CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck.

CD45 is a tyrosine phosphatase expressed in all hematopoietic cells which is important for signal transduction through the T cell antigen receptor (TCR). Studies using CD45-deficient cells have revealed that Lck, a tyrosine kinase thought to be essential for TCR signaling, is hyperphosphorylated on Y505 in the absence of CD45. This site of tyrosine phosphorylation negatively regulates the function of the Src family of kinases. Here we provide evidence that CD45 can modulate the binding of the Lck to an 11 amino acid tyrosine phosphorylated peptide containing the carboxy-terminus of Lck (lckP). Significantly, CD45 did not influence the binding of Fyn, PLC gamma 1, GAP and Vav to the same phosphopeptide. Lck protein which bound the peptide was dephosphorylated on Y505 and consisted of only 5-10% of the total cellular Lck. Interestingly, there was a marked increase in binding 15-30 min after CD4 or TCR cross-linking. Taken together, our data suggest that CD45 specifically modulates the conformation of Lck in a manner consistent with the intramolecular model of regulation of Src-like kinases.     

Activation of the lck tyrosine-protein kinase by the binding of the tip protein of herpesvirus saimiri in the absence of regulatory tyrosine phosphorylation.

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.     

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.     

The Gammaherpesvirus m2 protein manipulates the Fyn/Vav pathway through a multidocking mechanism of assembly

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell.     

Herpes simplex virus requires VP11/12 to activate Src family kinase-phosphoinositide 3-kinase-Akt signaling

We previously showed that the herpes simplex virus 1 (HSV-1) tegument protein VP11/12 activates the lymphocyte-specific Src family kinase (SFK) Lck and is tyrosine phosphorylated in an Lck-dependent manner during T cell infection. We now extend these findings to show that ectopic expression of Lck induces robust tyrosine phosphorylation of VP11/12 in Vero cells, strongly suggesting that VP11/12 participates in an Lck-mediated signaling pathway as a substrate of Lck or a kinase activated by Lck. We sought to elucidate signaling events downstream of VP11/12-SFK interactions. SFKs lie upstream of the canonical phosphoinositide 3-kinase (PI3K)-Akt pathway in signaling emanating from immune receptors, growth factor receptors, and polyomavirus middle T antigen. Here, we show that VP11/12 is required for virus-induced activation of PI3K-Akt signaling in HSV-infected Jurkat T cells and primary fibroblasts. VP11/12 interacts with PI3K or PI3K signaling complexes during infection, suggesting that VP11/12 activates PI3K directly. SFK activity is required for tyrosine phosphorylation of VP11/12, VP11/12-PI3K interactions, and Akt activation in infected fibroblasts, suggesting that SFK-dependent phosphorylation of VP11/12 is required for interactions with downstream signaling effectors. Akt controls many biological functions, including cell survival, cell motility, and translation, but it is currently unclear which Akt targets are modulated by VP11/12 during infection. Although the Akt target mTORC1 is activated during HSV-1 infection, VP11/12 is not required for this effect, implying that one or more additional viral proteins regulate this pathway. Further studies are therefore required to determine which Akt targets and associated biological functions are uniquely modulated by VP11/12.     

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.     

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.     

Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor zeta chain by the protein tyrosine kinase Lck.

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor zeta chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the zeta chain. Preliminary 1H-NMR studies of recombinant zeta chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six zeta chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor zeta chain. After in vitro phosphorylation by Lck, the partially phosphorylated zeta chain was digested with trypsin. Separation and identification of the zeta chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six zeta chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.     

Membrane-targeting is critical for the phosphorylation of Vav2 by activated EGF receptor

Vav2 is a member of the Vav family that serves as guanine nucleotide exchange factors (GEFs) for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the EGF receptor. Here, we show that in EGF-treated COS7 cells Vav2 is phosphorylated on tyrosine residues and associates with the EGF receptor. In addition, introducing point mutations into the SH2 domain of green fluorescens protein (GFP)-Vav2 fusion protein leads to the loss of Vav2 tyrosine phosphorylation in response to EGF. To investigate further the mechanism of Vav2 phosphorylation, N-terminal (NT) domain of Vav2 was transiently expressed in COS7 cells as GFP fusion protein. Whereas the NT domain of Vav2 is a preferred substrate for the activated EGF receptor in vitro, we could not detect tyrosine phosphorylation of the GFP-NT construct in EGF-treated cells. However, when the SH2 domain of Vav2 was fused to its NT domain, NT domain proved to be a substrate for the EGF receptor in vivo. These data suggest that membrane-targeting of Vav2 through its SH2 domain is an important event in the phosphorylation and activation of Vav2 in response to EGF.     

Vav1 modulates protein expression during ATRA-induced maturation of APL-derived promyelocytes: a proteomic-based analysis

Overexpression of Vav1 promotes the overcoming of the differentiation blockade that characterizes acute promyelocytic leukemia cells. At variance, down-modulation of Vav1 prevents ATRA-induced maturation, and in particular, the inhibition of its tyrosine phosphorylation prevents the neutrophil differentiation-related changes of cell morphology. These findings allowed to identify Vav1 as a crucial protein in the ATRA-dependent differentiation of tumoral promyelocytes. By means of a proteomic approach, here we have investigated a possible role for Vav1 in modulating protein expression during ATRA treatment of tumoral promyelocytes. We have performed high-resolution 2-DE coupled with mass spectra analysis of HL-60 and NB4 promyelocytic cell lines induced to differentiate with ATRA when the amounts or the tyrosine phosphorylation of Vav1 were forcedly reduced. We have found that the down-regulation of Vav1 affects the expression level of a number of proteins, including cell cycle/apoptosis- and cytoskeleton-related proteins. In particular, the expression of 14-3-3epsilon, alpha-enolase, alpha-tubulin and splice isoform 2 of alpha3 proteasome subunit changed as a consequence of the down-modulation of Vav1 during the differentiation of both HL-60 and NB4 cell lines, suggesting that these proteins may constitute a common part of the ATRA-induced pathway during maturation of APL-derived promyelocytes. These results indicate an unprecedented role for Vav1 in the maturation of myeloid cells as a regulator of protein expression.     

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.     

Removal of C-terminal SRC kinase from the immune synapse by a new binding protein

The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an approximately 72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this "parasynaptic" location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.