A herpesvirus saimiri membrane protein required for interleukin-2 independence forms a stable complex with p56lck.

ORF-2, a 32-kDa viral protein expressed by herpesvirus saimiri-transformed lymphocytes, is essential for transformation and is expressed on the plasma membrane of transformed cells. The current work now shows that most (approximately 80%) of ORF-2 resides in the cytoplasm, while only a small portion protrudes from the cell surface. Expressed as a glutathione S-transferase fusion protein, ORF-2 was found to interact with a 56-kDa cellular protein in untransformed, herpesvirus saimiri-transformed, and Jurkat lymphocytes. Microsequencing proved that this protein is the lymphocyte-specific tyrosine protein kinase p56lck. Two regions of ORF-2 were found to be required for p56lck interaction. Current evidence suggests that the interaction of ORF-2 with p56lck plays a key role in the specific transformation of T lymphocytes to an interleukin-2-independent phenotype.     

Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells.

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src.     

Activated p56lck directs maturation of both CD4 and CD8 single-positive thymocytes

p56(lck) is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56(lck) in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56(lck) (p56(lck)F505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56(lck)F505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56(lck) constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56(lck) in regulating TCR signaling.     

Demonstration that LSTRA cells have an elevated level of proteins phosphorylated on tyrosine residues

The LSTRA cell line has been shown to have an exceptionally high level of a tyrosine protein kinase (pp56lck). We now report that LSTRA cells also have a much higher level of proteins phosphorylated on tyrosine residues in comparison to several other cell lines with normal levels of pp56lck. The level of phosphotyrosine-containing proteins in LSTRA cells was comparable to that seen in K562 cells, a cell line known to have a constitutively active tyrosine protein kinase. These results provide evidence that LSTRA cells have an elevated level of in vivo tyrosine protein kinase activity, probably due to the overexpression and activation of pp56lck.     

Differential requirement for p56lck in HIV-tat versus TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal kinase, and apoptosis

HIV-tat protein, like TNF, activates a wide variety of cellular responses, including NF-kappa B, AP-1, c-Jun N-terminal kinase (JNK), and apoptosis. Whether HIV-tat transduces these signals through the same mechanism as TNF is not known. In the present study we investigated the role of the T cell-specific tyrosine kinase p56lck in HIV-tat and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, an isogeneic lck-deficient T cell line. Treatment with HIV-tat protein activated NF-kappa B, degraded I kappa B alpha, and induced NF-kappa B-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56lck kinase. These effects were specific to HIV-tat, as activation of NF-kappa B by PMA, LPS, H2O2, and TNF was minimally affected. p56lck was also found to be required for HIV-tat-induced but not TNF-induced AP-1 activation. Similarly, HIV-tat activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. HIV-tat also induced cytotoxicity, activated caspases, and reactive oxygen intermediates in Jurkat cells, but not in JCaM1 cells. HIV-tat activated p56lck activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56lck tyrosine kinase reversed the HIV-tat-induced NF-kappa B activation and cytotoxicity. Overall, our results demonstrate that p56lck plays a critical role in the activation of NF-kappa B, AP-1, JNK, and apoptosis by HIV-tat protein but has minimal or no role in activation of these responses by TNF.     

TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56(lck) residues

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56(lck) ) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56(lck) phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56(lck) or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H(2) O(2) or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56(lck) . This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56(lck) -specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56(lck) residues. This implies that dephosphorylation of Y505 is not crucial for p56(lck) activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56(lck) signaling pathways in T cells at single cell level. ? 2012 International Society for Advancement of Cytometry.     

The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity.

Protein tyrosine kinases participate in the transduction and modulation of signals that regulate proliferation and differentiation of cells. Excessive or deregulated protein tyrosine kinase activity can cause malignant transformation. The catalytic activity of the T cell protein tyrosine kinase p56lck is normally suppressed by phosphorylation of a carboxyl-terminal tyrosine, Tyr-505, by another cellular protein tyrosine kinase. Here we characterize a human cytosolic 50 kDa protein tyrosine kinase, p50csk, which specifically phosphorylates Tyr-505 of p56lck and a synthetic peptide containing this site. Phosphorylation of Tyr-505 suppressed the catalytic activity of p56lck. We suggest that p50csk negatively regulates p56lck, and perhaps other cellular src family kinases.     

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.     

Regulation of pp56lck during T-cell activation: functional implications for the src-like protein tyrosine kinases.

The lymphocyte-specific protein tyrosine kinase pp56lck, encoded by a member of the src gene family, is implicated in the control of T-cell growth and differentiation. Purified resting human T lymphocytes contain appreciable levels of lck mRNA and of pp56lck. Upon activation of these T cells, levels of lck mRNA and of pp56lck promptly decline. These reductions in lck mRNA and protein expression are closely correlated with the induction of lymphokine production. Both require identical stimuli and follow a similar time course of response. Down-regulation of lck expression, however, is not correlated with proliferation. Our results provide an example of regulation of a src-like protein tyrosine kinase in a normal fully differentiated cell population and suggest that modulation of lck RNA and protein expression is an important feature of the lymphocyte activation sequence leading to lymphokine production.     

The anti-apoptotic effect of Notch-1 requires p56lck-dependent, Akt/PKB-mediated signaling in T cells

The Notch family of transmembrane receptors have been implicated in a variety of cellular decisions in different cell types. Here we investigate the mechanism underlying Notch-1-mediated anti-apoptotic function in T cells using model cell lines as the experimental system. Ectopic expression of the intracellular domain of Notch-1/activated Notch (AcN1) increases expression of anti-apoptotic proteins of the inhibitors of apoptosis (IAP) family, the Bcl-2 family, and the FLICE-like inhibitor protein (FLIP) and inhibits death triggered by multiple stimuli that activate intrinsic or extrinsic pathways of apoptosis in human and murine T cell lines. Numb inhibited the AcN1-dependent induction of anti-apoptotic proteins and anti-apoptotic function. Using pharmacological inhibitors and dominant-negative approaches, we describe a functional role for phosphatidylinositol 3-kinase (PI3K)-dependent activation of the serine-threonine kinase Akt/PKB in the regulation of AcN1-mediated anti-apoptotic function and the expression of FLIP and IAP family proteins. Using a cell line deficient for the T cell-specific, Src family protein, the tyrosine kinase p56(lck) and by reconstitution approaches we demonstrate that p56(lck) is required for the Notch-1-mediated activation of Akt/PKB function. Furthermore, the Src tyrosine kinase inhibitor, PP2, abrogated ectopically expressed AcN1-mediated anti-apoptotic function and phosphorylation of p56(lck). We present evidence that endogenous Notch-1 associates with p56(lck) and PI3K but that Akt/PKB does not co-immunoprecipitate with the Notch1.p56(lck).PI3K complex. Finally, we demonstrate that the Notch1.p56(lck).PI3K complex is present in primary T cells that have been activated in vitro and sustained in culture with the cytokine interleukin-2.     

Specific deficiency of p56lck expression in T lymphocytes from type 1 diabetic patients

Peripheral T lymphocyte activation in response to TCR/CD3 stimulation is reduced in type 1 diabetic patients. To explore the basis of this deficiency, a comprehensive analysis of the signal transduction pathway downstream of the TCR/CD3 complex was performed for a cohort of patients (n = 38). The main result of the study shows that T cell hyporesponsiveness is positively correlated with a reduced amount of p56(lck) in resting T lymphocytes. Upon CD3-mediated activation, this defect leads to a hypophosphorylation of the CD3zeta-chain and few other polypeptides without affecting the recruitment of ZAP70. Other downstream effectors of the TCR/CD3 transduction machinery, such as phosphatidylinositol 3-kinase p85alpha, p59(fyn), linker for activation of T cells (LAT), and phospholipase C-gamma1, are not affected. In some patients, the severity of this phenotypic deficit could be linked to low levels of p56(lck) mRNA and resulted in the failure to efficiently induce the expression of the CD69 early activation marker. We propose that a primary deficiency in human type 1 diabetes is a defect in TCR/CD3-mediated T cell activation due to the abnormal expression of the p56(lck) tyrosine kinase.     

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.     

Herpesvirus saimiri Tip-484 membrane protein markedly increases p56lck activity in T cells.

Herpesvirus saimiri (HVS) is a T-cell-specific transforming and oncogenic virus. A protein encoded by HVS known as Tip-484 (for tyrosine kinase interacting protein from HVS strain 484) is required for this transformation. Tip-484 binds specifically to the nonreceptor protein tyrosine kinase p56lck. By transfecting Tip-484 into T cells, we now show that this interaction leads to a several hundred-fold increase in the kinase activity of p56lck. Tip-484 is part of a protein complex which is dependent on the presence of p56lck and is phosphorylated. We also show that two of the complexed proteins represent two phosphorylated forms of Tip-484. Furthermore, the p56lck kinase activity in HVS-infected human peripheral blood T lymphocytes was at least ninefold higher than that in noninfected control cells and significantly decreased in cells infected with a Tip-484 deletion mutant virus. Finally, we report that Tip-484 is required for oncogenesis in rabbits by the survival of rabbits inoculated with Tip-484 deletion mutant HVS. The data demonstrate dramatic stimulation of the signaling pathway of p56lck. This effect can contribute to the molecular mechanisms that lead to sustained autocrine secretion of growth factors, permanent T-cell growth, and ultimately lymphocytic tumor formation.     

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.     

Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene.

Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.