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)     

Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is a transmembrane protein with a short extracellular domain (123 amino acids) and two cytoplasmically localized protein-tyrosine phosphatase (PTP) domains. Here we report that RPTP alpha is constitutively phosphorylated on tyrosine in NIH 3T3 mouse fibroblasts. The in vivo tyrosine phosphorylation site was localized to the C-terminus of RPTP alpha by phosphopeptide mapping experiments using in vivo and in vitro 32P-labeled RPTP alpha. The identity of this site as Tyr789, located five residues from the C-terminus, was confirmed by site-directed mutagenesis. Transient overexpression of c-Src together with RPTP alpha in human embryonic kidney 293 cells increased phosphorylation of Tyr789, suggesting that c-Src may phosphorylate RPTP alpha in vivo. RPTP alpha had autodephosphorylation activity in vitro. When expressed in 293 cells the level of Tyr789 phosphorylation was higher in a non-functional mutant of RPTP alpha than in wild type RPTP alpha, indicating that RPTP alpha may have autodephosphorylation activity in vivo as well. The sequence on the C-terminal side of Tyr789 (YANF) fits the consensus binding site for the SH3-SH2-SH3 adaptor protein GRB2 (YXNX). We show that RPTP alpha, but not a mutant of RPTP alpha with a Tyr-->Phe mutation at position 789, bound to GRB2 in vitro. In addition, RPTP alpha co-immunoprecipitated with GRB2 from NIH 3T3 cells, demonstrating that GRB2 bound to RPTP alpha in vivo. The guanine nucleotide releasing factor for the Ras GTPase, Son of sevenless (Sos), which associates with GRB2 via its SH3 domains, was not detected in RPTP alpha immunoprecipitates. Our results suggest a role for RPTP alpha in attenuation of GRB2-mediated signaling.     

Involvement of SH2-SH2-SH3 domain of phospholipase cgamma1 in NF-kappaB signaling

To directly define the role of phospholipase Cgamma1 (PLCgamma1) in NF-kappaB activation, NF-kappaB promoted luciferase reporter gene plasmid (pNF-kappaB-Luc) was transfected into rat-3Y1 fibroblasts that overexpress whole PLCgamma1 (PLCgamma1-3Y1), src homology domains SH2-SH2-SH3 of PLCgamma1 (SH223-3Y1) and v-src (Src-3Y1). Transient transfection with pNF-kappaB-Luc remarkably increased the luciferase activity in all three transformants compared with normal rat-3Y1 cells. Pretreatment with inhibitors of protein tyrosine kinase reduced this increase in luciferase activity, but U73122 (a PLC inhibitor) did not. While PD98059, an inhibitor of mitogen activated protein kinase (MAPK), significantly reduced the luciferase activity, there was no effect by wortmannin and Ro-31-8220, inhibitors of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC), respectively. This study shows a direct evidence that the SH2-SH2-SH3 region of PLCgamma1 contributes to the NF-kappaB signaling and that MAPK, but not PI3K and PKC, is involved in SH2-SH2-SH3 mediated NF-kappaB activation in these cells.     

Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains

The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions.     

Signalling through SH2 and SH3 domains

In 1986, Pawson's group recognized a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed this the Src homology 2, or SH2, domain. In the ensuing years, SH2 domains have been found in an impressive variety of proteins, as has a second region of homology, inevitably termed SH3. These domains appear to mediate controlled protein-protein interactions. Many proteins that contain SH2 and SH3 domains are involved in signal transduction, suggesting a new paradigm for regulation of intracellular signalling pathways.     

An alliance between Ras GTPase-activating protein, filamin C, and Ras GTPase-activating protein SH3 domain-binding protein regulates myocyte growth

We have previously reported that Ras GTPase-activating protein (RasGAP) is involved in a pathway that regulates total cellular mRNA and protein synthesis in cardiac myocytes. A yeast two-hybrid screen resulted in identification of filamin C (FLN-C) as one of its targets. Knockdown of RasGAP or FLN-C, or severing their interaction, resulted in down-regulation of the RNA polymerase II kinase, cyclin-dependent kinase 7 (Cdk7). This appeared to be provoked by the release of cdk7 mRNA from RasGAP SH3 domain-binding protein, G3BP, and its subsequent degradation. In parallel, myocyte growth was also inhibited. On the other hand, overexpression of RasGAP induced a Cdk7- and FLN-C-dependent growth. Thus, we propose that the physical interaction between RasGAP and FLN-C facilitates an interaction between G3BP and cdk7 mRNA. This results in stabilization of cdk7 mRNA, an increase in its protein, which is required for cell growth.     

Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.     

The SH2-SH2-SH3 domain of phospholipase C-gamma1 directly binds to translational elongation factor-1alpha

Phospholipase C-gamma1 (PLC-gamma1) is a lipase that hydrolyzes PIP2 to generate two second messengers, IP3 and DAG. By using the yeast two-hybrid system, we identified the translational elongation factor-1alpha (EF-1alpha) as a binding protein of PLC-gamma1 from the human B-lymphocyte library. Direct interaction between EF-1alpha and PLC-gamma1 was confirmed by the in vitro binding experiment using purified PLC-gamma1. Furthermore, from the in vitro binding experiment, we could demonstrate that the carboxyl terminal region of EF-1alpha is involved in the interaction with PLC-gamma1, and that both SH2 and SH3 domains of PLC-gamma1 are required for the interaction with EF-1alpha. In vivo interaction between EF-1alpha and PLC-gamma1 was confirmed by the immunoprecipitation experiment using anti-EF-1alpha antibody. The interaction between EF-1alpha and PLC-gamma1 was enhanced by EGF-treatment. Taken together, we suggest that EF-1alpha might play a role in PLC-gamma1-mediated signal transduction.     

Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies

Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleckstrin homology (PH) domain followed by Tec homology (TH) domain, Src homology 3 and 2 (SH3 and SH2) domains, and a C-terminal kinase domain. Mutations in the human BTK gene cause the severe immunodeficiency disease X-linked agammaglobulinemia (XLA). The structural and functional basis of several XLA-causing mutations remains unknown, since only the structures of the PH and SH3 domains of human Btk are currently available. In this study, we overexpressed and purified a protein consisting of the SH3 and SH2 domains of human Btk for biochemical and structural analysis. The purified protein was only partially soluble and had a tendency to dimerize, which made it unsuitable for further studies. To overcome the problems of low solubility and dimerization, subdomain interactions were engineered without altering the function of the protein.     

The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains.

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.     

SH3-SH2 domain orientation in Src kinases: NMR studies of Fyn

The regulatory domains of Src family kinases SH3 and SH2 suppress Src activity when bound to the catalytic domain. Here, the isolated SH3-SH2 fragment from the Src family member Fyn (FynSH32) is studied by NMR. The properties of this fragment are expected to be similar to the domains in the active state, where they are dissociated from the catalytic domain. Crosscommunication between SH3 and SH2 of FynSH32, measured by chemical shift perturbation, was found to be small. Diffusion and alignment anisotropy measurements showed that SH3 and SH2 of peptide-bound FynSH32 are significantly coupled but still exhibit some interdomain flexibility. The observed average domain orientation indicates that a large SH3-SH2 domain closure is required to reach the inactive state. The implications of these results for Src regulation are discussed.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

Crystal structure of the C-terminal SH3 domain of the adaptor protein GADS in complex with SLP-76 motif peptide reveals a unique SH3-SH3 interaction

The Grb2-like adaptor protein GADS is essential for tyrosine kinase-dependent signaling in T lymphocytes. Following T cell receptor ligation, GADS interacts through its C-terminal SH3 domain with the adaptors SLP-76 and LAT, to form a multiprotein signaling complex that is crucial for T cell activation. To understand the structural basis for the selective recognition of GADS by SLP-76, herein is reported the crystal structure at 1.54 Angstrom of the C-terminal SH3 domain of GADS bound to the SLP-76 motif 233-PSIDRSTKP-241, which represents the minimal binding site. In addition to the unique structural features adopted by the bound SLP-76 peptide, the complex structure reveals a unique SH3-SH3 interaction. This homophilic interaction, which is observed in presence of the SLP-76 peptide and is present in solution, extends our understanding of the molecular mechanisms that could be employed by modular proteins to increase their signaling transduction specificity.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

Both the SH2 and SH3 domains of human CRK protein are required for neuronal differentiation of PC12 cells.

Human CRK protein is a homolog of the chicken v-crk oncogene product and consists mostly of src homology region 2 (SH2) and SH3, which are shared by many proteins, in particular those involved in signal transduction. SH2 has been shown to bind specifically to phosphotyrosine-containing peptides. We report here that both SH2 and SH3 are required for signaling from CRK protein. Microinjection of the CRK protein induced neurite formation of rat pheochromocytoma cell line PC12. This activity was abolished by mutation of the CRK protein in either SH2 or SH3. The neuronal differentiation induced by the CRK protein was blocked by an excess amount of peptides containing CRK SH3. Moreover, we identified three proteins, of 118, 125, and 136 kDa, which bound specifically to CRK SH3. The CRK-induced neuronal differentiation was also suppressed by monoclonal antibodies against either CRK SH2 or p21ras. These results suggest that both SH2 and SH3 of the CRK protein mediate specific protein-protein binding and that the resulting multimolecular complex generates a signal for neurite differentiation through activation of p21ras.