CD45 phosphotyrosine phosphatase and p56lck protein tyrosine kinase: a functional complex crucial in T cell signal transduction.

Tyrosine phosphorylation of several intracellular proteins is observed very early during T cell activation. p56lck, a non-receptor src-like protein tyrosine kinase (PTK) which is associated with the intracellular domains of CD4 and CD8 co-receptors, has been implicated in these early signal transduction events. Furthermore, recent experiments indicate that the receptor phosphotyrosine phosphatase, CD45, might be important in the regulation of p56lck PTK activity and that its expression is required for the generation of second messenger molecules following TCR triggering. Here, using co-capping experiments and double indirect immunofluorescence microscopy in functional human T lymphocytes, a specific co-distribution of a significant fraction of p56lck with CD45, but not with several other cell surface proteins, has been revealed. This is the first demonstration of a physical interaction between a receptor phosphotyrosine phosphatase and a PTK under physiologically relevant conditions. In addition, after antibody-induced capping of CD4, both a co-localization of p56lck and CD4, and concomitantly a significant increase in intracellular phosphotyrosine at the sites of CD4 caps were observed. In strong contrast to these results, co-clustering of CD4 with CD45 did not result in any detectable intracellular phosphotyrosine at the cap sites. These data indicate that CD45 can act on CD4-associated phosphoproteins in viable human T lymphocytes. Further, this provides evidence that p56lck PTK is a substrate of CD45 phosphotyrosine phosphatase in vivo and thereby supports the idea that CD45 is an early regulator of T cell activation involved in the modulation of the coupling of receptor-triggered events to intracellular signalling pathways.     

The tyrosine kinase activity of p56lck is increased in human T cells activated via CD2.

An early biochemical event associated with T cell activation is tyrosine phosphorylation. We have previously shown that p56lck, a lymphocyte-specific protein tyrosine kinase, is hyperphosphorylated on serine and tyrosine residues 15 minutes after activation via CD2 with a concomitant shift to a higher molecular mass. We now demonstrate that the tyrosine kinase activity of p56lck is increased within seconds following CD2 triggering. This activity decreases thereafter correlating with the appearance of changes in phosphorylation previously described. These results suggest that p56lck may play an important role in the CD2 activation pathway.     

Requirement for p56(lck) tyrosine kinase activation in Th subset differentiation

The lymphocyte-specific protein tyrosine kinase p56(lck) (Lck) is well documented with regard to its role in regulating T cell activation and thymocyte development through delivery of signals via the mature alphabeta TCR as well as the pre-TCR. Little is known, however, about the role of Lck in Th cell subset differentiation in the periphery. Here, we assess the requirement for tyrosine kinase activation of Lck in Th1 and Th2 cell differentiation by using a dominant-negative Lck (DLGKR) transgenic (Tg) mice under the control of a lck distal promoter that directs high expression in mature T cells, in which splenic CD4 T cells developed normally. This Tg mouse provides a good experimental model system to investigate the roles of Lck in mature T cell function in vivo. We show that the catalytically inactive Lck protein at about twice-normal concentrations inhibits Th2 subset differentiation in vivo and in vitro, whilst leaving the maturation of the other T cell subset, Th1, intact. These data indicate a requirement for Lck activity in Th2 cell differentiation, and a differential dependence for Lck activity between Th2 and Th1 cell differentiation.      

p59fyn tyrosine kinase regulates p56lck tyrosine kinase activity and early TCR-mediated signaling

To study the role of p59^fyn in T cell activation, we used antisenseRNA to inhibit p59^fyn expression in a T cell clone. Transfectantswith reduced levels of p59^fyn were functionally impaired intheir responses to antigen, Con A + recombinant IL-1 and cross-linkingwith anti-TCR mAb. Induction of tyrosine phosphorylation onmost intracellular substrates was greatly reduced. We also notedthat the Ick kinase activity was greatly reduced even thoughthe amount of Ick protein was equivalent to that present inparental D10 cells. Our results suggest that the protein tyrosinekinase p59^fyn is critical in TCR-mediated signaling and alsosuggests that p59^fyn may regulate p56^fyn tyrosine kinase activity.     

Interleukin-7 activates p56lck and p59fyn,two tyrosine kinases associated with the p90 interleukin-7 receptor in primary human T cells

We have investigated signaling events associated with the cloned 90-kDa (p90) interleukin-7 receptor (IL-7R) to determine whether changes in the signaling pathways initiated by this molecule can explain the ability of T cells to proliferate to IL-7 following activation. Using in vitro kinase assays we find that the p90 IL-7R in both unstimulated and activated human T cells is physically associated with two molecules with intrinsic kinase activity. Western blotting analysis reveals these proteins to be the src kinase enzymes, p59^fyn and p56^lck. Binding of human recombinant IL-7 to the p90 IL-7R results in increased activity of both receptor-associated kinases in both resting and activated mature T cells. Thus, the signaling pathways initiated via the p90 IL-7R-associated src kinases are unlikely to be solely responsible for the proliferation of only activated T cells in response to IL-7. Additional signals, which may derive from other IL-7R-associated molecules such as the γc, are clearly required for IL-7-driven proliferation of activated primary T cells.     

Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.     

Reciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS.      

Activation of phosphatidylinositol-3-kinase in Jurkat T cells depends on the presence of the p56lck tyrosine kinase

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56^lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56^lck, JCaM1, these responses were absent. We also show that p56^lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56^lck phosphorylates three tyrosine residues in the p85α subunit of PI3K and two in p110 of PI3K. Our results suggest that p56^lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.     

The lymphocyte-specific protein tyrosine kinase p56lck is hyperphosphorylated on serine and tyrosine residues within minutes after activation via T cell receptor or CD2

Human T cells can be activated and induced to proliferate through either the antigen-specific receptor complex (TcR-CD3) or the CD2 surface molecule. Following stimulation, both serine and tyrosine phosphorylation of cellular protein have been demonstrated to occur. p56lck, a protein tyrosine kinase associated to the inner face of the plasma membrane, is almost exclusively expressed in lymphoid cells, especially T cells. Within minutes after activation of a human T cell-derived line (Jurkat) via stimulation of either the TcR-CD3 complex or the CD2 glycoprotein, we observed a hyperphorphosylation of p56lck. A concomitant shift to a higher molecular weight in sodium dodecyl sulfate-polyacrylamide gel was also observed. Similar changes were obtained with phorbol 12-myristate 13-acetate. Tryptic phosphopeptide analysis of the hyperphosphorylated form of p56lck yielded new phosphorylated sites in serine residues and an increased tyrosine phosphorylation. These results suggest that p56lck may be intimately connected to the signaling pathway in T cell activation.     

Anti-CD45 isoform antibodies enhance phagocytosis and gene expression of IL-8 and TNF-alpha in human neutrophils by differential suppression on protein tyrosine phosphorylation and p56lck tyrosine kinase

To determine the biological functions of membrane expressed CD45 isoforms on polymorphonuclear neutrophils (PMN), the monoclonal IgG F(ab')2 antibody against CD45, CD45RA or CD45RO was used as surrogate ligand for binding with these molecules on PMN. We found 99.5 +/- 3.2%, 42.3 +/- 5.8% and 96.7 +/- 2.6% PMN expressed CD45, CD45RA and CD45RO molecules on the cell surface, respectively. The interaction of CD45, CD45RA or CD45RO with its specific antibody on PMN enhanced phagocytosis markedly (34-83% increase), mainly via increased expression of complement receptor type 3 (CR3, CD11b) on the cells. The production of IL-8 by PMN was also increased significantly after binding with antibodies (anti-CD45 > anti-CD45RO > anti-CD45RA). Anti-CD45RA and anti-CD45RO, but not anti-CD45, enhanced TNF-alpha mRNA expression and decreased protein tyrosine phosphorylation of PMN. However, only anti-CD45RO suppressed Src family protein tyrosine kinase p56lck expression in the cells. These results suggest that the cross-linking of CD45 isoforms by their specific antibodies stimulated different PMN activities by differential suppression on protein tyrosine phosphorylation and Src family tyrosine kinase p56lck.     

Dysregulation of p56lck kinase in patients with systemic lupus erythematosus.

In this study we investigated one of the possible mechanisms of p56lck down-regulation in peripheral blood lymphocytes (PBLs) from Systemic Lupus Erythematosus (SLE) patients and we correlated p56lck dysregulation with accelerated apoptosis in SLE PBLs. PBLs from SLE patients and healthy donors were isolated. p56lck protein expression and lck mRNA level were estimated by immunoblotting and RT-PCR, respectively. FACS analysis was used to evaluate the apoptosis and p56lck levels in apoptotic and non-apoptotic PBLs. A non-radioactive Tyrosine Kinase Assay Kit was used to measure p56lck activity. Our results demonstrated that PBLs from SLE patients displayed lower levels of lck mRNA and p56lck protein as compared to healthy donors. The apoptosis of fresh or cultured PBLs was enhanced in SLE patients, especially in anti-DNA negative group. The expression of p56lck was inverse correlated with apoptosis of fresh and cultured SLE PBLs, especially in anti-DNA negative patients. Double staining FACS analysis showed that p56lck expression was lower in apoptotic than in non-apoptotic PBLs. p56lck specific activity was directly correlated to apoptosis in SLE PBLs. While the low expression of p56lck may be the result of lower degree of synthesis, the increased specific activity could directly correlated to the extent of apoptosis in SLE PBLs. Based on our observations, we assume that the p56lck dysregulation could play a role in SLE pathogenesis.     

Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70-kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)-ζ chain, and the FcεRIγ chain. Cross-linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56^lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56^lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA-associated proteins are identified in immune complex kinase assays including the TCR-ζ, subunit, a p70–90 ζ-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56^lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56^lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.     

Lack of evidence for aggregation-dependent enhancement of p56lck in the signal transduction upon major histocompatibility complex recognition by mature T cells

The kinase activity of lymphocyte-specific tyrosine kinase p56lck (Lck) upon physiological major histocompatibility complex (MHC) recognition by normal mature T cells was examined. Recognition of the target MHC molecules by T cells induced phosphorylation of the zeta-chain without obvious enhancement of the background Lck activity. There was no sign of enhancement of Lck through putative T-cell receptor (TCR)-independent class II MHC/CD4 interactions either. As has been reported, cross-linking of CD4 molecules by antibodies induced a marked enhancement of Lck activity. However, it did not have an immediate relevance to TCR-mediated signal transduction, as judged from the lack of detectable de novo phosphorylation of zeta-chain and the absence of functional responses of T cells. These results strongly favour the model in which TCR-mediated signal transduction does not involve aggregation-dependent enhancement of Lck, suggesting that the signal can be triggered simply by the recruitment of already active Lck with basal kinase activity through the formation of a TCR/MHC/CD4 ternary complex.     

Lipopolysaccharide-induced cytokine production in human monocytes: Role of tyrosine phosphorylation in transmembrane signal transduction

The signal transduction events that follow the binding of lipopolysaccharide (LPS) to the macrophage cell surface are not well defined. In the current studies LPS was found to induce alterations in phosphorylation of monocyte proteins on tyrosine. Herbimycin A and genistein, inhibitors of tyrosine kinases, markedly attenuated LPS-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) protein and mRNA production. Reciprocally, the tyrosine phosphatase inhibitor sodium orthovanadate enhanced LPS-induced production of TNF-α. LPS induced a concentration-dependent increase in tyrosine phosphorylation of several proteins, which paralleled and preceded the onset of LPS-induced TNF-α production. LPS stimulation had different but reproducible effects on three members of the src family of tyrosine kinases. Both Hck and Lyn kinase activity increased before the onset of TNF-α production, consistent with their participation in the observed LPS-induced tyrosine phosphoprotein accumulation. In contrast, Yes kinase activity was not affected. These observations were made at concentrations of LPS that required serum rich in LPS-binding protein and the monocyte surface antigen CD14 for TNF-α production. These data indicate that tyrosine kinases and phosphatases are involved in the signal transduction cascade by which LPS induces production of TNF-α and IL-6 by human monocytes, and suggest that Lyn and Hck are candidate participants in this process.     

Interaction of human immunodeficiency virus glycoprotein 160 with CD4 in Jurkat cells increases p56lck autophosphorylation and kinase activity.

The tyrosine protein kinase p56lck, specifically expressed in lymphoid cells, undergoes modifications of its autophosphorylation and kinase activity when these cells are triggered by mAbs to the T cell determinants. The kinase activity and the autophosphorylation of p56lck were analysed following triggering Jurkat cells with the human immunodeficiency virus (HIV) glycoprotein gp160 which interacts with CD4: both the autophosphorylation and the kinase activity are increased within 1-5 min following addition of gp160, this increase is maximum at 5 min and is followed by a gradual return to the basal level within 2 h. Similar to observations made with anti-CD4 mAbs the increase in kinase activity of p56lck is not associated with changes in the gel mobility nor is it associated with T cell activation. Triggering of T cells with a combination of anti-CD3 mAbs which activate T cells but not p56lck and gp160 greatly potentiated the increase of p56lck autophosphorylation and kinase activity.     

Physical association of the cytoplasmic domain of CD2 with the tyrosine kinases p56lck and p59fyn

In T lymphocytes, CD2 forms part of a loosely associated membrane complex which includes the T cell receptor (TcR) for antigen, the CD3 subunits, CD4 or CD8, CD5 and the protein tyrosine kinases p56^lck and p59^fyn. The interaction of CD2 with tyrosine kinases in this complex provides a possible mechanism for transmembrane signal transduction by CD2. We have investigated whether the interaction of CD2 with the kinases is dependent on other known members of the complex, or whether an independent association can be observed. Using in vitro kinase assays with immune complexes precipitated from cell lysates, we demonstrate that CD2 can associate with p56^lck and p59^fyn in a rat thymoma line that does not express CD4 or CD8, and in a TcR-negative Jurkat cell line. In TcR-positive Jurkat cells that express rat CD2, interaction of CD2 with p56^lck and p59^fyn _Was clearly seen, but it was absent in cells where the cytoplasmic tail of CD2 is truncated, indicating that the interactions are mediated by the cytoplasmic region of CD2. Furthermore, using cells expressing CD2 molecules with partial truncations in the cytoplasmic domain, we show that the association of CD2 with p56^lck: is progressively lost as the cytoplasmic domain is shortened, and that the capacity of the mutants to associate with p56^lck correlates with their capacity to transduce transmembrane signals.     

Interactions between the tyrosine kinases p56lck,p59fyn and p50csk in CD4 signaling in T cells

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.     

T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56^lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56^lck. When CD8 bound to class I, phosphorylation of p56^lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56^lck activity.     

Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of zeta-chains and p59fyn/p56lck in human T cells

The expression and catalytic activity of the protein tyrosine kinase (PTK) ZAP-70 are needed for normal intracellular signaling through the T-cell receptor (TCR)/CD3 complex. However, the possible effect of aging on the catalytic activity of ZAP-70 in human peripheral blood T cells stimulated via the TCR/CD3 complex is unknown. The current studies show that T cells from a substantial proportion of elderly humans (12) exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. In addition, the reduced catalytic activity of ZAP-70 in T cells from elderly subjects was not restored to the normal levels in response to ligation of CD4 receptors, suggesting defects in PTKs linked to both CD3 and CD4 receptors. Other experiments demonstrated that the age-related impairments of ZAP-70 activation in anti-CD3-stimulated T cells were accompanied by decreased tyrosine phosphorylations of zeta-chains and autophosphorylations of the PTKs p561ck/p59fyn. Moreover, the age-related defects in these early TCR/CD3-mediated phosphorylation events were readily detectable in both CD45RO+ memory and CD45RA+ naive T cells. Thus, these results suggest that defects in early TCR/CD3-mediated phosphorylation events among CD45RO+ memory and CD45RA+ naive T cells from certain elderly humans may con tribute to impaired induction of ZAP-70 catalytic activity.