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)     

Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.     

Activation of c-Src in cells bearing v-Crk and its suppression by Csk.

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.     

A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner.

p47v-crk (v-Crk), a transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125-135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v-src (v-Src), as well as by v-Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2-containing molecules.     

Physiological Requirement for Both SH2 Domains for Phospholipase C-γ1 Function and Interaction with Platelet-Derived Growth Factor Receptors

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.     

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.     

SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.

src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-gamma and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosine-containing regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BIAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 x 10(7) to 40 x 10(7)/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.     

The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner.

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation.     

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of individual SH2 domains in mediating association of phospholipase C-gamma1 with the activated EGF receptor.

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 > pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 > pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.     

The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors.

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.     

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.     

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.     

The role of the Src homology domains in morphological transformation by v-src.

The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.     

Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor.

src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.     

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.     

Tyrosine phosphorylation enhances the SH2 domain-binding activity of Bcr and inhibits Bcr interaction with 14-3-3 proteins.

The cellular Bcr protein consists of an N-terminal serine/threonine kinase domain, a central guanine nucleotide exchange factor homology region and a C-terminal GTPase-activating protein domain. Previous work in our laboratory established that Bcr is a major transformation-related substrate for the v-Fps tyrosine kinase, and tyrosine phosphorylation of Bcr induces Bcr-Grb-2/SOS association in vivo through the Src homology 2 (SH2) domain of Grb-2. In the present study, we mapped the region of Bcr tyrosine phosphorylation by c-Fes, the human homologue of v-Fps, to Bcr N-terminal amino acids 162-413 by using a baculovirus/Sf-9 cell co-expression system. Tyrosine phosphorylation of Bcr by Fes greatly enhanced the binding of Bcr to the SH2 domains of multiple signalling molecules in vitro, including Grb-2, Ras GTPase activating protein, phospholipase C-gamma, the 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, and the Abl tyrosine kinase. In contrast with SH2 binding, tyrosine phosphorylation of Bcr reduced its ability to associate with the 14-3-3 protein Bap-1 (Bcr-associated protein-1), a Bcr substrate and member of a family of phosphoserine-binding adaptor proteins. These experiments provide in vitro evidence that tyrosine phosphorylation may modulate the interaction of Bcr with multiple growth-regulatory signalling pathways.     

Solution structure of the SH2 domain of Grb2 complexed with the Shc-derived phosphotyrosine-containing peptide.

The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.     

Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.     

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.