Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.

Recent studies have shown that ligand-activated growth factor receptors as well as transforming versions of nonreceptor protein-tyrosine kinases physically associate with phosphatidylinositol-3 kinase (PI-3 kinase). Reasoning that PI-3 kinase might also play a role in the normal functions of nonreceptor kinases, we sought to determine whether association with PI-3 kinase might serve as a measure of nonreceptor protein-tyrosine kinase activation under physiological conditions. We found that p60c-src as well as p59fyn, the product of another member of the src family of proto-oncogenes, physically associated with a PI kinase activity within 5 s after exposure to thrombin. Furthermore, PI kinase reaction products generated in p60v-src, p60c-src or p59fyn containing immunoprecipitates were indistinguishable, demonstrating the identity of the associated enzyme as PI-3 kinase. These findings demonstrate a thrombin-dependent interaction between p60c-src or p59fyn and PI-3 kinase and suggest a role for nonreceptor protein-tyrosine kinases in human platelet signal transduction.     

p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain.

Several lines of evidence link the protein tyrosine kinase p59fyn to the T-cell receptor. The molecular basis of this interaction has not been established. Here we show that the tyrosine kinase p59fyn can associate with chimeric proteins that contain the cytoplasmic domains of CD3 epsilon, gamma, zeta (zeta), and eta. Mutational analysis of the zeta cytoplasmic domain demonstrated that the membrane-proximal 41 residues of zeta are sufficient for p59fyn binding and that at least two p59fyn binding domains are present. The association of p59fyn with the zeta chain was specific, as two closely related Src family protein tyrosine kinases, p60src and p56lck, did not associate with a chimeric protein that contained the cytoplasmic domain of zeta. Mutational analysis of p59fyn revealed that a 10-amino-acid sequence in the unique amino-terminal domain of p59fyn was responsible for the association with zeta. These findings support evidence that p59fyn is functionally and structurally linked to the T-cell receptor. More importantly, these studies support a critical role for the unique amino-terminal domains of Src family kinases in the coupling of tyrosine kinases to the signalling pathways of cell surface receptors.     

Multiple features of the p59fyn src homology 4 domain define a motif for immune-receptor tyrosine-based activation motif (ITAM) binding and for plasma membrane localization

The src family tyrosine kinase p59fyn binds to a signaling motif contained in subunits of the TCR known as the immune-receptor tyrosine- based activation motif (ITAM). This is a specific property of p59fyn because two related src family kinases, p60src and p56lck, do not bind to ITAMs. In this study, we identify the residues of p59fyn that are required for binding to ITAMs. We previously demonstrated that the first 10 residues of p59fyn direct its association with the ITAM. Because this region of src family kinases also directs their fatty acylation and membrane association (Resh, M.D. 1993, Biochim. Biophys. Acta 1155:307-322; Resh, M.D. 1994. Cell. 76:411-413), we determined whether fatty acylation and membrane association of p59fyn correlates with its ability to bind ITAMs. Four residues (Gly2, Cys3, Lys7, and Lys9) were required for efficient binding of p59fyn to the TCR. Interestingly, the same four residues are present in p56lyn, the other src family tyrosine kinase known to bind to the ITAM, suggesting that this set of residues constitutes an ITAM recognition motif. These residues were also required for efficient fatty acylation (myristoylation at Gly2 and palmitoylation at Cys3), and plasma membrane targeting of p59fyn. Thus, the signals that direct p59fyn fatty acylation and plasma membrane targeting also direct its specific ability to bind to TCR proteins.     

Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif.

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.     

Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn)

Engagement of the clonotypic antigen receptor (TCR) on T lymphocytes provokes an activation response leading to cell proliferation and lymphokine secretion. To examine the molecular basis of T cell signaling, we generated transgenic animals in which a lymphocyte-specific nonreceptor protein-tyrosine kinase p59fyn(T) is 20-fold overexpressed in developing T lineage cells. Thymocytes from these mice, analyzed using both cellular and biochemical assays, were remarkably hyperstimulable. Moreover, the responsiveness of normal thymocytes to TCR-derived signals correlated well with the extent to which p59fyn was expressed in these cells. Overexpression of a catalytically inactive form of p59fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes. These effects are unique to p59fyn; overexpression of a closely related T cell-specific tyrosine kinase, p56lck, elicits dramatically different phenotypes. Our results suggest that p59fyn is a critically important component of the TCR signal transduction apparatus.     

RPTPalpha is essential for NCAM-mediated p59fyn activation and neurite elongation

The neural cell adhesion molecule (NCAM) forms a complex with p59fyn kinase and activates it via a mechanism that has remained unknown. We show that the NCAM140 isoform directly interacts with the intracellular domain of the receptor-like protein tyrosine phosphatase RPTPalpha, a known activator of p59fyn. Whereas this direct interaction is Ca2+ independent, formation of the complex is enhanced by Ca2+-dependent spectrin cytoskeleton-mediated cross-linking of NCAM and RPTPalpha in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTPalpha-deficient brains and is disrupted by dominant-negative RPTPalpha mutants, demonstrating that RPTPalpha is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTPalpha-deficient neurons, and disruption of the NCAM-p59fyn complex in RPTPalpha-deficient neurons or with dominant-negative RPTPalpha mutants blocks NCAM-dependent neurite outgrowth, implicating RPTPalpha as a major phosphatase involved in NCAM-mediated signaling.     

Association of Src-like protein tyrosine kinases with the CD2 cell surface molecule in rat T lymphocytes and natural killer cells.

The cell surface molecule CD2 has a signaling role in the activation of T lymphocytes and natural killer cells. Because perturbation of CD2 leads to the appearance of tyrosine-phosphorylated proteins, we investigated the possibility that CD2 associates with cytoplasmic protein tyrosine kinases. As determined by in vitro kinase assays and phosphoamino acid analysis, protein tyrosine kinase activity coprecipitated with CD2 from rat T lymphocytes, T lymphoblasts, thymocytes, interleukin-2-activated natural killer cells, and RNK-16 cells (a rat natural killer cell line). In each case, both p56lck and p59fyn were identified in the CD2 immunoprecipitate. In the thymus, the association between CD2 and these kinases occurred predominately in a small subset of thymocytes that had the cell surface phenotype of mature T cells, indicating that the association is a regulated event and occurs late in T-cell ontogeny. The finding that CD2 is associated with p56lck and p59fyn in detergent lysates suggests that interactions with these Src-like protein kinases play a critical role in CD2-mediated signal transduction.     

CSK: a protein-tyrosine kinase involved in regulation of src family kinases.

The functions of src family protein-tyrosine kinases are thought to be regulated negatively by the phosphorylation of highly conserved tyrosine residues close to their carboxyl termini. Recently we have purified and cloned a protein-tyrosine kinase (designated as CSK) that can specifically phosphorylate the negative regulatory site of p60c-src. To elucidate the relationship between CSK and other types of src family kinases, we investigated the tissue distribution of CSK and examined whether CSK could phosphorylate the negative regulatory sites of src family kinases other than p60c-src. Western blot analysis indicated that CSK was enriched at the highest level in lymphoid tissues in which the expression of p60c-src is considerably lower than those of other types of src family kinases. CSK phosphorylated p56lyn and p59fyn, which are known to be expressed in lymphoid tissues at a relatively high level. The putative regulatory site, tyrosine 508, was found to be essential for phosphorylation in p56lyn, and the kinase activities of these src family kinases were repressed by phosphorylation with CSK. These findings raise the possibility that CSK might act as a universal regulator for src family kinases.     

Src-related protein tyrosine kinases and T-cell receptor signalling.

Upon antigen stimulation, the T-cell receptor for antigen transduces an intracellular protein tyrosine phosphorylation signal that is critical for subsequent T-lymphocyte activation. As the antigen receptor does not possess an intrinsic protein tyrosine kinase activity, the mechanism by which it regulates protein tyrosine phosphorylation is unconventional. Evidence is increasing that the Src-related protein tyrosine kinases P56lck and p59fyn, as well as the protein tyrosine phosphatase CD45, are involved in this process.     

The CD45 tyrosine phosphatase regulates specific pools of antigen receptor-associated p59fyn and CD4-associated p56lck tyrosine in human T-cells.

A newly isolated T-cell line (CB1) derived from a T-acute lymphoblastic leukaemia (T-ALL) patient contained cells (40% of total) which did not express the CD45 phosphotyrosine phosphatase. The cells were sorted into CD45- and CD45+ populations and shown to be clonal in origin. T-cell receptor (TCR) cross-linking or coligation of the TCR with its CD4/CD8 co-receptors induced tyrosine phosphorylation and calcium signals in CD45+ but not in CD45- cells. Unexpectedly, whole cell p56lck and p59fyn tyrosine kinase activities were not reduced in CD45- compared to CD45+ cells. A novel technique was therefore developed to isolated specific pools of aggregated receptors expressed at the cell surface, together with their associated tyrosine kinases. Using this technique it was shown that cell surface CD4-p56lck kinase activity was 78% lower in CD45- than in CD45+ cells. Phosphorylation of TCR zeta- and gamma-chains occurred in TCR immunocomplexes from CD45+ but not CD45- cells, despite comparable levels of p59fyn and TCR proteins. Furthermore, TCR-associated tyrosine kinase activity towards an exogenous substrate was 84% lower in CD45- than in CD45+ cells. Addition of recombinant p59fyn to TCR immunocomplexes isolated from CD45-cells restored the phosphorylation of the TCR zeta- and gamma-chains. Our results demonstrate that CD45 selectively regulates the pools of p59fyn and p56lck kinases which are associated with the TCR and CD4 at the cell surface. Activation by CD45 of these receptor-associated kinase pools correlates with the ability of the TCR and its coreceptors to couple to intracellular signalling pathways.     

Differential association of cellular proteins with family protein-tyrosine kinases

We have sought to identify candidate substrates for src family protein-tyrosine kinases potentially important for transformation. Transfected NIH/3T3 cells, each overexpressing a normal or activated version of the fyn, fgr, or src translational product, were examined using antibody to phosphotyrosine as a probe. Expression of each cDNA induced similar but distinct patterns of tyrosine phosphorylated cellular proteins, with the extent of phosphorylation being greatest in cells expressing an activated kinase. A 70-kDa tyrosine-phosphorylated protein was found to associate with the activated fyn gene product. A protein designated p130, tyrosine phosphorylated in vitro, and in vivo, was found to physically associate with the activated product of each src family gene examined. Physical interaction of three different highly transforming tyrosine kinases with a common cellular protein suggests that p130 may play an important role in transformation induced by src family kinases.     

Transient tyrosine phosphorylation of human ryanodine receptor upon T cell stimulation

The ryanodine receptor of Jurkat T lymphocytes was phosphorylated on tyrosine residues upon stimulation of the cells via the T cell receptor/CD3 complex. The tyrosine phosphorylation was transient, reaching a maximum at 2 min, and rapidly declined thereafter. In co-immunoprecipitates of the ryanodine receptor, the tyrosine kinases p56(lck) and p59(fyn) were detected. However, only p59(fyn) associated with the ryanodine receptor in a stimulation-dependent fashion. Both tyrosine kinases, recombinantly expressed as glutathione S-transferase (GST) fusion proteins, phosphorylated the immunoprecipitated ryanodine receptor in vitro. In permeabilized Jurkat T cells, GST-p59(fyn), but not GST-p56(lck), GST-Grb2, or GST alone, significantly and concentration-dependently enhanced Ca(2+) release by cyclic ADP-ribose. The tyrosine kinase inhibitor PP2 specifically blocked the effect of GST-p59(fyn). This indicates that intracellular Ca(2+) release via ryanodine receptors may be modulated by tyrosine phosphorylation during T cell activation.     

Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking.

Activation of T cells by specific antigens in the context of major histocompatibility complex encoded proteins is mediated by the T cell antigen receptor (TcR), consisting of a variable (Ti) and an invariant (CD3) subunits. Tyrosine phosphorylation is considered to be one of the earliest steps in TcR-mediated signal transduction. There are indications that the p60fyn protein tyrosine kinase is involved in signaling via TcR. However, enzymatic activation of the Src-related tyrosine kinases upon TcR triggering has not been shown yet, therefore the identity of TcR-activated tyrosine kinase(s) remains unclear. We demonstrate that cross-linking of CD3 activates p60fyn and induces tyrosine phosphorylation of cellular proteins in human T cells (resting peripheral T cells, a helper T cell clone, a helper T cell clone immortalized with Herpesvirus saimiri, and a leukemic T cell line). Activation of p60fyn was fast, and its maximum (2-4-fold activation as compared with the basal activity) was followed by a decline. The amount of p60fyn in the cells remained unchanged. None of the other T cell Src-related tyrosine kinases was activated after cross-linking of CD3. Activation of p60fyn was induced by anti-CD3, but not by anti-CD4, anti-CD2, or anti-CD28. The activation was correlated with an increase of the phosphotyrosine content of p60fyn. These studies provide direct proof for the functional association between p60fyn and the TcR.     

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.     

Activation of phosphatidylinositol 3-kinase by a complex of p59fyn and the receptor tyrosine kinase Xmrk is involved in malignant transformation of pigment cells.

Malignant melanoma in the fish Xiphophorus is induced by overexpression of the Xmrk-oncogene, encoding a subclass I receptor tyrosine kinase. The mutationally activated Xmrk protein triggers constitutive mitogenic signalling in fish melanoma cells. In recent studies we showed that in melanoma cells phosphatidylinositol (PtdIns) 3-kinase, as well as p59fyn, has elevated levels of kinase activity. Both bind directly to different phosphotyrosine residues in the Xmrk receptor C-terminus through their SH2 domains. To analyse the mechanism of regulation of these Xmrk-associated kinases in melanoma we characterized the protein-protein interactions between PtdIns 3-kinase, p59fyn and the Xmrk receptor in detail. A ternary complex in which the p85 subunit of PtdIns 3-kinase is associated with p59fyn as well as with Xmrk was identified. Contrary to complexes described for other receptors, the adaptor protein p120Cbl was not involved in these interactions. Thus, we describe here a new mechanism of activation of PtdIns 3-kinase by a receptor of the epidermal growth factor receptor family in which p59fyn acts as an adaptor as well as an activator of PtdIns 3-kinase. Activation of PtdIns 3-kinase activity by fyn was also found in vivo. The fact that this was only detectable in highly transformed Xmrk overexpressing melanomas but not in benign lesions points to the essential role of the Xmrk receptor in this mechanism of regulation.     

Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.     

Multimolecular associations of the T-cell antigen receptor

T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).     

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.