The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.

A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.     

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.     

Src homology region 2 domains direct protein-protein interactions in signal transduction.

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.     

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.     

Direct analysis of the binding of Src-homology 2 domains of phospholipase C to the activated epidermal growth factor receptor.

A number of proteins involved in intracellular signaling contain regions of homology to the product of the src oncogene that are termed Src-homology (SH) 2 domains. SH2 domains are believed to mediate the association of these proteins with various tyrosine-phosphorylated receptors in a growth factor-dependent manner. We have examined the kinetic characteristics of one of these interactions, the binding of the SH2 domains of phospholipase C gamma 1 with the receptor for epidermal growth factor (EGF). Bacterial fusion proteins were prepared containing the two SH2 domains of PLC gamma 1 and labeled metabolically with [35S]methionine/cysteine. A fusion protein containing both SH2 domains bound to the purified EGF receptor from EGF-treated cells, whereas no binding to receptors from control cells was detected. Binding was rapid, reaching apparent equilibrium by 10 min. Dissociation of the complex occurred only in the presence of excess unlabeled SH2 protein and exhibited two kinetic components. Similarly, analysis of apparent equilibrium binding revealed a nonlinear Scatchard plot, further indicating complex binding kinetics that may reflect cooperative behavior. The binding of the fusion protein containing both SH2 domains was inhibited by a fusion protein containing only the amino-terminal SH2 domain, although at concentrations an order of magnitude higher than that observed with the complete fusion protein. Fusion proteins containing SH2 domains from the GTPase-activating protein, the p85 regulatory subunit of phosphatidylinositol 3'-kinase, or the Abl oncoprotein competed less effectively. Binding of the PLC gamma 1 SH2 fusion protein to a mutant EGF receptor lacking the two carboxyl-terminal tyrosine phosphorylation sites exhibited a significantly lower affinity than that observed with the wild type, suggesting that this region of the receptor may play an important role. This binding assay represents a means with which to evaluate the pleiotropic nature of growth factor action.     

Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth.     

Structure of a regulatory complex involving the Abl SH3 domain,the Crk SH2 domain,and a Crk-derived phosphopeptide

On phosphorylation of Y221 by Abelson (Abl) kinase, the Crk-II adapter protein undergoes an intramolecular reorganization initiated by the binding of its own Src homology 2 (SH2) domain to the pY221 site. Conformational changes induced by phosphotyrosine recognition promote the binding of the Src homology 3 (SH3) domain of the Abl tyrosine kinase to a proline-rich loop located between the betaD and betaE strands of the SH2 domain (DE loop). We have determined the NMR solution structure of the ternary complex of the Abl SH3 domain with the Crk SH2 domain bound to a Crk pY221 phosphopeptide. The SH2 domain bridges two ligands that bind at distinct sites. The interaction between the Abl SH3 domain and the Crk SH2 domain is localized to a canonical eight-residue site within the DE loop. From (15)N relaxation experiments, the DE loop of the SH2 domain in the complex displays a significant degree of conformational freedom. The structural and dynamic data therefore indicate that these SH2 and SH3 domains do not assume a unique orientation with respect to one another; rather, they appear to be only tethered via the DE loop. Thus, SH2 domain-SH3 domain interactions do not require additional tertiary contacts or restriction of domain orientation when a recognition motif is presented in a mobile loop. This complex between the Abl SH3 domain, Crk SH2 domain, and Crk phosphopeptide is an example of the extremely modular nature of regulatory proteins that provides a rich repertoire of mechanisms for control of biological function.     

v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation

v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF.     

The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity.

Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.     

Cloning of ASH, a ubiquitous protein composed of one Src homology region (SH) 2 and two SH3 domains, from human and rat cDNA libraries.

The protein ASH (for abundant Src homology), composed of one Src homology region (SH) 2 and two SH3 domains, was cloned by screening human and rat cDNA libraries with an oligonucleotide probe directed to a consensus sequence of the SH2 domains. The rat-derived ASH peptide was comprised of 217 amino acids with a molecular mass of 25-28 kDa and was found to be ubiquitous in rat tissues. A human cDNA clone was also found to code for part of the same protein, suggesting that ASH is common to human and rat. The amino acid sequence of ASH was strikingly similar to Sem-5, the product of a nematode cell-signaling gene, and ASH is most probably a mammalian homologue of Sem-5. ASH bound in vitro to phosphotyrosine-containing proteins, including activated epidermal growth factor receptor, the ASH SH2 domain being responsible for the binding. Induced expression of an antisense ASH cDNA led to a reduction in cell growth. Considering these observations and the structural homology to Sem-5, ASH is likely to function as a ubiquitous signal transducer, possibly resembling Sem-5, which communicates between a receptor protein tyrosine kinase and a Ras protein.     

Felic (CIP4b), a novel binding partner with the Src kinase Lyn and Cdc42, localizes to the phagocytic cup

Through its Src homology 3 (SH3) and SH2 domains, the Src kinase Lyn interacts with a small number of phosphoproteins, such as Shc, Cbl, and Vav, which regulate cell cycle and the cytoskeleton. Using Lyn's Unique, SH3, and SH2 domains as bait in a yeast 2-hybrid screen, we isolated a novel gene product with features of a scaffolding protein. We named it Felic because it contains a domain homologous to the tyrosine kinase Fes and the cytoskeletal protein ezrin and forms a Lyn interaction with the GTPase Cdc42 (Felic). Felic was expressed in both hematopoietic and nonhematopoietic tissues. Because it represents an alternative splice product related to the Cdc42-interacting protein 4, CIP4, we also refer to Felic as CIP4b. Felic contains an SH3 recognition site RXPXXP and multiple tyrosine residues. In insulin or serum-stimulated HEK293 cells, Felic became tyrosine phosphorylated. Like CIP4, Felic associated with Cdc42 in its activated form only. Unlike CIP4, Felic does not possess a C-terminal SH3 domain. Coprecipitation studies show that Felic bound to Lyn or activated forms of Cdc42. Overexpression of Felic or CIP4 inhibited NIH 3T3 cell invasiveness in a Matrigel assay. Because Lyn and Cdc42 are involved in phagocytosis, we examined the distribution of Felic in RAW macrophages during particle ingestion. Felic was recruited more efficiently than CIP4 to the phagocytic cups. Altogether, these data suggest that CIP4/Felic constitute a novel family of cytoskeletal scaffolding proteins, integrating Src and Cdc42 pathways. The absence of an SH3 domain in Felic provides a structural basis for functional differences.     

High-efficiency expression/cloning of epidermal growth factor-receptor-binding proteins with Src homology 2 domains.

Src homology 2 domains bind to tyrosine-phosphorylated growth factor receptors and are found in proteins that serve as substrates for tyrosine kinases, such as phospholipase C-gamma 1 and ras GTPase-activating protein. We have previously described the cloning of phosphatidylinositol 3'-kinase-associated p85 from expression libraries with the tyrosine-phosphorylated epidermal growth factor receptor as a probe. We have now modified this technique by using T7 polymerase-based expression libraries, which significantly improves sensitivity of the method. In one screening of such a library, we identified five different murine Src homology 2 domain-containing proteins, which we call GRBs (growth factor receptor-bound proteins). Two of these proteins represented the tyrosine kinase fyn and the mouse homologue of phospholipase C-gamma 1, whereas two genes encoded proteins similar to v-crk and NCK. We also isolated the gene for GRB-7, which encodes a protein of 535 amino acids. In addition to a Src homology 2 domain, GRB-7 also has a region of similarity to the noncatalytic domain of ras GTPase-activating protein and is highly expressed in liver and kidney. Use of this expression/cloning system should increase our ability to identify downstream modulators of growth factor action.     

Involvement of pp60c-src with two major signaling pathways in human breast cancer.

The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.     

Identification of the SH2 domain binding protein of Bruton's tyrosine kinase as BLNK--functional significance of Btk-SH2 domain in B-cell antigen receptor-coupled calcium signaling.

Bruton's tyrosine kinase (Btk) is a critical component in the B-cell antigen receptor (BCR)-coupled signaling pathway. Its deficiency in B cells leads to loss or marked reduction in the BCR-induced calcium signaling. It is known that this BCR-induced calcium signaling depends on the activation of phospholipase Cgamma (PLCgamma), which is mediated by Btk and another tyrosine kinase Syk and that the SH2 and pleckstrin homology (PH) domains of Btk play important roles in this activation process. Although the importance of the PH domain of Btk has been explained by its role in the membrane targeting of Btk, the functional significance of the SH2 domain in the calcium signaling has remained merely a matter of speculation. In this report, we identify that one of the major Btk-SH2 domain-binding proteins in B cells is BLNK (B-cell linker protein) and present evidences that the interaction of BLNK and the SH2 domain of Btk contributes to the complete tyrosine phosphorylation of PLCgamma.     

Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region.

A previously undescribed 62-kDa protein (p62) that does not contain phosphotyrosine but, nevertheless, binds specifically to the isolated src homology 2 (SH2) domain of p56lck has been identified. The additional presence of the unique N-terminal region of p56lck prevents p62 binding to the SH2 domain. However, phosphorylation at Ser-59 (or alternatively, its mutation to Glu) reverses the inhibition and allows interaction of the p56lck SH2 domain with p62. Moreover, p62 is associated with a serine/threonine kinase activity and also binds to ras GTPase-activating protein, a negative regulator of the ras signaling pathway. Thus, phosphotyrosine-independent binding of p62 to the p56lck SH2 domain appears to provide an alternative pathway for p56lck signaling that is regulated by Ser-59 phosphorylation.     

Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation

STE20-related kinases play significant regulatory roles in a range of cellular responses to environmental stimuli. GCKR (also referred to as KHS1) is a serine/threonine protein kinase that has an STE20-like protein kinase domain and that stimulates the stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. GCKR has a large C-terminal regulatory domain that provides sites for interactions with other proteins. Adaptor proteins mediate the interactions between signaling molecules. In this study we showed that the adaptor proteins Crk and CrkL associated with GCKR. When Crk-I, Crk-II, or CrkL was transiently expressed in HEK 293T cells along with GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr-Abl transformed cell line, K562 endogenous GCKR and CrkL coimmunoprecipitated, indicating a constitutive association. Detection of the CrkL-GCKR interaction required the SH3 domains of CrkL and 2 regions in GCKR-1 between amino acids 387 and 395 that contains a consensus SH3 binding motif and the other between amino acids 599 and 696. Crk or CrkL overexpression increased GCKR catalytic activity. A dominant negative form of Ras abolished Crk- or CrkL-induced GCKR activation, suggesting a dependence on Ras activation for their activation of GCKR. Finally, we showed impairment of the known ability of CrkL to activate the SAPK pathway by a catalytically inactive form of GCKR or by a GCKR antisense construct. Thus, GCKR associates with other proteins through interactions mediated by SH2/SH3 adaptor proteins, which can lead to GCKR and SAPK activation.     

Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells

Stem cell factor (SCF) plays critical roles in proliferation, survival, migration, and function of hematopoietic progenitor and mast cells through binding to Kit receptor. Previous studies have implicated the adaptor protein Lnk as an important negative regulator of SCF signaling. However, the molecular mechanism underlying this regulation is unclear. Here, we showed that the Src homology 2 domain (SH2) of Lnk binds directly and preferentially to phosphorylated tyrosine 567 in Kit juxtamembrane domain. Using Lnk(-/-) bone marrow mast cells (BMMCs) transduced with different Lnk proteins, we demonstrated that Lnk down-regulates SCF-induced proliferation with attenuation of mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase signaling. Furthermore, we showed that Lnk(-/-) BMMCs displayed increased SCF-dependent migration compared with wild-type cells, revealing a novel Lnk-mediated inhibitory function. This correlated with enhanced Rac and p38 MAPK activation. Finally, we found that Lnk domains and carboxy-terminal tyrosine contribute differently to inhibition of in vitro expansion of hematopoietic progenitors. Altogether, our results demonstrate that Lnk, through its binding to Kit tyrosine 567, negatively modulates specific SCF-dependent signaling pathways involved in the proliferation and migration of primary hematopoietic cells.     

Src homology 2 domain as a specificity determinant in the c-Abl-mediated tyrosine phosphorylation of the RNA polymerase II carboxyl-terminal repeated domain.

The Src-homology (SH) 2 domain, found in a variety of proteins, has a binding site for phosphotyrosine-containing peptides. In adaptor proteins such as Grb2, the SH2 domain plays an important role in the assembly of signal transducer complexes. Many nonreceptor tyrosine kinases--e.g., Abl and Src--also contain SH2 domains. Without a functional SH2 domain, these tyrosine kinases retain catalytic activity but lose their biological function. This result suggests that the SH2 domain may be involved in the selection of biologically relevant substrates. We have previously shown that the carboxyl-terminal repeated domain (CTD) of the mammalian RNA polymerase II is a substrate for the Abl but not the Src tyrosine kinase. This specificity is conferred in part by the SH2 domain. The Abl SH2 domain binds the tyrosine-phosphorylated [Tyr(P)] CTD and is required for the processive and stoichiometric phosphorylation of the 52 tyrosines in the CTD. Mutation of the Abl SH2 or exchanging it with that of Src, which does not bind the Tyr(P)-CTD, abolished processivity and reduced the CTD kinase activity without any effect on autophosphorylation or the phosphorylation of nonspecific substrates. These results demonstrate that the SH2 domain of the Abl tyrosine kinase plays an active role in catalysis and suggests that SH2 domain and the tyrosine kinase domain may act in concert to confer substrate specificity.     

Disease specificity of kinase domains: the src-encoded catalytic domain converts erbB into a sarcoma oncogene.

src and erbB are two tyrosine kinase-encoding oncogenes carried by retroviruses, which have distinct disease specificities. The former induces predominantly sarcomas, and the latter, leukemias. Src and ErbB have similar catalytic domains but have very different regulatory domains. A wealth of information exists concerning how different regulatory domains [Src homology 2 (SH2) and SH3 domains and autophosphorylation sites] control substrate and disease specificities. Whether the catalytic domain helps determine these specificities remains to be explored. Here we show that the Src catalytic domain is enzymatically active when substituted into the ErbB backbone and interacts with the ErbB regulatory domain. This ErbB/Src chimera displays autophosphorylation and substrate phosphorylation patterns different from those of both Src and ErbB. Neither SH2 and SH3 nor autophosphorylation sites are required for the Src catalytic domain to exert its fibroblast transforming ability. Most significantly, the catalytic domain can convert erbB from a leukemogenic oncogene into a sarcomagenic oncogene, suggesting that the leukemogenic determinants in part reside within the ErbB catalytic domain.     

Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.