SMART MODE:

NORMAL GENOMIC

• Lau AG, Hall RA
• Oligomerization of nherf-1 and nherf-2 pdz domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation.
• Biochemistry. 2001; 40: 8572-80
• Display abstract

• PDZ domains bind to the carboxyl-termini of target proteins, and some PDZ domains are capable of oligomerization to facilitate the formation of intracellular signaling complexes. The Na(+)/H(+) exchanger regulatory factor (NHERF-1; also called "EBP50") and its relative NHERF-2 (also called "E3KARP", "SIP-1", and "TKA-1") both have two PDZ domains. We report here that the PDZ domains of NHERF-1 and NHERF-2 bind specifically to each other but not to other PDZ domains. Purified NHERF-2 PDZ domains associate with each other robustly in the absence of any associated proteins, but purified NHERF-1 PDZ domains associate with each other only weakly when examined alone. The oligomerization of the NHERF-1 PDZ domains is greatly facilitated when they are bound with carboxyl-terminal ligands, such as the carboxyl-termini of the beta(2)-adrenergic receptor or the platelet-derived growth factor receptor. Oligomerization of full-length NHERF-1 is also enhanced by mutation of serine 289 to aspartate (S289D), which mimics the phosphorylated form of NHERF-1. Co-immunoprecipitation experiments with differentially tagged versions of the NHERF proteins reveal that NHERF-1 and NHERF-2 form homo- and hetero-oligomers in a cellular context. A point-mutated version of NHERF-1 (S289A), which cannot be phosphorylated on serine 289, exhibits a reduced capacity for co-immunoprecipitation from cells. These studies reveal that both NHERF-1 and NHERF-2 can oligomerize, which may facilitate NHERF-mediated formation of cellular signaling complexes. These studies furthermore reveal that oligomerization of NHERF-1, but not NHERF-2, is highly regulated by association with other proteins and by phosphorylation.

• Stein EG, Gustafson TA, Hubbard SR
• The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors.
• FEBS Lett. 2001; 493: 106-11
• Display abstract

• Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling.

• Barnes H, Larsen B, Tyers M, van Der Geer P
• Tyrosine-phosphorylated low density lipoprotein receptor-related protein 1 (Lrp1) associates with the adaptor protein SHC in SRC-transformed cells.
• J Biol Chem. 2001; 276: 19119-25
• Display abstract

• v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta-amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.

• Dans M, Gagnoux-Palacios L, Blaikie P, Klein S, Mariotti A, Giancotti FG
• Tyrosine phosphorylation of the beta 4 integrin cytoplasmic domain mediates Shc signaling to extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes.
• J Biol Chem. 2001; 276: 1494-502
• Display abstract

• Ligation of the alpha(6)beta(4) integrin induces tyrosine phosphorylation of the beta(4) cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of beta(4) that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr(1440), or secondarily Tyr(1422), interacts with the SH2 domain of Shc, whereas Tyr(1526), or secondarily Tyr(1642), interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by alpha(6)beta(4). In addition, mutation of beta(4) Tyr(1526), which binds to the PTB domain of Shc, but not of Tyr(1422) and Tyr(1440), which interact with its SH2 domain, abolishes the activation of ERK by alpha(6)beta(4). Phenylalanine substitution of the beta(4) tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of alpha(6)beta(4) in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of beta4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type beta(4). This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of beta(4) containing phenylalanine substitutions at Tyr(1422) and Tyr(1440), at Tyr(1526) and Tyr(1642), or at all four tyrosine phosphorylation sites. These results suggest that beta(4) Tyr(1526) interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.

• Jaulin-Bastard F et al.
• The ERBB2/HER2 receptor differentially interacts with ERBIN and PICK1 PSD-95/DLG/ZO-1 domain proteins.
• J Biol Chem. 2001; 276: 15256-63
• Display abstract

• Identification of protein complexes associated with the ERBB2/HER2 receptor may help unravel the mechanisms of its activation and regulation in normal and pathological situations. Interactions between ERBB2/HER2 and Src homology 2 or phosphotyrosine binding domain signaling proteins have been extensively studied. We have identified ERBIN and PICK1 as new binding partners for ERBB2/HER2 that associate with its carboxyl-terminal sequence through a PDZ (PSD-95/DLG/ZO-1) domain. This peptide sequence acts as a dominant retention or targeting basolateral signal for receptors in epithelial cells. ERBIN belongs to the newly described LAP (LRR and PDZ) protein family, whose function is crucial in non vertebrates for epithelial homeostasis. Whereas ERBIN appears to locate ERBB2/HER2 to the basolateral epithelium, PICK1 is thought to be involved in the clustering of receptors. We show here that ERBIN and PICK1 bind to ERBB2/HER2 with different mechanisms, and we propose that these interactions are regulated in cells. Since ERBIN and PICK1 tend to oligomerize, further complexity of protein networks may participate in ERBB2/HER2 functions and specificity.

• Kuo WN, Kreahling JM, Shanbhag VP, Shanbhag PP, Mewar M
• Protein nitration.
• Mol Cell Biochem. 2000; 214: 121-9
• Display abstract

• Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 microM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent Km of PKA for cAMP increased from approximately 10(-8) to 10(-6) M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.

• Pai JJ, Kirkup MP, Frank EA, Pachter JA, Bryant RW
• Compounds capable of generating singlet oxygen represent a source of artifactual data in scintillation proximity assays measuring phosphopeptide binding to SH2 domains.
• Anal Biochem. 1999; 270: 33-40
• Display abstract

• We developed scintillation proximity assays (SPA) to discover compounds which inhibit phosphopeptide binding to Src homology 2 (SH2) domain proteins Grb2 and Syk. An assay artifact is reported here as a caveat to others. The SPA used an antibody to couple glutathione-S-transferase SH2 domain fusion proteins to scintillant beads coated with protein A. A pyrazoloquinolone and indolocarbazole inhibited [3H]phosphopeptide binding in both assays. Their potency in the SPA increased with prolonged (2 to 24 h) assay exposure to ambient light. They were inactive in absence of light and in an alternate binding assay. Both compounds absorbed visible light and generated singlet oxygen based on 2-methylfuran-trapping experiments. Their inhibitory activity was suppressed by the singlet oxygen scavengers sodium azide and dithiothreitol. The results suggest that compounds, not previously considered photosensitizers, generated enough singlet oxygen to damage oxidant-sensitive SPA components. Therefore, this SPA should be protected from light to minimize occurrence of false positives.

• Fournier E, Blaikie P, Rosnet O, Margolis B, Birnbaum D, Borg JP
• Role of tyrosine residues and protein interaction domains of SHC adaptor in VEGF receptor 3 signaling.
• Oncogene. 1999; 18: 507-14
• Display abstract

• The VEGFR3/FLT4 receptor, which is involved in vasculogenesis and angiogenesis, binds and phosphorylates SHC proteins on tyrosine residues. SHC contains two phosphotyrosine interaction domains: a PTB (Phosphotyrosine Binding) and a SH2 (Src Homology 2) domain. Previous studies have shown that SHC proteins are phosphorylated on Y239/Y240 and Y313 (Y317 in humans) by tyrosine kinases such as the EGF and IL3 receptors. We have investigated which of the SHC tyrosine residues are targeted by the VEGFR3/ FLT4 kinase and the role of the SHC PTB and SH2 domains in this process. Our results show that Y239/ Y240 and Y313 are simultaneously phosphorylated by the kinase, creating GRB2 binding sites. Mutation of SHC PTB, but not SH2, domain interferes with the SHC phosphorylation by VEGFR3/FLT4. Soft agar assay experiments revealed that the VEGFR3/FLT4 transforming capacity is increased by the mutation of Y239/Y240 to phenylalanines in SHC, suggesting that these two residues mediate an inhibitory signal for cell growth. Mutation of the two phosphorylation sites increases this effect, suggesting that they have a synergistic role.

• Farooq A, Plotnikova O, Zeng L, Zhou MM
• Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner.
• J Biol Chem. 1999; 274: 6114-21
• Display abstract

• Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors.

• Siegal G
• The surprisingly flexible PTB domain.
• Nat Struct Biol. 1999; 6: 7-10

• Lupher ML Jr et al.
• Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323.
• J Biol Chem. 1998; 273: 35273-81
• Display abstract

• The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998) J. Biol. Chem. 273, 8867-8874). A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells. Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr323 in the negative regulation of Syk. Co-expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-zeta. Unexpectedly, these effects were largely due to the loss of Syk protein. Both the decrease in Syk and CD8-zeta phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk. These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.

• Liu SK, McGlade CJ
• Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc.
• Oncogene. 1998; 17: 3073-82
• Display abstract

• Shc proteins are important substrates of receptor and cytoplasmic tyrosine kinases that couple activated receptors to downstream signaling enzymes. Phosphorylation of Shc tyrosine residues 239 and 317 leads to recruitment of the Grb2-Sos complex, thus linking Shc phosphorylation to Ras activation. We have used phosphorylated peptides corresponding to the regions spanning tyrosine 239/240 and 317 of Shc in an expression library screen to identify additional downstream targets of Shc. Here we report the identification of Gads, a novel adaptor protein most similar to Grb2 and Grap that contains amino and carboxy terminal SH3 domains flanking a central SH2 domain and a 120 amino acid unique region. Gads is most highly expressed in the thymus and spleen of adult animals and in human leukemic cell lines. The binding specificity of the Gads SH2 domain is similar to Grb2 and mediates the interaction of Gads with Shc, Bcr-Abl and c-kit. Gads does not interact with Sos, Cbl or Sam68, although the isolated carboxy terminal Gads SH3 domain is able to bind these molecules in vitro. Our results suggest that the unique structure of Gads regulates its interaction with downstream SH3 domain-binding proteins and that Gads may function to couple tyrosine-phosphorylated proteins such as Shc, Bcr-Abl and activated receptor tyrosine kinases to downstream effectors distinct from Sos and Ras.

• Cattaneo E, Pelicci PG
• Emerging roles for SH2/PTB-containing Shc adaptor proteins in the developing mammalian brain.
• Trends Neurosci. 1998; 21: 476-81
• Display abstract

• In mammalian systems, SH2-containing cytoplasmic signalling molecules are known to play an important role in determining cell responsiveness to the environment. In particular, following activation of a receptor protein tyrosine kinase (RPTK), proteins like Shc and Grb2 bind to phosphotyrosine residues of stimulated receptors, thereby activating downstream components of specific signalling pathways. The ShcA gene was identified in 1992 and was found to encode three proteins with properties of adaptor molecules coupling RPTKs to Ras. Early data obtained in non-neuronal cells have revealed that Shc and Grb2 proteins are highly expressed and activated in all cells. However, recent analyses of ShcA mRNA and protein in the developing brain revealed progressive downregulation of their expression during differentiation from neuroblasts to neurons. Conversely, the two newly identified Shc homologues (ShcB/Sli and ShcC/Rai) are highly expressed in the mature brain.Thus, variations in the intracellular levels of adaptor proteins might represent one of the mechanisms by which a differentiating cell changes its ability to respond to a given factor, allowing a cell to choose between proliferation and differentiation.

• Li SC et al.
• Structure of a Numb PTB domain-peptide complex suggests a basis for diverse binding specificity.
• Nat Struct Biol. 1998; 5: 1075-83
• Display abstract

• The phosphotyrosine-binding (PTB) domain of Numb, a protein involved in asymmetric cell division, has recently been shown to bind to the adapter protein Lnx through an LDNPAY sequence, to the Numb-associated kinase (Nak) through a sequence that does not contain an NPXY motif and to GP(p)Y-containing peptides obtained from library screening. We show here that these diverse peptide sequences bind with comparable affinities to the Numb PTB domain at a common binding site on the surface of the protein. The NMR structure of the Numb PTB domain in complex with a GPpY-containing peptide reveals a novel mechanism of binding with the peptide in a helical turn that does not hydrogen bond to the PTB domain beta-sheet. These results suggest that PTB domains can potentially have multiple modes of peptide recognition and provide a structural basis from which the multiple functions of the Numb PTB domain during asymmetric cell division could arise.

• Xu Y, Guo DF, Davidson M, Inagami T, Carpenter G
• Interaction of the adaptor protein Shc and the adhesion molecule cadherin.
• J Biol Chem. 1997; 272: 13463-6
• Display abstract

• In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Using several experimental approaches we demonstrate that Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras.

• Ravichandran KS et al.
• Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo.
• Mol Cell Biol. 1997; 17: 5540-9
• Display abstract

• The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine-binding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.

• Giorgetti-Peraldi S, Ottinger E, Wolf G, Ye B, Burke TR Jr, Shoelson SE
• Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking.
• Mol Cell Biol. 1997; 17: 1180-8
• Display abstract

• Shc and insulin receptor substrate 1 (IRS-1) are cytoplasmic substrates of tyrosine kinase receptors that engage, localize, and activate downstream SH2 enzymes. Each contains a phosphotyrosine-binding (PTB) domain that is structurally unrelated to SH2 domains. We have designed high-affinity, cellular inhibitors of the Shc PTB domain by incorporating nonnatural, phosphatase-resistant amino acids into short peptides. None of the inhibitors bind the IRS-1 PTB domain, consistent with distinct specificities for domains. The best inhibitor of the Shc domain was introduced by electroporation into Rat1 fibroblasts that express human insulin receptors. Insulin-stimulated phosphorylation of Shc was inhibited, with no effect on IRS-1, and downstream effects on mitogen-activated protein kinase and DNA synthesis were both inhibited. The PTB domain inhibitor had less influence on epidermal growth factor-induced effects and essentially no impact on serum- or phorbol ester-induced effects. The inhibitor did not affect insulin internalization and its degradation. We conclude that the PTB domain of Shc is critical for its phosphorylation by the insulin receptor, that Shc is an important mediator of insulin's mitogenic effects, and that Shc is not central to insulin receptor cycling in these cells. PTB domains can be inhibited selectively in cells and represent potential targets for drug discovery.

• Lupher ML Jr, Songyang Z, Shoelson SE, Cantley LC, Band H
• The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70.
• J Biol Chem. 1997; 272: 33140-4
• Display abstract

• The Cbl protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cbl physically interacts with tyrosine kinases is of substantial interest. We recently identified a phosphotyrosine binding (PTB) domain residing within the N-terminal transforming region of Cbl (Cbl-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the Cbl-PTB domain selects a D(N/D)XpY motif, reminiscent of but distinct from the NPXpY motif recognized by the PTB domains of Shc and IRS-1/2. A phosphopeptide predicted by this motif and corresponding to the in vivo negative regulatory phosphorylation site of ZAP-70 (Tyr(P)292) specifically inhibited binding of ZAP-70 to Cbl-N. A ZAP-70/Y292F mutant failed to bind to Cbl-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cbl-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cbl-N or full-length Cbl in vivo. These results identify a potential Cbl-PTB domain-dependent role for Cbl in the negative regulation of ZAP-70 and predict potential Cbl-PTB domain binding sites on other protein tyrosine kinases known to interact with Cbl.

• Senderowicz L, Wang JX, Wang LY, Yoshizawa S, Kavanaugh WM, Turck CW
• 3-Phosphohistidine cannot replace phosphotyrosine in high-affinity binding to phosphotyrosine binding or Src homology 2 domains.
• Biochemistry. 1997; 36: 10538-44
• Display abstract

• Posttranslational phosphorylation of proteins is an important event in many cellular processes. Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors. Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms. Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction. We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains. From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.

• Olejniczak ET, Zhou MM, Fesik SW
• Changes in the NMR-derived motional parameters of the insulin receptor substrate 1 phosphotyrosine binding domain upon binding to an interleukin 4 receptor phosphopeptide.
• Biochemistry. 1997; 36: 4118-24
• Display abstract

• Proteins recognize ligands by forming specific intermolecular interactions that often involve solvent exposed residues. Changes in the motional properties of these residues upon binding can affect the conformational entropy of the system and thus are related to the energetics of binding. The role that dynamics plays in ligand recognition can be investigated by comparing the motional properties of a free and ligated protein. NMR relaxation studies are well suited for examining changes in dynamics, especially for motions on a nanosecond to picosecond time scale. Recently, we determined the solution structure of the phosphotyrosine binding (PTB) domain of the insulin receptor substrate (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the interleukin 4 (IL-4) receptor [Zhou et al., (1996) Nat. Struct. Biol. 3, 388-393]. The peptide binds tightly to the protein in a surface exposed pocket, resulting in the partial burial of many protein residues. Using NMR relaxation studies, the dynamics of the backbone nitrogens of IRS-1 PTB domain were studied in both the free protein and the protein when complexed to the IL-4 receptor phosphopeptide. The backbone nitrogens of many residues that make important contacts to the ligand are motionally restricted in the free and complexed protein. Additional residues become motionally restricted only after ligand binding, including several residues that do not make any direct contacts with the ligand. These observed changes in the dynamics are compared to structural features of the complex.

• Margolis B
• The PI/PTB domain: a new protein interaction domain involved in growth factor receptor signaling.
• J Lab Clin Med. 1996; 128: 235-41
• Display abstract

• In summary, a new domain called the PI/PTB domain has been identified in the Shc adapter protein. This motif binds the NPXpY motif that is found in a large number of signal transduction molecules. The presence in Shc of both a PI/PTB domain and an SH2 domain presumably gives Shc the ability to interact with a large number of tyrosine-phosphorylated proteins. The structure of the PI/PTB domain has been solved and is very similar to the PH domain. Highly related in binding specificity is the PTB domain of IRS-1 and IRS-2, which also binds an NPXpY motif. Several other PI domains have been identified that may also have binding specificity for the NPXY motif. The terminology used at present to define these domains is unclear. The original terms PI and PTB domain stood for phosphotyrosine interaction and phosphotyrosine binding domain, respectively. The name may be inappropriate for some members of this family in which phosphotyrosine may not be essential for binding. Furthermore, these domains are structurally related to the previously named PH domains. At present we feel the use of the name PTB domain should be reserved for Shc and IRS-1/IRS-2, where phosphotyrosine binding has been demonstrated. We place the other proteins we have identified in the PI domain family, with PI now representing the protein interaction rather than the phosphotyrosine interaction domain. The role of several of the PI domains in other proteins is beginning to be studied. It seems clear that our understanding of these domains and their function in cell biology will rapidly expand over the next several years.

• Laminet AA, Apell G, Conroy L, Kavanaugh WM
• Affinity, specificity, and kinetics of the interaction of the SHC phosphotyrosine binding domain with asparagine-X-X-phosphotyrosine motifs of growth factor receptors.
• J Biol Chem. 1996; 271: 264-9
• Display abstract

• The phosphotyrosine binding (PTB) domain specifically binds to tyrosine-phosphorylated proteins, but differs in structure and mechanism of action from the SH2 domain family. We quantitated the affinity, specificity, and kinetics of the interaction of the SHC PTB domain with a sequence motif, asparagine-X-X-phosphotyrosine (NXX(pY)), found in several receptor tyrosine kinases and oncogenic proteins. PTB domain-mediated interaction with the NXX(pY) motif of c-ErbB2 was characterized by similar overall affinity but slower kinetics than that reported for SH2 domains. This suggested that unlike SH2 domains, PTB domains may not rapidly exchange among associated proteins. Furthermore, when directly and quantitatively compared, PTB domain binding specificity did not significantly overlap with a panel of seven SH2 domains. Thus, signaling pathways involving PTB and SH2 domain-mediated interactions can be regulated separately. Finally, our data define the minimal SHC PTB domain binding motif as NXX(pY), not NPX(pY) as suggested by other authors, and suggest a high affinity motif, hydrophobic residue-(D/E)-N-X-X-pY-(W/F), found in the Trk and ErbB receptor tyrosine kinase families. We conclude that PTB domains mediate specific protein-protein interactions independent from those mediated by SH2 domains.

• Adams RM, Das S, Smith TF
• Multiple domain protein diagnostic patterns.
• Protein Sci. 1996; 5: 1240-9
• Display abstract

• We have implemented an iterative algorithm for the identification of diagnostic patterns from sets of multiple-domain proteins, where domains need not be common to all the proteins in the defining set. Our algorithm was applied to sequences gathered using a variety of methods, including BLAST, common keywords, and common E.C. numbers. In all cases, useful diagnostic patterns were obtained, possessing both high sensitivity and specificity. The patterns were found to correlate in several cases with both functional and structural domains. Patterns generated from a large number of sequence families were analyzed for probable multiple-domain structure.

• van der Geer P et al.
• Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.
• Proc Natl Acad Sci U S A. 1996; 93: 963-8
• Display abstract

• The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

• Catipovic B, Schneck JP, Brummet ME, Marsh DG, Rafnar T
• Csk is constitutively associated with a 60-kDa tyrosine-phosphorylated protein in human T cells.
• J Biol Chem. 1996; 271: 9698-703
• Display abstract

• The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase-activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP.

• Ravichandran KS, Igras V, Shoelson SE, Fesik SW, Burakoff SJ
• Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.
• Proc Natl Acad Sci U S A. 1996; 93: 5275-80
• Display abstract

• Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling.

• Yi T, Zhang J, Miura O, Ihle JN
• Hematopoietic cell phosphatase associates with erythropoietin (Epo) receptor after Epo-induced receptor tyrosine phosphorylation: identification of potential binding sites.
• Blood. 1995; 85: 87-95
• Display abstract

• Erythropoietin (Epo) binding to its receptor (EpoR) induces tyrosine phosphorylation in responsive cells and this ability is required for a mitogenic response. One of the substrates of tyrosine phosphorylation is the Epo receptor (EpoR). The carboxyl region of EpoR cytoplasmic domain is required for EpoR phosphorylation and has been shown to negatively affect the response to Epo both in vivo and in cell lines. Hematopoietic cell phosphatase (HCP) has also been hypothesized to negatively regulate erythropoiesis, based on the hypersensitivity to Epo of erythroid lineage cells in moth-eaten mice that genetically lack HCP. In the studies presented here, we show that HCP binds the tyrosine phosphorylated Epo receptor through the amino-terminal src-homology 2 (SH2) domain of HCP. Using a series of phosphotyrosine-containing peptides, potential HCP binding sites in the cytoplasmic domain of the EpoR are identified. The results support the concept that, after Epo stimulation, phosphorylation of EpoR provides a docking site for HCP in the receptor complex. Recruitment of HCP to the complex and its subsequent dephosphorylation of substrates and/or associated kinases may be important to mitigate the ligand-induced mitogenic response.

• Castagnino P, Biesova Z, Wong WT, Fazioli F, Gill GN, Di Fiore PP
• Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent.
• Oncogene. 1995; 10: 723-9
• Display abstract

• Several signal transducers bind through their SH2 domains to phosphotyrosine-containing motifs present in receptor tyrosine kinases (RTKs). However, the juxtamembrane regions of the epidermal growth factor receptor (EGFR) and of the related erbB-2 protein, while important in mitogenic signaling, lack demonstrable tyrosine phosphorylation sites, suggesting that other modalities of receptor-transducer interactions exist. A candidate for investigating this type of association is p97eps8, a recently described substrate for RTKs. p97eps8 is phosphorylated by several RTKs, associates with EGFR in vivo and, upon overexpression, enhances the transduction of EGFR-mediated mitogenic signals. Here we report that eps8 binds directly to the juxtamembrane region of EGFR through a domain that does not bear resemblance to SH2 domains and by a mechanism that does not require the presence of phosphotyrosine residues. Thus, the physical association between EGFR and eps8 represents a novel interaction between RTKs and their substrates.

• Wang LL, Richard S, Shaw AS
• P62 association with RNA is regulated by tyrosine phosphorylation.
• J Biol Chem. 1995; 270: 2010-3
• Display abstract

• The ras-GAP associated protein, p62, is a major tyrosine phosphoprotein in transformed and growth factor treated cells. Although its exact function is not known, it can bind directly to src-family tyrosine kinases and has been implicated as a linker protein bridging activated src family tyrosine kinases with downstream effectors. One novel feature of p62, revealed by its predicted amino acid sequence, is the presence of an RNA-binding region, the KH domain. As p62 becomes tyrosine phosphorylated when src-kinases become activated, we compared the RNA binding ability of p62 in both its phosphorylated and unphosphorylated state. The ability of p62 to bind RNA was severely impaired when p62 was tyrosine phosphorylated. This suggests that the ability of p62 to bind RNA is regulated by tyrosine phosphorylation and implicates the regulation of RNA as a component of tyrosine kinase signaling pathways.

• Giorgetti-Peraldi S, Peyrade F, Baron V, Van Obberghen E
• Involvement of Janus kinases in the insulin signaling pathway.
• Eur J Biochem. 1995; 234: 656-60
• Display abstract

• The adaptor molecule growth-factor-receptor-bound protein-2 (Grb2) plays a role in insulin action since it links tyrosine phosphorylated IRS-1 and Shc to the guanine-nucleotide-exchange factor, Sos, which initiates the mitogen-activated-protein (MAP) kinase cascade by producing Ras-GTP. Both IRS-1 and Shc are phosphorylated by the insulin-receptor tyrosine kinase. In the present study, we have investigated whether the tyrosine kinases of the Janus kinase family (JAK) could be involved in insulin signaling by acting on Grb2. In fibroblasts over-expressing insulin receptors we observed that two tyrosine-phosphorylated proteins interact with Grb2 and with a mutant of Grb2, which lacks the Src homology 2 (SH2) domain, indicating that these proteins associate with the SH3 domains of Grb2. Further, we found that both JAK1 and JAK2 constitutively associate with Grb2, through interaction with the SH3 domains of Grb2. Finally, insulin appears to induce the tyrosine phosphorylation of JAK1, but does not modify the tyrosine phosphorylation state of JAK2. In conclusion, our results suggest that the JAK proteins could participate in insulin signal transduction, and could therefore constitute an alternative pathway for mediating some of the pleiotropic responses induced by insulin.

• Zhou S et al.
• Catalytic specificity of protein-tyrosine kinases is critical for selective signalling.
• Nature. 1995; 373: 536-9
• Display abstract

• How do distinct protein-tyrosine kinases activate specific down-stream events? Src-homology-2 (SH2) domains on tyrosine kinases or targets of tyrosine kinases recognize phosphotyrosine in a specific sequence context and thereby provide some specificity. The role of the catalytic site of tyrosine kinases in determining target specificity has not been fully investigated. Here we use a degenerate peptide library to show that each of nine tyrosine kinases investigated has a unique optimal peptide substrate. We find that the cytosolic tyrosine kinases preferentially phosphorylate peptides recognized by their own SH2 domains or closely related SH2 domains (group I; ref. 3), whereas receptor tyrosine kinases preferentially phosphorylate peptides recognized by subsets of group III SH2 domains. The importance of these findings for human disease is underscored by our observation that a point mutation in the RET receptor-type tyrosine kinase, which causes multiple endocrine neoplasia type 2B, results in a shift in peptide substrate specificity.

• van der Geer P et al.
• A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.
• Curr Biol. 1995; 5: 404-12
• Display abstract

• BACKGROUND: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. RESULTS: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. CONCLUSION: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.

• Ricci A et al.
• Analysis of protein-protein interactions involved in the activation of the Shc/Grb-2 pathway by the ErbB-2 kinase.
• Oncogene. 1995; 11: 1519-29
• Display abstract

• In murine fibroblasts activation of the Shc/Grb-2 pathway by the ErbB-2 kinase involves tyrosine phosphorylation of Shc products and the formation of Shc/ErbB-2, Shc/Grb-2 and Grb-2/ErbB-2 complexes. Tyr 1139 of ErbB-2 bound to the Grb-2 SH2 domain in vitro as well as in intact cells. Tyr 1221 and 1248 are binding sites of gp185ErbB-2 for Shc SH2 domain in vitro whereas Tyr 1196 and 1248 are major binding sites of ErbB-2 for Shc PTB domain. Inhibition of Shc/ErbB-2 complex formation in intact cells was obtained by simultaneous mutational inactivation of Shc SH2 and Shc PTB binding sites of gp185ErbB-2. Shc/ErbB-2 complexes are formed upon activation of the ErbB-2 kinase and tyrosine phosphorylation of Shc proteins; they are located in both cytosol and cellular membranes. ErbB-2 activation induces also translocation of Grb-2 from cytosol to membranes. This network of protein-protein interactions may reflect the ability of the Shc/Grb-2 pathway to act as a molecular switch controlling different cellular functions regulated by RTK activation. In fact the Ras GDP exchanger mSOS was recruited in Grb-2/ErbB-2 complexes; furthermore besides mSOS, other polypeptides present in either cytosolic or membrane preparations were able to complex in vitro with Grb-2 SH3 domains.

• Shiue L et al.
• Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.
• Mol Cell Biol. 1995; 15: 272-81
• Display abstract

• Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif.(ABSTRACT TRUNCATED AT 250 WORDS)

• Sun XJ et al.
• Role of IRS-2 in insulin and cytokine signalling.
• Nature. 1995; 377: 173-7
• Display abstract

• The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.

• Weiss A
• Signal transduction. Zapping tandem SH2 domains.
• Nature. 1995; 377: 17-8

• Wolf G et al.
• PTB domains of IRS-1 and Shc have distinct but overlapping binding specificities.
• J Biol Chem. 1995; 270: 27407-10
• Display abstract

• PTB domains are non-Src homology 2 (SH2) phosphotyrosine binding domains originally described in the receptor tyrosine kinase substrate, Shc. By serial truncation, we show that a 174-residue region of Shc p52 (33-206) has full PTB activity. We also show that a 173-residue region of insulin receptor substrate-1 (IRS-1; residues 144-316) has related PTB activity. In vitro both domains bind directly to activated insulin receptors. Binding is abrogated by substitution of Tyr-960 and selectively inhibited by phosphopeptides containing NPXY sequences. Phosphopeptide assays developed to compare PTB domain specificities show that the Shc PTB domain binds with highest affinity to psi XN beta 1 beta 2 pY motifs derived from middle T (mT), TrkA, ErbB4, or epidermal growth factor receptors (psi = hydrophobic, beta = beta-turn forming); the IRS-1 PTB domain does not bind with this motif. In contrast, both the Shc and IRS-1 PTB domains bind psi psi psi XXN beta 1 beta 2pY sequences derived from insulin and interleukin 4 receptors, although specificities vary in detail. Shc and IRS-1 are phosphorylated by distinct but overlapping sets of receptor-linked tyrosine kinases. These differences may be accounted for by the inherent specificities of their respective PTB domains.

• Dikic I et al.
• Shc binding to nerve growth factor receptor is mediated by the phosphotyrosine interaction domain.
• J Biol Chem. 1995; 270: 15125-9
• Display abstract

• Shc is an adaptor protein that contains two phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the newly described phosphotyrosine interaction (PI) domain. Shc interacts with several tyrosine-phosphorylated proteins and is itself tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines. Upon phosphorylation, Shc binds to the Grb2.Sos complex leading to the activation of the Ras signaling pathway. Mutational analysis of the nerve growth factor (NGF) receptor (TrkA) suggested that the binding of Shc to the activated receptor is required for NGF-induced neuronal differentiation of PC12 cells. Here we report that the PI domain of Shc directly binds to tyrosine 490 on the autophosphorylated NGF receptor. The PI domain specifically recognizes an I/LXN-PXpY motif (where p indicates phosphorylation) as determined by phosphopeptide competition assay. In addition, the PI domain is able to efficiently compete for binding of full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc SH2 domain interacts with an unidentified tyrosine-phosphorylated protein of 115 kDa but not with the activated NGF receptor. The ability of Shc to interact with different tyrosine-phosphorylated proteins via its PI and SH2 domains may allow Shc to play a unique role in tyrosine kinase signal transduction pathways.

• Mora N, Lacombe JM, Pavia AA
• A new approach to phosphoserine, phosphothreonine and phosphotyrosine synthons and to thiophospho analogs. Stepwise synthesis of mono- and multiphosphorylated phosphopeptides related to src-protein kinase.
• Int J Pept Protein Res. 1995; 45: 53-63
• Display abstract

• Several phosphoserine, phosphothreonine and phosphotyrosine synthons suitable for the stepwise synthesis of phosphopeptides were prepared. Treatment of methylthiomethyl (MTM) esters of either Z-, Boc-, Allocserine and threonine with phosphochloridate in pyridine followed by MgBr2 cleavage of MTM in diethyl ether afforded the title compounds in good yield. Thiophosphoserine and phosphotyrosine synthons were also obtained by the phosphoramidite method using di-(2,2,2-trichloroethyl)-N,N-diisopropylphosphoramidite and MCPBA as oxidizing reagent. Trichloroethyl proved valuable as phosphate protecting group especially in phosphotyrosine derivatives owing to its stability in acidic conditions. These synthons were involved in the liquid-phase synthesis of several phospho and/or thiophosphopeptides related to either src-protein kinase or rat liver pyruvate kinase.

• Lopez CA, Davis RL, Mou K, Denhardt DT
• Activation of a signal transduction pathway by osteopontin.
• Ann N Y Acad Sci. 1995; 760: 324-6

• Velazquez L, Mogensen KE, Barbieri G, Fellous M, Uze G, Pellegrini S
• Distinct domains of the protein tyrosine kinase tyk2 required for binding of interferon-alpha/beta and for signal transduction.
• J Biol Chem. 1995; 270: 3327-34
• Display abstract

• tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-alpha/beta binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-alpha 8 and partial signaling. While no contribution of this protein toward interferon-beta binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-alpha and -beta binding and signaling.

• Xiao S et al.
• Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction.
• J Biol Chem. 1994; 269: 21244-8
• Display abstract

• Syp (SH-PTP2) was recently identified as a phosphotyrosine phosphatase containing two SH2 domains within its primary structure. In response to appropriate growth factor stimulation, Syp becomes phosphorylated on tyrosine residues and associates with insulin receptor substrate 1 (IRS-1) and/or the corresponding growth factor receptor via its SH2 domains, leading to increased Syp activity. To assess the importance of Syp in mitogenic signaling, we microinjected mammalian fibroblasts with several reagents designed to interfere with Syp SH2/phosphotyrosine interaction in vivo. Insulin-, insulin-like growth factor-1-, and epidermal growth factor-stimulated DNA synthesis, indicated by bromodeoxyuridine (BrdUrd) incorporation, was dramatically decreased following microinjection of a Syp antibody (Ab) (65-85%) or a Syp GST-SH2 fusion protein (approximately 90%) in comparison with cells microinjected with control IgG or glutathione S-transferase (GST), respectively. In addition, microinjection of an IRS-1-derived phosphonopeptide, which inhibits in vitro binding of Syp-SH2 to IRS-1 with an ED50 value of approximately 23 microM, also decreased BrdUrd incorporation in vivo by approximately 50-75%. Microinjection of the Syp Ab, Syp GST-SH2 fusion protein, or the phosphonopeptide had no effect on serum-stimulated BrdUrd incorporation. In conclusion, disruption of Syp function in living cells inhibited cell cycle progression in response to growth factor stimulation, indicating that Syp is a critical positive regulator of mitogenic signal transduction.

• Songyang Z et al.
• Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.
• Mol Cell Biol. 1994; 14: 2777-85
• Display abstract

• Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

• Munoz GE, Marshall SH
• Selective phosphoamino acid enrichment by organic solvent fractionation.
• Biotechniques. 1994; 17: 1044-6

• Johansson MW, Larsson E, Luning B, Pasquale EB, Ruoslahti E
• Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin.
• J Cell Biol. 1994; 126: 1299-309
• Display abstract

• We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY beta 1 peptide, but not to the non-phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways.

• Blaikie P, Immanuel D, Wu J, Li N, Yajnik V, Margolis B
• A region in Shc distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors.
• J Biol Chem. 1994; 269: 32031-4
• Display abstract

• Shc is a ubiquitously expressed Src homology 2 (SH2) domain protein that can transform fibroblasts and differentiate PC12 cells in a Ras-dependent fashion. Shc binds a variety of tyrosine-phosphorylated growth factor receptors presumably via its carboxyl-terminal SH2 domain. We cloned a fragment of Shc when screening a bacterial expression library with tyrosine-phosphorylated epidermal growth factor (EGF) receptor. Surprisingly, this fragment encodes the amino terminus of Shc, a region that has no significant similarity to an SH2 domain. When expressed as a glutathione S-transferase fusion protein, this amino-terminal domain binds to autophosphorylated EGF receptor, as well as HER2/neu and TrkA receptors. This fragment acts like an SH2 domain in that it does not bind non-phosphorylated EGF receptor or EGF receptor with all tyrosine phosphorylation sites mutated or deleted. Our data define a novel domain in Shc that has the potential to interact with growth factor receptors and other tyrosine-phosphorylated proteins.

• Yu H, Schreiber SL
• Signalling an interest.
• Nat Struct Biol. 1994; 1: 417-20

• Jaramillo ML, Afar DE, Almazan G, Bell JC
• Identification of tyrosine 620 as the major phosphorylation site of myelin-associated glycoprotein and its implication in interacting with signaling molecules.
• J Biol Chem. 1994; 269: 27240-5
• Display abstract

• Myelin-associated glycoprotein (MAG) is a myelin-specific cell adhesion molecule of the immunoglobulin supergene family and is tyrosine-phosphorylated in the developing brain. To define the role of MAG in signal transduction, the tyrosine phosphorylation sites were analyzed. The major tyrosine phosphorylation residue was identified as Tyr-620, which was found to interact specifically with the SH2 domains of phospholipase C (PLC gamma). This domain may represent a novel protein binding motif that can be regulated by tyrosine phosphorylation. MAG also specifically bound the Fyn tyrosine kinase, suggesting that MAG serves as a docking protein that allows the interaction between different signaling molecules.

• Malek SN, Desiderio S
• A cyclin-dependent kinase homologue, p130PITSLRE is a phosphotyrosine-independent SH2 ligand.
• J Biol Chem. 1994; 269: 33009-20
• Display abstract

• Src-homology 2 (SH2) domains are conserved, globular protein modules that mediate assembly of multicomponent signaling complexes. Phosphoproteins from the B-lymphoid cell line A20 were isolated by SH2 affinity chromatography; the peptide sequence from one of these proteins was used to molecularly clone several related complementary DNAs whose predominant protein product, p130PITSLRE, is an abundant serine/threonine kinase with ubiquitous expression in murine tissues. The sequence of a previously described cyclin-dependent kinase homologue, p58clk-1, is entirely contained within the p130PITSLRE sequence. Specific binding of p130PITSLRE to SH2 domains is mediated by a serine- and glutamic acid-rich cluster of amino acids in the N-terminal region. This interaction is dependent on serine/threonine phosphorylation but independent of tyrosine phosphorylation. Binding is inhibited by free phosphotyrosine and by a phosphotyrosine-containing peptide from polyoma middle T antigen, suggesting that the p130PITSLRE binding site in the SH2 domain overlaps the region that binds phosphotyrosine-containing peptides. Bacterially expressed p130PITSLRE fragments acquire the ability to bind an SH2 domain when phosphorylated in vitro with casein kinase II. A subset of casein kinase II phosphorylation sites may therefore constitute a phosphotyrosine-independent class of SH2 ligands.

• Koyasu S et al.
• Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.
• Proc Natl Acad Sci U S A. 1994; 91: 6693-7
• Display abstract

• To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.

• Pawson T, Olivier P, Rozakis-Adcock M, McGlade J, Henkemeyer M
• Proteins with SH2 and SH3 domains couple receptor tyrosine kinases to intracellular signalling pathways.
• Philos Trans R Soc Lond B Biol Sci. 1993; 340: 279-85
• Display abstract

• The targets of receptor protein-tyrosine kinases are characterized by Src homology 2 (SH2) domains, that mediate specific interactions with receptor autophosphorylation sites. SH2-mediated interactions are important for the activation of biochemical signalling pathways in cells stimulated with growth factors. A distinct protein module, the SH3 domain, is frequently found in polypeptides that contain SH2 domains, and is also implicated in controlling protein-protein interactions in signal transduction. Evidence suggesting that SH2 and SH3 domains act synergistically in stimulation of the Ras pathway is discussed.

• Greenleaf AL
• A positive addition to a negative tail's tale.
• Proc Natl Acad Sci U S A. 1993; 90: 10896-7

• Stradley SJ, Rizo J, Gierasch LM
• Conformation of a heptapeptide substrate bound to protein farnesyltransferase.
• Biochemistry. 1993; 32: 12586-90
• Display abstract

• Protein farnesyltransferase catalyzes isoprenylation of the cysteine four residues from the C-terminus of several proteins including p21ras. Farnesylation is required for the transforming activity of Ras, and many efforts are underway to develop inhibitors of farnesyltransferase. We have used nuclear magnetic resonance spectroscopy to determine the farnesyltransferase-bound conformation of a heptapeptide substrate, KTKCVFM, which competes for the modification of p21Ha-ras in an in vitro assay. Analysis of transferred nuclear Overhauser effects reveals that the CVFM sequence of the peptide substrate is directly involved in binding to the enzyme and adopts a type I beta-turn conformation in the bound state. The present structural information should aid in the design of more effective inhibitors of the enzyme and in understanding the nature of the peptide binding site.

• Myers MG Jr, White MF
• The new elements of insulin signaling. Insulin receptor substrate-1 and proteins with SH2 domains.
• Diabetes. 1993; 42: 643-50
• Display abstract

• Since the discovery of insulin and its receptor, the downstream elements responsible for the pleiotropic insulin signal have been difficult to define. The recently discovered insulin receptor substrate, IRS-1, provides an innovative and simple way to think about this problem: IRS-1 may mediate the control of various cellular processes by insulin. Overexpression of IRS-1 enhances insulin-stimulated DNA synthesis in Chinese hamster ovary cells, and microinjection of IRS-1 protein potentiates the maturation of Xenopus oocytes. We suspect that insulin signals are enabled when the activated insulin receptor kinase phosphorylates specific tyrosine residues in IRS-1. These phosphorylated sites associate with high affinity to cellular proteins that contain SH2 (src homology-2) domains. This association is specific and depends on the amino acid sequence surrounding the phosphotyrosine residue and the isoform of the SH2 domain. A growing number of SH2 domain-containing proteins have been identified, and we suspect that IRS-1 has the potential to simultaneously regulate many of them. We have only begun to identify the specific proteins that associate with phosphorylated IRS-1. One of them, the phosphatidylinositol 3'-kinase, is activated when the SH2 domains in its 85,000-M(r) regulatory subunit bind to phosphorylated IRS-1. IRS-1 also interacts with other proteins such as SHPTP2, a novel SH2 domain-containing Tyr phosphatase, and GRB-2/sem-5, a protein that is implicated in p21ras signaling. The interaction between phosphorylated IRS-1 and multiple SH2 domain-containing proteins may ultimately explain the pleiotropic effects of insulin.

• Patstone G, Maher PA
• Phosphotyrosine-containing proteins are concentrated in differentiating cells during chicken embryonic development.
• Growth Factors. 1993; 9: 243-52
• Display abstract

• Protein tyrosine phosphorylation may be an important indicator of both the proliferative status and differentiation status of cells during embryonic development. To determine how each of these factors contributes to the level of phosphotyrosine-containing proteins detectable in embryonic tissues we have used immunohistochemistry with anti-phosphotyrosine antibodies on sections of developing chicken embryos. In contrast to an earlier study (Takata and Singer, 1988) we found proteins phosphorylated on tyrosine residues to be present in many different cells of the developing chicken embryo. The successful detection of phosphotyrosine-containing proteins in many cell types required the presence of sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, during fixation. Despite the fact that the majority of tyrosine kinases identified to date are growth factor receptors, the highest levels of phosphotyrosine-containing proteins in many tissues were localized to populations of cells which were differentiating or migrating rather than dividing.

• Nishimura R et al.
• Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.
• Mol Cell Biol. 1993; 13: 6889-96
• Display abstract

• Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

• Pawson T
• Cell signalling. Conviction by genetics.
• Nature. 1992; 356: 285-6

• Clark SG, Stern MJ, Horvitz HR
• C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains.
• Nature. 1992; 356: 340-4
• Display abstract

• The induction of the hermaphrodite vulva and the migration of the sex myoblasts in the nematode Caenorhabditis elegans are both controlled by intercellular signalling. The gonadal anchor cell induces formation of the vulva from nearby hypodermal cells, and a set of somatic gonadal cells attract the migrating sex myoblasts to their final positions. Many genes required for vulval induction have been identified, including the let-23 receptor tyrosine kinase gene and the let-60 ras gene. We report here the identification and characterization of a new gene, sem-5 (sem, sex muscle abnormal), that acts both in vulval induction and in sex myoblast migration. On the basis of its DNA sequence, sem-5 encodes a novel 228-amino-acid protein which consists almost entirely of one SH2 (SH, src homology region) and two SH3 domains. SH2 and SH3 domains are present in many signalling proteins regulated by receptor and non-receptor tyrosine kinases. Mutations that impair sem-5 activity alter residues that are highly conserved among different SH2 and SH3 domains. Our results indicate that the sem-5 gene encodes a novel protein that functions in at least two distinct cell-signalling processes.

• Marengere LE, Pawson T
• Identification of residues in GTPase-activating protein Src homology 2 domains that control binding to tyrosine phosphorylated growth factor receptors and p62.
• J Biol Chem. 1992; 267: 22779-86
• Display abstract

• Ras GTPase-activating protein (GAP) contains two Src homology 2 (SH2) domains which are implicated in binding to tyrosine-phosphorylated sites in specific activated growth factor receptors and to a cytoplasmic tyrosine-phosphorylated protein, p62. We have used site-directed mutagenesis of the two GAP SH2 domains (SH2-N and SH2-C) to identify residues involved in receptor and p62 binding. A bacterial fusion protein containing the precise SH2-N domain, as defined by sequence homology, associated with both the activated beta platelet-derived growth factor receptor and epidermal growth factor receptor, and p62 in vitro. However, short deletions at either the N or C termini of the SH2-N domain abolished binding, suggesting that the entire SH2 sequence is required for formation of an active domain. Conservative substitutions of 2 highly conserved basic residues in the SH2-N domain, an arginine and a histidine, resulted in complete loss of receptor and p62 binding, whereas other basic residues, and residues at variable SH2 sites, were more tolerant of substitution. The conserved arginine and histidine therefore appear critical for association with phosphotyrosine-containing proteins, possibly through an interaction with phosphotyrosine. The GAP SH2-C domain, unlike SH2-N, does not bind efficiently to activated receptors or p62 in vitro. The SH2-C domain lacks 3 residues which are otherwise well conserved, and contribute to high affinity SH2-N binding. Replacement of 1 of these residues, a cysteine, with the consensus glycine, conferred SH2-C binding activity toward tyrosine-phosphorylated p62 and epidermal growth factor receptor. Loss-of-function and gain-of-function mutations in the GAP SH2 domains can therefore be used to identify residues that are critical for receptor and p62 binding.

• Margolis B
• Proteins with SH2 domains: transducers in the tyrosine kinase signaling pathway.
• Cell Growth Differ. 1992; 3: 73-80

• Mayer BJ, Jackson PK, Van Etten RA, Baltimore D
• Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.
• Mol Cell Biol. 1992; 12: 609-18
• Display abstract

• We have constructed a series of point mutations in the highly conserved FLVRES motif of the src homology 2 (SH2) domain of the abl tyrosine kinase. Mutant SH2 domains were expressed in bacteria, and their ability to bind to tyrosine-phosphorylated proteins was examined in vitro. Three mutants were greatly reduced in their ability to bind both phosphotyrosine itself and tyrosine-phosphorylated cellular proteins. All of the mutants that retained activity bound to the same set of tyrosine-phosphorylated proteins as did the wild type, suggesting that binding specificity was unaffected. These results implicate the FLVRES motif in direct binding to phosphotyrosine. When the mutant SH2 domains were inserted into an activated abl kinase and expressed in murine fibroblasts, decreased in vitro phosphotyrosine binding correlated with decreased transforming ability. This finding implies that SH2-phosphotyrosine interactions are involved in transmission of positive growth signals by the nonreceptor tyrosine kinases, most likely via the assembly of multiprotein complexes with other tyrosine-phosphorylated proteins.

• Pelicci G et al.
• A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.
• Cell. 1992; 70: 93-104
• Display abstract

• A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.

• Turck CW, Herrmann J, Escobedo JA, Williams LT
• Identification of phosphotyrosine residues during protein sequence analysis.
• Pept Res. 1991; 4: 36-9
• Display abstract

• Synthetic tyrosine-phosphorylated peptides were subjected to protein sequence analysis using a gas-phase sequencer and on-line phenylthiohydantoin (PTH) amino acid analysis. Our data show that phosphotyrosine is stable to the gas-phase sequencing chemistry and can be detected as its PTH-derivative during routine sequence analysis without the need of prior tyrosine radiolabeling.

• Koch CA, Anderson D, Moran MF, Ellis C, Pawson T
• SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.
• Science. 1991; 252: 668-74
• Display abstract

• Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

• Munoz GE, Marshall SH
• Naturally occurring free phosphotyrosine in human liver.
• Cell Mol Biol. 1991; 37: 735-8
• Display abstract

• We report an endogenous tyrosine-driven phosphorylating activity in human liver extracts. The detection is achieved after selective enrichment of soluble components present in a post-mitochondrial supernatant fraction (PMS) while measuring in vitro kinase activity. A putative functional role is inferred from the competence displayed by exogenous free target amino acids when added to the reaction. We demonstrate that exogenous tyrosine is specifically phosphorylated. In view of the close association between protein phosphorylation and cell function, our observations broader the scope of interpretation for the pivotal role phosphoamino acids might have in cell metabolism.

• Ringer DP, Etheredge JL, Dalrymple BL, Niedbalski JS
• Fluorescence of phosphotyrosine--terbium(III) complexes.
• Biochem Biophys Res Commun. 1990; 168: 267-73
• Display abstract

• Phosphotyrosine, a biologically important protein residue, was investigated for the ability to enhance terbium (Tb3+) fluorescence. Spectroscopic analysis of the Tb3+: phosphotyrosine interaction indicated the development of a new excitation peak at 275 nm and strong Tb+ fluorescence enhancement at 488 and 540 nm that was linear over a range from 0.5 to 100 microM amino acid. Subsequent experiments comparing the ability of phosphotyrosine, phosphothreonine, phosphoserine and 20 other common non-phosphorylated amino acids showed that only phosphotyrosine produced significant Tb3+ fluorescence enhancement. Analysis of various phospho-sugars and nucleotides showed (with the expected exception of GMP) that they produced little or no significant fluorescence enhancement, indicating a further selectiveness for the phosphotyrosine: Tb3+ fluorescence enhancement event. These results establish a basis for the future use of Tb3+ fluorescence enhancement as a unique probe for the investigation of phosphotyrosine residues.

• Hille A, Braulke T, von Figura K, Huttner WB
• Occurrence of tyrosine sulfate in proteins--a balance sheet. 1. Secretory and lysosomal proteins.
• Eur J Biochem. 1990; 188: 577-86
• Display abstract

• 1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/or long-term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein-bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.

• Matsuda M, Mayer BJ, Fukui Y, Hanafusa H
• Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins.
• Science. 1990; 248: 1537-9
• Display abstract

• Although the oncogene product of CT10 virus, P47gag-crk, does not itself phosphorylate proteins at tyrosine residues, it elevates phosphotyrosine in transformed cells. The P47gag-crk oncoprotein contains SH2 and SH3 domains, which are conserved in several proteins involved in signal transduction, including nonreceptor tyrosine kinases. P47gag-crk bound in vitro to phosphotyrosine-containing proteins from crk-transformed cells and from cells transformed by oncogenic tyrosine kinases. The association between P47gag-crk and p60v-src, a phosphotyrosine-containing protein, was abolished by dephosphorylation of p60v-src. This suggests that the SH2 and SH3 regions function to regulate protein interactions in a phosphotyrosine-dependent manner.

• Moran MF et al.
• Src homology region 2 domains direct protein-protein interactions in signal transduction.
• Proc Natl Acad Sci U S A. 1990; 87: 8622-6
• Display abstract

• Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.

• Hille A, Huttner WB
• Occurrence of tyrosine sulfate in proteins--a balance sheet. 2. Membrane proteins.
• Eur J Biochem. 1990; 188: 587-96
• Display abstract

• 1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class.

• Jenne D, Hille A, Stanley KK, Huttner WB
• Sulfation of two tyrosine-residues in human complement S-protein (vitronectin).
• Eur J Biochem. 1989; 185: 391-5
• Display abstract

• Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.

• Tillotson ML, Wood JG
• Phosphotyrosine antibodies specifically label ameboid microglia in vitro and ramified microglia in vivo.
• Glia. 1989; 2: 412-9
• Display abstract

• Using an affinity-purified, polyclonal antibody to phosphotyrosine (Wang: Molecular and Cellular Biology 5:3640-3643, 1985) we have previously demonstrated that phosphotyrosine immunoreactivity is restricted to a population of multipolar GFAP-negative neuroglia in adult rat brain (Tillotson and Wood: Journal of Comparative Neurology 282:133-141, 1989) and retina (Tillotson and Wood: Journal of Cell Biology 107:724a, 1988). In this study, we show that the phosphotyrosine-immunoreactive cells are microglia. This conclusion is supported by numerous morphological and ultrastructural similarities between the phosphotyrosine-immunoreactive cells and microglia. Furthermore, phosphotyrosine co-localizes with the microglial-specific B4 isolectin of Bandeiraea simplifolia-1 lectin. Phosphotyrosine antibodies also stain ameboid microglia in primary cultures of neonatal rat brain. In addition, after 7 days in vitro, microglia are the only phosphotyrosine-immunoreactive element in the cultures. This temporal pattern of staining in vitro mimics the developmental progression of phosphotyrosine immunoreactivity in situ, in which a variety of structures stain during postnatal neural development (Tillotson and Wood: Journal of Comparative Neurology 282:133-141, 1989), but only microglia stain in mature brain. The significance of phosphotyrosine-containing proteins potentially expressed in a microglial-specific manner is discussed.

• Wang JY
• Antibodies for phosphotyrosine: analytical and preparative tool for tyrosyl-phosphorylated proteins.
• Anal Biochem. 1988; 172: 1-7

• Martensen TM
• Phosphotyrosine in proteins. Stability and quantification.
• J Biol Chem. 1982; 257: 9648-52
• Display abstract

• The acid and base stability of the phosphoryl bond of phosphotyrosine (Tyr-P) was studied using conditions for rapid and complete hydrolysis of protein peptide bonds. A method was developed for the quantification of Tyr-P in proteins using rapid base hydrolysis and an amino acid analyzer equipped with a fluorometric detection system. The recovery of [32P]Tyr-P from base digests of radiolabeled samples of phosphotyrosyl glutamine synthetase, transforming protein of Rous sarcoma virus, casein, and rabbit anti-sarcoma IgG was 80 +/- 2%. Phosphotyrosine could not be detected in several commercial histone samples, but Tyr-P was detected in phosvitin samples. The putative Tyr-P from the phosvitin hydrolysate was separated from normal amino acids by Dowex 50-H+ chromatography. Treatment of the partially purified Tyr-P with bacterial alkaline phosphatase produced tyrosine in near equivalent quantities to the measured level of Tyr-P. These results show that basic hydrolysis of phosphotyrosyl proteins yields Tyr-P in constant and good yields which can be quantified in amounts greater than or equal to 100 pmol or radiochemically detected in smaller amounts with an amino acid analyzer.

© 2021 EMBL | Privacy Policy