Fyn SH3 Domain Research Articles

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains.

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

An SH3-binding Site Conserved in Bruton's Tyrosine Kinase and Related Tyrosine Kinases Mediates Specific Protein Interactions in Vitro and in Vivo

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

The Ras GTPase-activating Protein (GAP) Is an SH3 Domain-binding Protein and Substrate for the Src-related Tyrosine Kinase, Hck

The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the co-infected cells, indicative of Hck.GAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (delta CAT) or the Src homology region (delta SH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, delta CAT, and delta SH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain.

Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins.

ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.

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.

The protein tyrosine kinase Fyn activates transcription from the HIV promoter via activation of NF kappa B-like DNA-binding proteins.

Protein tyrosine kinase p59fyn (Fyn) associates with the TCR-CD3 complex, which suggests that Fyn plays a significant role in the signal transduction involving TCR complex. In addition to cellular genes, viral promoters such as the HIV long terminal repeat (LTR) are also activated upon T cell activation. To elucidate the functional significance of Fyn in the expression of viral promoters, we transfected a Fyn-expression vector together with a reporter plasmid containing the chloramphenicol acetyltransferase gene driven by HIV LTR into a human T cell line, Jurkat. In this assay, Fyn stimulated the promoter in HIV LTR when the transfected cells were treated with both concanavalin A and PMA as an antigen-mimic stimulation. This activation required the intact SH2 domain of Fyn. Mutational analysis of HIV LTR showed that the NF kappa B binding sites were responsible for this effect. Electrophoretic mobility shift assays and UV cross-linking experiments showed that activation of T cells by anti-CD3 antibody induced four kappa B-binding proteins (50, 60, 65 and 100 kDa) in Fyn-overexpressing cells more efficiently than in the parental cells. Our results suggested that Fyn was able to regulate expression of a subset of genes via kappa B-binding proteins upon T cell activation.

Binding of the nicotinic acetylcholine receptor to SH2 domains of Fyn and Fyk protein tyrosine kinases.

The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine both in vitro and in vivo. To identify the protein tyrosine kinase that phosphorylates the receptor, we have previously cloned and characterized two protein tyrosine kinases, Fyn and Fyk, that are highly expressed in Torpedo electric organ, a tissue enriched in the AChR. Both kinases were shown by coimmunoprecipitation to be specifically associated with the AChR. In this study, we examined the molecular basis for the interaction of Fyn and Fyk with the AChR using fusion proteins containing the SH2 domains of the two kinases as affinity reagents. The AChR bound specifically and in a protein concentration-dependent manner to the SH2 domain fusion proteins of Fyn and Fyk. Quantitation of the association revealed that the binding of the AChR to Fyn and Fyk SH2 domain fusion proteins was to a single class of saturable high affinity sites. In addition, the association of the AChR with the SH2 domain fusion proteins was dependent on tyrosine phosphorylation of the AChR and was mediated by the delta subunit of the receptor. Furthermore, upon dissociation of the AChR into subunits, the delta subunit coimmunoprecipitated with both Fyn and Fyk. These data suggest that the association of the AChR with Fyn and Fyk is mediated by an interaction of the tyrosine-phosphorylated delta subunit of the receptor with the SH2 domains of the protein tyrosine kinases.

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.

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.

The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor.

Tyrosine phosphorylation of multiple cellular proteins is a critical event in T cell receptor (TCR)-mediated activation. This pathway has also been implicated in cellular transformation in multiple systems. The viral oncogene v-cbl is the transforming gene of a murine retrovirus that induces pre-B cell lymphomas and myelogenous leukemias. The product of its cellular homolog, p120cbl, is a 120-kDa cytoplasmic protein that is non-transforming when overexpressed. Here we show that the 120-kDa protein tyrosine phosphorylated in Jurkat T cells upon TCR engagement is p120cbl. Following stimulation through the TCR, this tyrosine phosphorylation is rapid and reversible. Tyrosine-phosphorylated p120cbl binds to glutathione S-transferase fusion proteins generated from SH2 domains of the Fyn, Lck, and Blk protein tyrosine kinases, GTPase-activating protein and phospholipase C gamma. The p120cbl from unactivated and activated cells also binds to full-length glutathione S-transferase-Grb2 and the Grb2 N-terminal SH3 domain, but not to the Grb2 C-terminal SH3 domain. Additionally, p120cbl binds to SH3 domains of Fyn and Lck, but not Blk. These data expand our knowledge of protein tyrosine kinase signaling pathways in T cells by identifying a prominent tyrosine kinase substrate. This protein, the product of the cellular homolog of a transforming oncogene, can interact with several known signaling molecules.

Identification of a Src SH3 domain binding motif by screening a random phage display library.

A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.

Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.

Activation-dependent tyrosine phosphorylation of Fyn-associated proteins in T lymphocytes.

While previous studies have implicated the tyrosine protein kinase p60fyn in antigenic activation of T lymphocytes, it is clear that signal transduction initiated through the antigen receptor requires the concerted actions of several proteins. Here, we report our finding that the activation of p60fyn following TcR cross-linking results in the tyrosine phosphorylation of two Fyn-associated proteins of 82 and 116 kDa. In the cells analyzed, p116 appears to represent one of the major substrates of T-cell antigen receptor-mediated tyrosine phosphorylation. In activated T-cells, the interaction of these proteins is specific for p60fyn since neither p56lck nor p62c-yes were found to detectably associate with p82 or p116. Furthermore, the p60fyn-p82/p116 complex could be dissociated and then reconstituted in vitro using purified recombinant Fyn. Using this technique we demonstrated that both p82 and p116 were capable of binding to the Fyn SH2 domain while p82 was to some extent capable of independent binding to the Fyn SH3 domain. An association between p60fyn and phosphoproteins possibly related to the T-cell p82 and p116 was also observed in other hematopoietic cells. Thus, the activation-induced phosphorylation of the p60fyn-associated proteins p116 and p82 and the wide distribution of potentially similar p60fyn-associated proteins in hematopoietic cells suggest that p116 and p82 may play a role as physiological substrates and/or regulators of p60fyn.

T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

SH2 domains of the protein-tyrosine kinases Blk, Lyn, and Fyn(T) bind distinct sets of phosphoproteins from B lymphocytes.

Several members of the Src family, including Blk, Lyn, Fyn(T), and Lck, are expressed in B cells. These kinases associate with the antigen receptor complex, and the activities of Blk, Fyn(T), and Lyn increase upon receptor engagement. Differences in the amino acid sequences and patterns of expression of these kinases suggest that they serve distinct functions. In this communication it is shown that the SH2 domains from Blk, Lyn, and Fyn(T) preferentially bind distinct sets of phosphoproteins from the mature B cell line A20. These interactions were found to depend on recognition of phosphotyrosine. The Blk SH2 domain bound more than 10 distinct phosphoprotein species, most of which reacted with an antiphosphotyrosine antibody; the phosphotyrosine content of these proteins was increased if surface immunoglobulin was cross-linked before extracts were made. Phosphoproteins of 72, 76, 115, and 130 kDa bound to the SH2 domains of Blk, Lyn, and Fyn(T). Phosphoamino acid analysis of these four proteins revealed that each contained phosphoserine, phosphothreonine, and phosphotyrosine. Proteins of 90 kDa, 130 kDa, and 150 kDa were preferentially bound by the Blk SH2 domain, while the Fyn(T) SH2 domain showed preferential binding to proteins of 76 and 180 kDa. The Lyn SH2 binding profile resembled that of Blk, but differences in the binding specificities of these kinases were also observed. Thus, among proteins that exhibit increased tyrosine phosphorylation following antigen receptor cross-linking, several have been identified that bind preferentially to SH2 domains of Blk, Fyn(T), or Lyn, suggesting that these kinases serve distinct functions. In addition, chimeric Fyn(T)-Blk SH2 domains were shown to be functional in binding assays and to exhibit binding specificities intermediate between those of the parent domains, consistent with the interpretation that the differences we observe in phosphoprotein binding by Fyn(T) and Blk SH2 domains reflect differences in their native structures.

Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex.

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.