p68 Sam is a substrate of the insulin receptor and associates with the SH2 domains of p85 PI3K.

The 68 kDa Src substrate associated during mitosis is an RNA binding protein with Src homology 2 and 3 domain binding sites. A role for Src associated in mitosis 68 as an adaptor protein in signaling transduction has been proposed in different systems such as T-cell receptors. In the present work, we have sought to assess the possible role of Src associated in mitosis 68 in insulin receptor signaling. We performed in vivo studies in HTC-IR cells and in vitro studies using recombinant Src associated in mitosis 68, purified insulin receptor and fusion proteins containing either the N-terminal or the C-terminal Src homology 2 domain of p85 phosphatidylinositol-3-kinase. We have found that Src associated in mitosis 68 is a substrate of the insulin receptor both in vivo and in vitro. Moreover, tyrosine-phosphorylated Src associated in mitosis 68 was found to associate with p85 phosphatidylinositol-3-kinase in response to insulin, as assessed by co-immunoprecipitation studies. Therefore, Src associated in mitosis 68 may be part of the signaling complexes of insulin receptor along with p85. In vitro studies demonstrate that Src associated in mitosis 68 associates with the Src homology 2 domains of p85 after tyrosine phosphorylation by the activated insulin receptor. Moreover, tyr-phosphorylated Src associated in mitosis 68 binds with a higher affinity to the N-terminal Src homology 2 domain of p85 compared to the C-terminal Src homology 2 domain of p85, suggesting a preferential association of Src associated in mitosis 68 with the N-terminal Src homology 2 domain of p85. This association may be important for the link of the signaling with RNA metabolism.     

Sam68 associates with the SH3 domains of Grb2 recruiting GAP to the Grb2-SOS complex in insulin receptor signaling

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) that translocates from the nucleus to the cytoplasm and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K and GAP, in vivo and in vitro. In the present work, we have further demonstrated the cytoplasmic localization of Sam68, which is increased in cells overexpressing IR. Besides, we sought to further study the association of Sam68 with the Ras-GAP pathway by assessing the interactions with SH3 domains of Grb2. We employed GST-fusion proteins containing the SH3 domains of Grb2 (N or C), and recombinant Sam68 for in vitro studies. In vivo studies of protein-protein interaction were assessed by co-immunoprecipitation experiments with specific antibodies against Sam68, GAP, Grb2, SOS, and phosphotyrosine; and by affinity precipitation with the fusion proteins (SH3-Grb2). Insulin stimulation of HTC-IR cells promotes phosphorylation of Sam68 and its association with the SH2 domains of GAP. Sam68 is constitutively associated with the SH3 domains of Grb2 and it does not change upon insulin stimulation, but Sam68 is Tyr-phosphorylated and promotes the association of GAP with the Grb2-SOS complex. In vitro studies with fusion proteins showed that Sam68 association with Grb2 is preferentially mediated by the C-terminal SH3 domains of Grb2. In conclusion, Sam68 is a substrate of the IR and may have a role as a docking protein in IR signaling, recruiting GAP to the Grb2-SOS complex, and in this way it may modulate Ras activity.     

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The association of Sam68 with Vav1 contributes to tumorigenesis

Vav1 functions in the hematopoietic system as a specific GDP/GTP nucleotide exchange factor regulated by tyrosine phosphorylation. An intact C-terminal SH3 domain of Vav1 (Vav1SH3C) was shown to be necessary for Vav1-induced transformation, yet the associating protein(s) necessary for this activity have not yet been identified. Using a proteomics approach, we identified Sam68 as a Vav1SH3C-associating protein. Sam68 (Src-associated in mitosis of 68 kD) belongs to the heteronuclear ribonucleoprotein particle K (hnRNP-K) homology (KH) domain family of RNA-binding proteins. The Vav1/Sam68 interaction was observed in vitro and in vivo. Mutants of Vav1SH3C previously shown to lose their transforming potential did not associate with Sam68. Co-expression of Vav1 and Sam68 in Jurkat T cells led to increased localization of Vav1 in the nucleus and changes in cell morphology. We then tested the contribution of Sam68 to known functions of Vav1, such as focus-forming in NIH3T3 fibroblasts and NFAT stimulation in T cells. Co-expression of oncogenic Vav1 with Sam68 in NIH3T3 fibroblasts resulted in a dose-dependent increase in foci, yet no further enhancement of NFAT activity was observed in Jurkat T cells, as compared to cells overexpressing only Vav1 or Sam68. Our results strongly suggest that Sam68 contributes to transformation by oncogenic Vav1.     

A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.     

Comprehensive analysis of interactions between the Src-associated protein in mitosis of 68 kDa and the human Src-homology 3 proteome

The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6) as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85) and seven already known high-confidence Sam68-ligands (mainly from the Src-kinase family), as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down) and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins). Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains any more, as subsequently demonstrated by FRET-analyses. In conclusion, we present a thorough characterization of Sam68's interplay with the SH3 proteome. The observed interaction between Sam68 and OSF complements the known Sam68-Src and OSF-Src interactions. Thus, we propose, that Sam68 functions as a classical scaffold protein in this context, assembling components of an osteoclast-specific signalling pathway.     

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.     

Functional cooperation between c-Cbl and Src-like adaptor protein 2 in the negative regulation of T-cell receptor signaling

Adaptor proteins assemble multiprotein signaling complexes, enabling the transduction of intracellular signals. While many adaptor proteins positively regulate signaling in this manner, a subgroup of adaptors function as negative regulators. Here we report the identification of a hematopoiesis-specific adaptor protein that we have designated Src-like adaptor protein 2 (SLAP-2). SLAP-2 is most closely related to SLAP and contains a Src homology 3 (SH3) domain and an SH2 domain, as well as an amino-terminal myristoylation site that mediates SLAP-2 association with membranes. Following stimulation of primary thymocytes with anti-CD3 and anti-CD28, SLAP-2 coimmunoprecipitates with tyrosine-phosphorylated c-Cbl and an unidentified protein of approximately 72 kDa. In activated Jurkat T cells, SLAP-2 also binds an additional 70-kDa phosphoprotein, identified as ZAP-70. Binding of SLAP-2 to both p72 and ZAP-70 is dependent on its SH2 domain, while c-Cbl interacts with the carboxy-terminal region. Overexpression of wild-type SLAP-2 alone or in combination with c-Cbl in Jurkat T cells leads to inhibition of T-cell antigen receptor-induced activation of nuclear factor of activated T cells. The inhibitory effect of SLAP-2 requires the carboxy-terminal c-Cbl binding region. Expression of SLAP-2 with SYK or ZAP-70 in COS cells or Jurkat T cells causes the degradation of these kinases, and SLAP-2 overexpression in Jurkat T cells reduces the surface expression of CD3. These results suggest that the mechanism of action of SLAP-2 and the related protein SLAP is to promote c-Cbl-dependent degradation of the tyrosine kinases SYK and ZAP-70 and down-regulation of CD3 at the cell surface.     

Identification of an amino-terminal substrate-binding domain in the Yersinia tyrosine phosphatase that is required for efficient recognition of focal adhesion targets

YopH is a protein tyrosine phosphatase (PTP) that is delivered into host mammalian cells via a type III secretion pathway in pathogenic Yersinia species. Although YopH is a highly active PTP, it preferentially targets a subset of tyrosine-phosphorylated proteins in host cells, including p130^Cas. Previous in vitro studies have indicated that the carboxy-terminal PTP domain contributes specificity to the interaction of YopH with substrates. However, it is not known if the PTP domain is sufficient for substrate recognition by YopH. Here, we have identified paxillin as an additional substrate of YopH in HeLa cells. In addition, we have identified a domain in the amino-terminal region of YopH that binds to both p130^Cas and paxillin and is required for the efficient recognition of substrates by the wild-type enzyme. This 'substrate-binding' domain exhibits a ligand specificity that is similar to that of the Crk Src homology 2 (SH2) domain, and it binds substrates directly in a phosphotyrosine-dependent manner. The substrate-binding domain of YopH may represent a novel type of protein–protein interaction module, as it lacks significant sequence similarity with any known SH2 or phosphotyrosine-binding (PTB) domain.     

Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells.

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.     

Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.     

Crystal structures of the armadillo repeat domain of adenomatous polyposis coli and its complex with the tyrosine-rich domain of Sam68

Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68?kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm and?Sam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.     

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.     

A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.     

cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor.

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.     

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.     

Multiple SH2-mediated interactions in v-src-transformed cells.

The Src homology 2 (SH2) domain is a noncatalytic region which is conserved among a number of signaling and transforming proteins, including cytoplasmic protein-tyrosine kinases and Ras GTPase-activating protein (GAP). Genetic and biochemical data indicate that the SH2 domain of the p60v-src (v-Src) protein-tyrosine kinase is required for full v-src transforming activity and may direct the association of v-Src with specific tyrosine-phosphorylated proteins. To test the ability of the v-Src SH2 domain to mediate protein-protein interactions, v-Src polypeptides were expressed as fusion proteins in Escherichia coli. The bacterial v-Src SH2 domain bound a series of tyrosine-phosphorylated proteins in a lysate of v-src-transformed Rat-2 cells, including prominent species of 130 and 62 kDa (p130 and p62). The p130 and p62 tyrosine-phosphorylated proteins that complexed v-Src SH2 in vitro also associated with v-Src in v-src-transformed Rat-2 cells; this in vivo binding was dependent on the v-Src SH2 domain. In addition to binding soluble p62 and p130, the SH2 domains of v-Src, GAP, and v-Crk directly recognized these phosphotyrosine-containing proteins which had been previously denatured and immobilized on a filter. In addition, the SH2 domains of GAP and v-Crk bound to the GAP-associated protein p190 immobilized on a nitrocellulose membrane. These results show that SH2 domains bind directly to tyrosine-phosphorylated proteins and that the Src SH2 domain can bind phosphorylated targets of the v-Src kinase domain.(ABSTRACT TRUNCATED AT 250 WORDS)