. These oncogenes, which are frequently expressed in human malignancies, code for proteins (p21ras) that are locked in the activated GTP-bound state because their GTPase is refractory to the ras-specific GTPase activating protein (GAP). In other cases p21ras-GTP levels have been found to be elevated as a result of an increase in GDP/GTP exchange rate. In neurofibromatosis v. Recklinghausen, a mutated gene (NF1) is detectable. The protein encoded by NF1 contains a GAP homology region, binds p21ras-GTP, and stimulates the hydrolysis of p21ras-bound GTP. Both ras-GAP (p120 GAP) and NF1-GAP are inhibited by acidic lipids. Elevated levels of these lipids may exert growth-stimulatory or perhaps tumor-promoting activity by increasing p21ras GTP. The function of transforming p21ras is under control of tumor suppressor genes. Putative suppressor genes isolated from revertants from ras-transformed cells include rsp-1 and the ras recision gene (rrg). Experimentally, an overexpression of Rap 1A/Krev-1 is able to antagonize transformation by p21ras. This mechanism also may be relevant under normal conditions. p53 also is capable of inhibiting transforming p21ras. It is postulated that p105-RB exerts a similar anti-ras effect. The mechanism by which retinoic acid suppresses transformation by ras is discussed. Current strategies for a pharmacological interference of p21ras function are described.

• Cox AD, Graham SM, Solski PA, Buss JE, Der CJ
• The carboxyl-terminal CXXX sequence of Gi alpha, but not Rab5 or Rab11, supports Ras processing and transforming activity.
• J Biol Chem. 1993; 268: 11548-52
• Display abstract

• Although the heterotrimeric Gi alpha subunit terminates in an apparent CXXX prenylation signal (CGLF), it is not modified by isoprenylation. To determine if the Gi alpha CXXX sequence can signal prenylation when placed at the carboxyl termini of normally prenylated proteins, we have characterized the processing and biological activity of chimeric oncogenic Ras proteins that terminate in the Gi alpha CXXX sequence (Ras/Gi alpha). Surprisingly, these chimeras were prenylated both in vivo and in vitro, demonstrated significant membrane association, exhibited transforming activity, and induced transcriptional transactivation from Ras-responsive elements. We then extended these studies to determine if, unlike the CC or CXC carboxyl-terminal sequences of other Rab proteins, the carboxyl-terminal CXXX sequences of the Ras-related Rab5 and Rab11 proteins represent conventional CXXX prenylation signals that can support Ras processing and transforming activity. Unexpectedly, these Ras/Rab chimeras were nonprenylated, were cytosolic, and lacked detectable transforming or transcriptional transactivation activity. Taken together, these results suggest that the context within which a CXXX sequence occurs may also critically control the modification of a protein by prenylation, and that the Rab5 and Rab11 carboxyl termini do not possess conventional CXXX sequences. Instead, their CCXX and CCXXX motifs may represent additional classes of protein prenylation signals.

• Newman CM, Magee AI
• Posttranslational processing of the ras superfamily of small GTP-binding proteins.
• Biochim Biophys Acta. 1993; 1155: 79-96

• Self AJ, Paterson HF, Hall A
• Different structural organization of Ras and Rho effector domains.
• Oncogene. 1993; 8: 655-61
• Display abstract

• Ras regulates proliferation and differentiation signals in cells, and activation of the protein can lead to malignant transformation. Activation of the related protein, Rho, affects cell morphology, and it has been suggested that it may also have some oncogenic potential. We show here that Rho does not induce a malignant phenotype in NIH3T3 cells but instead is a potent activator of actin stress fibre formation. The limited homology between Ras and Rho has enabled us to determine the amino acids specifying their different biological activities and GTPase-activating protein (GAP) protein sensitivities using chimeras. Rho substituted with amino acids 23-46 of Ras induces transformed foci in NIH3T3 monolayers, and we conclude that Ras has a single effector domain required for downstream signalling. Although mutational analysis of Rho has revealed that residues 32-42 are also essential for its biological activity, Ras substituted with amino acids 25-48 of Rho does not induce actin stress fibre formation. On the basis of these experiments, we propose that Rho may have two effector domains: one at amino acids 32-42 and corresponding to the effector region of Ras and the second located elsewhere in the carboxy-terminal two-thirds of the molecule.

• Culine S et al.
• A possible role for the Ras-related Rab2 protein in the immunological events associated with hematological malignancies.
• Nouv Rev Fr Hematol. 1993; 35: 41-4
• Display abstract

• The Rab branch of the Ras-related guanine nucleotide (GTP/GDP)-binding proteins currently includes at least thirty related members which are involved in the intracellular vesicular transport along the secretory and endocytic pathways in eukaryotic cells. We have demonstrated the overexpression of the Rab2 protein in peripheral blood mononuclear cells from patients exhibiting Sezary syndromes and other lymphoid and myeloid malignancies. Several lines of evidence suggest that the Rab2 overexpression can be related not to leukemic cells but to a subset of peripheral lymphocytes with a CD2+ phenotype. Our results provide strong evidences for the implication of a small GDP/GTP binding protein in immunological events associated with neoplastic states. The precise cellular population involved in this process remains to be determined.

• Ligeti E, Pizon V, Wittinghofer A, Gierschik P, Jakobs KH
• GTPase activity of small GTP-binding proteins in HL-60 membranes is stimulated by arachidonic acid.
• Eur J Biochem. 1993; 216: 813-20
• Display abstract

• The GTPase activity of membranes isolated from differentiated HL-60 cells was investigated to obtain information about the possible involvement of membrane-bound GTP-binding proteins in the regulation of the NADPH oxidase. A more than tenfold increase in the rate of hydrolysis of membrane-bound GTP was observed when cytosol and arachidonic acid were added simultaneously, i.e. under the same conditions where NADPH oxidase becomes activated. There were parallel changes in GTPase and NADPH oxidase activities when the concentration of arachidonic acid or the species of the fatty acid was varied or different detergents were applied. Separation of the GTP-binding proteins of the solubilized membrane by sucrose density gradient centrifugation, allowed us to ascribe the observed effect to the stimulation of the GTPase activity of small GTP-binding proteins by cytosolic component(s). Indirect evidence suggests that, in contrast to the effect upon recombinant ras and ras-GTPase-activating protein, in intact HL-60 membranes the interaction of rap1A with rap-GTPase-activating protein, is strongly enhanced by arachidonic acid.

• Lowy DR et al.
• Cell transformation by ras and regulation of its protein product.
• Ciba Found Symp. 1993; 176: 67-80
• Display abstract

• We are studying the biological activity and regulation of mammalian Ras protein in tumours and in physiological signalling. We have shown that GAP (the GTPase-activating protein) is a potent negative regulator of normal Ras in cells. Reduction or loss of the NF1 gene product neurofibromin, in association with genetic abnormalities of the NF1 locus, has been identified in schwannoma cell lines from patients with neurofibromatosis and in melanoma and neuroblastoma lines from patients without neurofibromatosis. Although loss of neurofibromin in the schwannoma lines was associated with a high proportion of normal Ras protein in the active GTP-bound state, Ras-GTP appeared to be appropriately regulated in the melanoma and neuroblastoma lines, which contain normal levels of GAP. Therefore the GTPase-activating activity of neurofibromin is not essential for negative regulation of Ras in some cell types and the putative tumour suppressor function of neurofibromin in such cell types is independent of its GTPase-activating activity. Mitogen activation of Ras in fibroblasts is mediated primarily by exchange factors, which probably interact with a region on the Ras protein distinct from the region required for interaction with GAP. Multiple full-length cDNAs have identified a mouse gene whose products are related to yeast CDC25 guanine nucleotide exchange factor.

• Burstein ES, Macara IG
• Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A.
• Proc Natl Acad Sci U S A. 1992; 89: 1154-8
• Display abstract

• The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.

• Jena BP, Brennwald P, Garrett MD, Novick P, Jamieson JD
• Distinct and specific GAP activities in rat pancreas act on the yeast GTP-binding proteins Ypt1 and Sec4.
• FEBS Lett. 1992; 309: 5-9
• Display abstract

• Previous studies have demonstrated that Sec4, a 23.5 kDa guanine nucleotide-binding protein of the ras superfamily is required for exocytosis in the budding yeast Saccharomyces cerevisiae. Ypt1, another ras-like 23 kDa guanine nucleotide-binding protein in yeast has been found to be involved in ER-Golgi transport. A mammalian homologue of Ypt1 called rab1 has also been identified. Recent studies using purified Sec4 protein have identified a component of yeast lystate that specifically stimulates the hydrolysis of GTP bound to Sec4. In the present study, purified recombinant Sec4 and Ypt1 proteins expressed in E. coli have been used as substrates to determine if GTPase activating proteins (GAPs) directed toward these proteins are present in rat pancreas. Our studies showed that 65% of Sec4-GAP activity was associated with the 150,000 x g pancreatic particulate fraction with approximately 35% being found in the cytosol. On the other hand, more than 95% of Ypt1-GAP activity was found to associate with the particulate fraction. Sec4 and Ypt1 competition assays further demonstrated the specificity of the Sec4 and Ypt1 GAPs. The results from the present study suggest the presence of a distinct GAP in the pancreas that interacts with Sec4, and another that interacts with Ypt1.

• Downward J
• Signal transduction. Rac and Rho in tune.
• Nature. 1992; 359: 273-4

• Farrell FX, Lapetina EG
• Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes.
• Biochem Biophys Res Commun. 1992; 189: 717-21
• Display abstract

• Rap2b is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a approximately 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.

• Sassone-Corsi P, Borrelli E
• Mutations in signal transduction pathways and inherited diseases.
• Curr Opin Genet Dev. 1992; 2: 455-8
• Display abstract

• Intracellular signal transduction pathways have central roles in processes such as growth, differentiation, neurotransmission and development. The aberrant expression of components of various signal transduction pathways has profound consequences for cellular functions. Recent findings indicate that many cases of neoplasia and inherited diseases have, at their roots, mutations in key steps of signalling pathways.

• Kitayama H, Matsuzaki T, Sugimoto Y, Ikawa Y, Noda M
• Genetic analysis of the K-rev-1 transformation-suppressor gene.
• Environ Health Perspect. 1991; 93: 73-7
• Display abstract

• Flat revertants with reduced malignancy in vivo can be isolated from Kirsten sarcoma virus-transformed NIH 3T3 cells (DT line) following transfection with a normal human fibroblast cDNA expression library. We have recovered from one such revertant a 1.8-kb cDNA clone, K-rev-1, that exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when transfected into DT cells. The K-rev-1 cDNA has the capacity to encode a protein with a calculated molecular weight of 21,000, having strong structural similarity to ras proteins (approximately 50% homology), especially in their guanosine triphosphate/guanosine diphosphate-binding, effector-binding, and membrane-attachment domains. Toward understanding the mechanism of action of K-rev-1 protein, we constructed a series of point mutants of K-rev-1 cDNA and tested their biological activities. 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 including Gly12 to Val12, Ala59 to Thr59, and Gln63 to Glu63 were found to significantly increase the transformation-suppressor activity of K-rev-1. These findings are consistent with the idea that K-rev-1 protein is regulated like many other G-proteins by guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.

• Rubinfeld B et al.
• A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange.
• Int J Pept Protein Res. 1991; 38: 47-53
• Display abstract

• Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.

• Chardin P
• Small GTP-binding proteins of the ras family: a conserved functional mechanism?
• Cancer Cells. 1991; 3: 117-26
• Display abstract

• Mutated ras genes can acquire a transforming potential and are frequently detected in human tumors. The mammalian ras gene family includes at least 35 distinct members that can be divided into three main groups on the basis of their sequence similarity to ras, rho, or rab genes. All these genes encode small GTP-binding proteins. Rho proteins are implicated in actin organization and control of cell shape, probably by interacting with the cytoskeleton and intracellular membranes. Rab proteins are involved in vesicular traffic, and appear to control the translocation of vesicles from donor to acceptor membranes. The precise function of ras proteins is unknown, although the prevailing view is that they act as transducers of mitogenic signals. We propose that ras proteins, by analogy with rho and rab, are involved in the lateral segregation of multi-protein complexes at the plasma membrane, and we suggest how this process may be important for mitogenic signal transduction.

• Polakis PG, Rubinfeld B, Evans T, McCormick F
• Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells.
• Proc Natl Acad Sci U S A. 1991; 88: 239-43
• Display abstract

• rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.

• Nelson TJ, Sanchez-Andres JV, Schreurs BG, Alkon DL
• Classical conditioning-induced changes in low-molecular-weight GTP-binding proteins in rabbit hippocampus.
• J Neurochem. 1991; 57: 2065-9
• Display abstract

• Classical conditioning of Hermissenda, involving paired light-rotation events, results in a 30-35% decrease in the levels of a 20-kDa G protein (cp20). To test whether a similar protein exists in vertebrates, rabbits were trained to associate a tone with periorbital electrical stimulation and G proteins were analyzed by photoaffinity labeling with [alpha-32P]GTP-azidoanilide. A 20-kDa G protein similar to cp20 decreased by 36% in the hippocampus of rabbits subjected to paired tone and electrical stimulation, but not in unpaired controls. Learning-specific decreases were also found in the amount of ras protein.

• Hall A
• The cellular functions of small GTP-binding proteins.
• Science. 1990; 249: 635-40
• Display abstract

• A substantial number of novel guanine nucleotide binding regulatory proteins have been identified over the last few years but the function of many of them is largely unknown. This article will discuss a particular family of these proteins, structurally related to the Ras oncoprotein. Approximately 30 Ras-related small guanosine triphosphate (GTP)-binding proteins are known, and from yeast to man they appear to be involved in controlling a diverse set of essential cellular functions including growth, differentiation, cytoskeletal organization, and intracellular vesicle transport and secretion.

• Frech M et al.
• Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product.
• Science. 1990; 249: 169-71
• Display abstract

• Krev-1 is known to suppress transformation by ras. However, the mechanism of the suppression is unclear. The protein product of Krev-1, Rap1A-p21, is identical to Ras-p21 proteins in the region where interaction with guanosine triphosphatase (GTPase) activating protein (GAP) is believed to occur. Therefore, the ability of GAP to interact with Rap1A-p21 was tested. Rap1A-p21 was not activated by GAP but bound tightly to GAP and was an effective competitive inhibitor of GAP-mediated Ras-GTPase activity. Binding of GAP to Rap1A-p21 was strictly guanosine triphosphate (GTP)-dependent. The ability of Rap1A-p21 to bind tightly to GAP may account for Krev-1 suppression of transformation by ras. This may occur by preventing interaction of GAP with Ras-p21 or with other cellular proteins necessary for GAP-mediated Ras GTPase activity.

• Bar-Sagi D, Feramisco JR
• Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins.
• Science. 1986; 233: 1061-8
• Display abstract

• Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins.

© 2021 EMBL | Privacy Policy