Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.     

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.     

Computational and functional analysis of the putative SH2 domain in Janus Kinases

Src homology 2 (SH2) domains interact in a highly specific manner with phosphorylated tyrosine residues on other signaling molecules. Protein tyrosine kinases (PTK) frequently contain SH2 domains, which often control signaling specificity. The Janus Kinases (JAKs) are a family of PTKs involved in signal transduction pathways mediated by various cytokines. Initial characterization of JAKs showed no identifiable SH2 domain. However, we have found substantial evidence supporting the existence of an SH2 domain in JAKs through the use of various web-based computational analysis programs. Predictive secondary and tertiary structures recognize an SH2 domain in JAKs. In addition, a three-dimensional homology model was constructed using the SH2 domains of Src tyrosine kinase and Syp tyrosine phosphatase as templates. These results, in conjunction with preliminary binding studies showing interactions with tyrosine phosphorylated proteins in activated splenocytes, suggest a functional role for this domain in JAKs.     

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.     

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.     

The v-Src SH3 domain facilitates a cell adhesion-independent association with focal adhesion kinase

Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activation and the formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signaling complex occurs. Co-expression studies in human 293T cells showed that v-Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S-transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) by in vitro pull-down assays, and these sites are composed of a PXXPXXPhi (where Phi is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular proteins to a glutathione S-transferase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98) could associate in vivo with Phe-397 FAK in an SH2-independent manner. These studies establish a functional role for the v-Src SH3 domain in stabilizing an adhesion-independent signaling complex with FAK.     

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.     

Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases

When cells are treated with Ca(2+) and Ca(2+)-ionophore, c-Src kinase activity increases, whereas c-Yes kinase activity decreases. This opposite modulation can be reproduced in an in vitro reconstitution assay and is dependent on Ca(2+) and on soluble factors present in cell lysates. Since c-Src and c-Yes share a high degree of homology, with the exception of their N-terminal "unique" domains, their activity was thought to be coordinately regulated. To assess the mechanism of regulation we generated stable cell lines expressing eight different constructs containing wild type c-Src and c-Yes, as well as swaps of the unique domain alone, unique and Src homology 3 (SH3) domains together and the SH3 domain alone. Swapping of the unique domains was not sufficient to reverse the regulation of the chimeric molecules. On the other hand, chimeras containing swaps of the unique plus the SH3 domains displayed reverse regulation, implicating both domains in the regulation of kinase activity by Ca(2+). To rule out the participation of the unique domain, we used chimeric molecules with swapped SH3 domains only and found that the SH3 domain is necessary and sufficient to confer Ca(2+)-mediated regulation of Src and Yes tyrosine kinases.     

Fibroblast growth factor receptor-1-mediated endothelial cell proliferation is dependent on the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-alpha/FGFR-1 (alphaR/FR) wild type and mutant Y463F receptors. The kinase activity of alphaR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant alphaR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type alphaR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.     

The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.     

Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.

Many cellular signaling proteins contain SH3 (Src homology 3) domains that mediate protein interactions via specific proline-containing peptides. Unlike SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 binding site, the regulatory mechanism for SH3 interactions is unclear. p120 RasGAP (GTPase-activating protein), which contains an SH3 domain flanked by two SH2 domains, forms an abundant SH2-mediated complex with p190 RhoGAP in cells expressing activated tyrosine kinases. We have identified two closely linked tyrosine-containing peptides in p190 that bind simultaneously to the RasGAP SH2 domains upon p190 phosphorylation. This interaction is expected to bring the two SH2 domains into close proximity. Consequently, RasGAP undergoes a conformational change that results in a 100-fold increase in the accessibility of the target binding surface of its SH3 domain. These results indicate that the tandem arrangement of SH2 and SH3 domains found in a variety of cellular signaling proteins can provide a conformational mechanism for regulating SH3-dependent interactions through tyrosine phosphorylation. In addition, it appears that the role of p190 in the RasGAP signaling complex is to promote additional protein interactions with RasGAP via its SH3 domain.     

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3''-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.     

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human B lymphoma cell surface triggers Cbl tyrosine phosphorylation, its association with p85 subunit, Crk-L and Syk and its dissociation with Vav

It is well established that CD21 activation on human B cell surface triggers B cell proliferation. We previously demonstrated that CD21 activation also triggers tyrosine phosphorylation of two components, p95 and p120, both interacting with SH2 domains of the p85 subunit of PI 3-kinase. We successively identified p95 as the nucleolin and the first signal transduction pathway specifically triggered by CD21 activation, i.e.: pp60Src activation, tyrosine phosphorylation of p95 nucleolin, its interaction with SH2 domains of p85 subunit and PI 3-kinase activation, followed by AKT-GSK-3 activations. We herein identified the p120 component as the protooncoprotein Cbl and the first steps associated to its activation. First, CD21 activation triggered Cbl tyrosine phosphorylation, which required c-Src kinase but not PI 3-kinase or Syk kinase activities. Involvement of Src kinase in this step was supported by inhibition of Cbl phosphorylation and its interactions with other components when cells were either preincubated with specific Src inhibitor or transfected with dominant-negative c-Src form. Second, once tyrosine phosphorylated, Cbl interacts with SH2 domains of p85 subunit, SH2 domains of Crk-L and with tyrosine phosphorylated Syk kinase. The third and unexpected feature was to found that, at the contrary of BCR or of CD19 (herein also analyzed for the first time), CD21 activation triggers dissociation of Cbl-Vav complex. Thus, these results provide the first molecular basis of a new signal transduction pathway specifically triggered by CD21 activation.     

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.     

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.     

HCV NS5A Protein Containing Potential Ligands for Both Src Homology 2 and 3 Domains Enhances Autophosphorylation of Src Family Kinase Fyn in B Cells

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin''s lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg¹⁷⁶ to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr³³⁴ was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.     

Biological and biochemical activity of v-Crk chimeras containing the SH2/SH3 regions of phosphatidylinositol-specific phospholipase C-gamma and Src.

The chicken CT10 virus oncogene product, P47gag-crk, contains SH2/SH3 domains that have been identified as conserved domains among proteins involved in signal transduction. We studied the functional similarity of the SH2/SH3 domains by replacing those of v-Crk with those of phosphatidylinositol-specific phospholipase C-gamma, v-Src, or c-Src. The transforming activity of v-Crk was partially retained in a mutant with a v-Src SH3 domain but not in the other mutants with heterologous SH2/SH3 domains. Mutant viruses with Crk-SH2/SH2' domains induced tyrosine phosphorylation of cellular proteins, but mutants with phosphatidylinositol-specific phospholipase C-gamma or Src SH2/SH2' domains did not. However, the mutant proteins with heterologous SH2/SH2' regions were able to weakly associate with some phosphotyrosine-containing proteins in vitro. These results indicate that in the context of the P47gag-crk structure, the requirement of Crk-SH2/SH3 is more stringent for its activity to induce cell transformation than to cause phosphorylation of cellular proteins. The substitution with heterologous sequences least perturbs the capacity to bind phosphotyrosine-containing proteins. In each case, the SH3 domain is more flexible to substitution than is the SH2 domain.     

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.