The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation

IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways.     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Role of TNF receptor-associated factor 2 and p38 mitogen-activated protein kinase activation during 4-1BB-dependent immune response

4-1BB is a costimulatory member of the TNFR family, expressed on activated CD4(+) and CD8(+) T cells. Previous results showed that 4-1BB-mediated T cell costimulation is CD28-independent and involves recruitment of TNFR-associated factor 2 (TRAF2) and activation of the stress-activated protein kinase cascade. Here we describe a role for the p38 mitogen-activated protein kinase (MAPK) pathway in 4-1BB signaling. Aggregation of 4-1BB alone induces p38 activation in a T cell hybridoma, whereas, in normal T cells, p38 MAPK is activated synergistically by immobilized anti-CD3 plus immobilized 4-1BB ligand. 4-1BB-induced p38 MAPK activation is inhibited by the p38-specific inhibitor SB203580 in both a T cell hybridoma and in murine T cells. T cells from TRAF2 dominant-negative mice are impaired in 4-1BB-mediated p38 MAPK activation. A link between TRAF2 and the p38 cascade is provided by the MAPK kinase kinase, apoptosis-signal-regulating kinase 1. A T cell hybrid transfected with a kinase-dead apoptosis-signal-regulating kinase 1 fails to activate p38 MAPK in response to 4-1BB signaling. To assess the role of p38 activation in an immune response, T cells were stimulated in an MLR in the presence of SB203580. In a primary MLR, SB203580 blocked IL-2, IFN-gamma, and IL-4 secretion whether the costimulatory signal was delivered via 4-1BB or CD28. In contrast, following differentiation into Th1 or Th2 cells, p38 inhibition blocked IL-2 and IFN-gamma without affecting IL-4 secretion. Nevertheless, IL-4 secretion by Th2 cells remained costimulation-dependent. Thus, critical T cell signaling events diverge following Th1 vs Th2 differentiation.     

Protein tyrosine phosphorylation and p56lck modification in IL-2 or phorbol ester-activated human natural killer cells.

Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.     

Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction.

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34-36, 50-60, 62-68, 77-78, 104-110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01-25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by approximately 20-50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.     

Tyrphostin B42 inhibits IL-12-induced tyrosine phosphorylation and activation of Janus kinase-2 and prevents experimental allergic encephalomyelitis

IL-12 is a macrophage-derived cytokine that induces proliferation, cytokine production, and cytotoxic activity of T and NK cells. Signaling through its receptor, IL-12 induces these cellular responses by tyrosine phosphorylation and activation of Janus kinase-2 (Jak-2), Tyk-2, Stat3, and Stat4. We have used tyrphostin B42 (AG490), a Jak-2 inhibitor, to determine the role of Jak-2 kinase in IL-12 signaling and IL-12-induced T cell functions. Treatment of activated T cells with tyrphostin B42 inhibited the IL-12-induced tyrosine phosphorylation and activation of Jak-2 without affecting Tyk-2 kinase. In contrast, treatment with tyrphostin A1 inhibited the tyrosine phosphorylation of Tyk-2 but not that of Jak-2 kinase. Inhibition of either Jak-2 or Tyk-2 leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat3, but not of Stat4, protein. While inhibition of Jak-2 lead to programmed cell death, the inhibition of Jak-2 or Tyk-2 resulted a decrease in IFN-gamma production. We have further tested the in vivo effects of tyrphostin B42 in experimental allergic encephalomyelitis, a Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin B42 decreased the proliferation and IFN-gamma production of neural Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced the incidence and severity of active and passive EAE. These results suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling and IL-12-mediated Th1 differentiation in vivo.     

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.     

Regulation and phenotype of an innate Th1 cell: role of cytokines and the p38 kinase pathway

We have explored the phenotype and regulation of Th1 cell activation by the cytokines IL-12 and IL-18. We demonstrate that these two cytokines selectively induce IFN-gamma in a differentiated Th1 cell population through the previously described p38 mitogen-activated protein (MAP) kinase pathway. Using a highly selective p38 MAP kinase inhibitor, we demonstrate that it is possible to block IFN-gamma induction from activated, differentiated Th1 cells via p38 MAP kinase without disrupting the activation and differentiation of naive T cells or the proliferation of naive or differentiated T cells. In addition, IL-12 and IL-18 provide an Ag and IL-2-independent survival signal to this uniquely differentiated Th1 cell population. We hypothesize that this Ag-independent survival of Th1 cells may participate in an innate inflammatory loop with monocytes at the sites of chronic inflammation. In addition, p38 MAP kinase inhibition of this cytokine-regulated pathway may be a unique mechanism to inhibit chronic inflammation without disruption of Ag-driven activation and function of naive T cells.     

p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis.

Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-gamma, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-gamma was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-gamma synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.     

Distinctly different sensitivity in the induction and reversal of anergy of Th1 and Th2 cells

T cell anergy is one of the mechanisms of immunological tolerance. We examined in this study the distinct responses of Th1 and Th2 cells to in vitro anergic stimulation using Th1 and Th2 cells from two strains of T cell receptor transgenic mice. Proliferation of the Th2 cells was difficult to suppress by anergic stimulation, while that of Th1 cells was significantly inhibited even by weak stimulation. However, IL-4 production by Th2 cells was definitely reduced by anergic stimulation, although the inhibition level of IL-4 was lower than that of IFN-gamma production by Th1 cells. We also examined the reversal of anergy in both subsets. While both the anergized Th1 and Th2 cells responded to IL-2 stimulation, only the anergy of the Th2 cells could be reversed. This result indicates that progression of the cell cycle was not sufficient for anergy reversal in Th1 cells. Our findings indicate that the induction and reversal of T cell anergy might be affected by the distinct signaling features of Th1 and Th2 cells.     

The 75,000-dalton interleukin-2 receptor transmits a signal for the activation of a tyrosine protein kinase

The high-affinity receptor for interleukin-2 (IL-2) is composed of two distinct subunits with molecular weights of 55,000 and 75,000 (p55 and p75). While the presence of the high-affinity receptor requires the simultaneous expression of p55 and p75, these subunits can also be expressed independently, resulting in IL-2 receptors with low and intermediate affinities, respectively. IL-2 can induce proliferation in cells expressing either the intermediate affinity p75 receptor or the p55.p75 high-affinity complex, suggesting that p75 is responsible for signal transduction. We have previously shown that signal transduction by the high-affinity IL-2 receptor involves the activation of a tyrosine protein kinase. In order to evaluate the role of p75 in the activation of this kinase we assessed the ability of IL-2 to induce the activation of a tyrosine protein kinase in the human leukemic cell lines Hut 78 and YT. These cells express p75 as the predominant IL-2 receptor. IL-2-dependent tyrosine phosphorylation was observed in both cell lines and the concentrations of IL-2 needed to stimulate this phosphorylation were similar to that required for binding to the p75 receptor. Antibodies that inhibit binding of IL-2 to p55 had no effect on the IL-2-induced tyrosine phosphorylations in YT cells, while antibodies that block the binding of IL-2 to p75 completely inhibited the phosphorylations. These results demonstrate that the signaling capacity for the IL-2-induced tyrosine phosphorylation resides in the p75 receptor.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Differential effects of interleukin-2 and interleukin-4 on protein tyrosine phosphorylation in factor-dependent murine T cells

Interleukin-2 (IL-2) is a requisite factor for growth and proliferation of IL-2-dependent T cells. At present, the mechanism by which the high-affinity IL-2-IL-2 receptor interaction transmits a mitogenic signal to the cellular interior remains unclear. In this report we have used three murine T cell clones to demonstrate that IL-2 stimulates rapid tyrosine phosphorylation of several proteins. Two of these clones, CTLL-2 and CT6, exhibit a cytotoxic T cell phenotype, while the third, HT-2, was derived from a helper T cell line. All three T cell clones proliferated in response to IL-2 stimulation, but HT-2 cells also proliferated in response to interleukin-4 (IL-4). We comparatively examined the effects of IL-2 and IL-4 on protein tyrosine phosphorylation in these cells by immunoaffinity purification of phosphotyrosyl substrates with an anti-phosphotyrosine monoclonal antibody. Stimulation with concentrations of IL-2 resulting in maximal (10-30 U/ml) or sub-maximal (1-5 U/ml) proliferation caused the rapid tyrosine phosphorylation of 97 and 57 kDa proteins in all three cell lines. The 97 kDa protein was localized in the cytosol, while the 57 kDa protein was detected in both cytosolic and crude membrane fractions. IL-2-dependent tyrosine phosphorylation of an 86 kDa cytosolic protein was observed only in CT6 cells. Tyrosine phosphorylation of 22, 23 and 200 kDa proteins was also observed, but only in the cytotoxic T cell clones. Phosphoamino acid analyses revealed that the 97, 86 and 57 kDa proteins contained phosphotyrosine and phosphoserine residues. Concentrations of IL-2 below the threshold concentration for induction of a proliferative response correspondingly failed to stimulate protein tyrosine phosphorylation. In contrast, growth stimulation of HT-2 cells by IL-4 was not preceded by early changes in protein tyrosine phosphorylation, suggesting that protein tyrosine phosphorylation may not be essential for the induction of IL-4-dependent cell-cycle progression. These results demonstrate that high-affinity IL-2 receptors are coupled to tyrosine kinase activity(s) in T cells. However, the failure of IL-4 to stimulate protein tyrosine phosphorylation in the same cells indicates that enhanced protein tyrosine phosphorylation may not be requisite for growth factor-dependent T cell proliferation.     

Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase.

Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.     

PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes

Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.     

Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.