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.      

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.     

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.     

A potential role for protein tyrosine kinase p56(lck) in rheumatoid arthritis synovial fluid T lymphocyte hyporesponsiveness

Rheumatoid arthritis (RA) synovial fluid (SF)-T lymphocytes appear relatively inactive in situ and respond only weakly to diverse stimuli ex vivo. To characterize the molecular defects underlying this hyporesponsiveness we analyzed the expression level of several proteins involved in TCR-proximal signal transduction. As compared to peripheral blood (PB)-T lymphocytes, SF-T cells from some (but not all) of the patients analyzed expressed lower levels of TCRalphabeta, CD3epsilon, TCRzeta, p56(lck) and LAT, while p59(fyn), phospholipase C-gamma1 and ZAP-70 expression was unaltered. Semi-quantitative analysis of T cells from several patients revealed that the degree of TCRzeta chain and p56(lck) modulation correlated statistically significantly with the level of SF-T cell hyporesponsiveness. The differential reactivity of p56(lck) specific monoclonal and polyclonal antibodies in SF-T but not PB-T lymphocytes indicated that p56(lck) modulation consists of a conformational change rather than loss of expression. Our results indicate that multiple signaling molecules can be modulated in RA SF-T cells and show for the first time a direct quantitative correlation between T cell hyporesponsiveness and modulation of TCRzeta and of p56(lck), a critical protein tyrosine kinase required for T cell activation.     

The lymphocyte-specific tyrosine protein kinase p56lck

The CD4 and CD8 T cell surface antigens are physically associated with the tyrosine protein kinase p56lck. Accumulating data indicate that p56lck transduces intracellular tyrosine protein phosphorylation signals upon engagement of CD4 and CD8 by major histocompatibility complex (MHC) determinants expressed on antigen-presenting cells (APCs). Recent studies show that these p56lck-related phosphorylation events enhance T cell receptor (TCR)-mediated functions and are critical for the proposed co-receptor roles of CD4 and CD8. p56lck is also capable of enhancing antigen receptor responsiveness in the absence of CD4 or CD8 expression, suggesting that it can directly contribute to the TCR-induced tyrosine phosphorylation signal.     

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.     

The cytoskeleton-associated TCR zeta chain is constitutively phosphorylated in the absence of an active p56(lck) form

The TCR recognizes peptide-MHC complexes and transmits activation signals leading to cellular responses. We have previously characterized two TCR populations expressed on the T cell surface; one is linked to the cytoskeleton via a detergent-insoluble cytoskeleton-associated zeta (cska-zeta) chain, while the other is detergent soluble and not linked to the cytoskeleton. The cska-zeta form displays unique properties: it is constitutively phosphorylated, does not undergo hyperphosphorylation upon TCR stimulation as opposed to its non-cytoskeleton-associated counterpart (non-cska-zeta) and it maintains a molecular mass of 16 kDa. It is well established that p56(lck) and possibly p59(fyn) are responsible for the generation of the 21/23-kDa phosphorylated detergent-soluble zeta form. We now demonstrate that the phosphorylation of cska-zeta does not require the activity of p56(lck). We also show that although Lck does not phosphorylate cska-zeta in vivo, it retains the capacity to phosphorylate cska-zeta in vitro. Moreover, differences in zeta-associated kinase activity were detected for non-cska-zeta and cska-zeta. Our results indicating that different kinases phosphorylate the two zeta forms are consistent with a growing consensus that each TCR form may regulate distinct cellular functions.     

Biochemical analysis of the CD45-p56(lck) complex in Jurkat T cells lacking expression of lymphocyte phosphatase-associated phosphoprotein

In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.      

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.     

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.     

p56lck is not essential for the T-cell response to allo-MHC antigens.

In mice lacking the src family protein tyrosine kinase, p 56lck (lck -/-), a greatly reduced number of peripheral T cells is observed due to a profound blockage of the thymocyte development. The peripheral T cells in lck -/- mice exhibit proliferative response after T-cell receptor (TCR)-crosslinking, but can not respond to viral antigens. In this report, we examined the allo-responses of peripheral T cells in the lck -/- mice and the following results were thus obtained. (1) After an intravenous injection of fully allogeneic [allo-major histocompatability complex (MHC)] spleen cells, an increase of interleukin (IL)-2R alpha+ cells was observed in both the CD4+ or CD8+ peripheral T cells of the lck -/- mice and the increase was similar to those in the lck +/+ littermate, with only a somewhat delayed and prolonged time kinetics observed in the CD4+ T cells of the lck -/- mice. (2) the lck -/- mice rejected the fully allogeneic trunk skin grafts several days later than the lck +/+ mice, but did not reject the minor allogeneic grafts. (3) The peripheral T cells of the graft-rejected lck -/- mice exhibited a weaker but significantly proliferative response, while the cytotoxic T lymphocyte (CTL) activities to allo-MHC antigens in vitro were comparable to those in lck +/+ mice. While the response to the minor allo-antigens was shown by the peripheral T cells in the lck +/+ mice with minor allogeneic skin grafts but not by those in the lck -/- mice with the grafts. These results thus suggest that p56lck is not essential for peripheral T cells to both respond and exhibit effector functions to allo-MHC antigens.     

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.     

Mitogenic signals through CD28 activate the protein kinase Ctheta-NF-kappaB pathway in primary peripheral T cells

Mitogenic anti-CD28 antibody stimulates all peripheral T cells to proliferate in the absence of TCR ligation, providing an exception to the two-signal requirement of T cell responses. This antibody preferentially recognizes a mobilized signaling-competent form of CD28, normally induced following TCR ligation, thus providing a unique non-physiological tool to dissect CD28-specific signals leading to T cell proliferation. The protein kinase C (PKC)theta-NF-kappaB pathway has recently been shown to integrate TCR- and CD28-derived signals in co-stimulation. We now demonstrate that this pathway is activated by mitogenic anti-CD28 antibody stimulation. In contrast to conventional anti-CD28 antibody, mitogenic anti-CD28 antibody induced activation of phospholipase Cgamma and Ca(2+) flux in peripheral rat T cells despite no or low levels of inducible tyrosine phosphorylation of TCRzeta chain, TCRzeta-associated protein of 70 kDa (ZAP-70) or linker for activation of T cells (LAT)-critical components of the TCR signaling machinery. Nevertheless, PKCtheta kinase activity in vitro was increased following mitogenic anti-CD28 antibody stimulation, as was membrane association of both PKCtheta and Bcl10. As downstream targets of PKCtheta activation, NF-kappaB components translocated to the nucleus at levels comparable to those after TCR-CD28 co-stimulation. NF-kappaB translocation was diminished by PKCtheta inhibition, as was induction of the NF-kappaB/AP-1 responsive activation marker CD69. We propose that co-stimulation is a sequential process in which appropriate TCR engagement is required to mobilize CD28 into a signaling-competent form which then activates the PKCtheta-NF-kappaB pathway necessary for IL-2 production and proliferation.     

CD4-independent signal transduction through the T-cell receptor (TCR/CD3).

The membrane-bound CD4 glycoprotein has been proposed to act like a co-receptor along with the T-cell antigen receptor (TCR/CD3) during ligand recognition and cell activation. Due to its association with the protein tyrosine kinase (PTK) p56lck, CD4 is believed to transduce a signal and support CD3 activation of T cells. In this study we have shown that CD3 ligation on murine T-cell hybridomas induces tyrosine phosphorylation of proteins, including phospholipase C-gamma 1 (PLC gamma 1), both in the presence as well as in the absence of CD4-linked p56lck. Furthermore, using HPB clones deficient in CD3/PTK association, it has been found that the presence of CD4/p56lck does not overcome the defect in signalling. Not even co-aggregation of CD4 with CD3 triggers tyrosine phosphorylation of proteins in these cells. Together, the present results indicate that CD3-linked PTK(s) plays a primary role in the induction of signalling through TCR/CD3, and the presence of CD4/p56lck is neither necessary nor sufficient to elicit these events. In the light of these results a possible role for CD4 in antigen presentation has been proposed.     

Interaction of CD4:lck with the T cell receptor/CD3 complex induces early signaling events in the absence of CD45 tyrosine phosphatase.

Antibody-mediated ligation of the CD3/T cell antigen receptor (TcR) activates phospholipase C (PLC) via a tyrosine kinase signaling pathway that requires expression of the transmembrane tyrosine phosphatase CD45. In normal T cells, CD3-mediated PLC activation is significantly augmented by co-ligation of CD3 with the CD4 co-receptor; however, unlike CD3-associated tyrosine kinases, antibody-induced activation of the CD4-associated tyrosine kinase p56lck does not require CD45 expression. To explore the role of CD45 in the CD3 and CD4 activation pathways further, we examined the effect of CD3/CD4 cross-linking on tyrosine phosphorylation and activation of phospholipase C in CD45- mutant cells of the T cell leukemia line HPB.ALL. In accord with previous observations, anti-CD3 stimulation of the CD45-deficient cells failed to activate tyrosine kinases, or PLC as measured by mobilization of intracellular calcium. However, we show here that ligation of CD3 with CD4 leads to tyrosine phosphorylation of PLC gamma 1 and elevation in the intracellular free Ca2+ concentration in CD45- cells that is in excess of that seen in CD45+ cells. Since CD4 stimulation alone did not activate PLC, a component of the CD3 signaling pathway must be independent of CD45. Anti-CD4-induced tyrosine phosphorylation and activation of CD4-associated lck was also enhanced in CD45- cells, suggesting that increased lck activation compensates for the defect in CD3/TcR signaling, such that interaction of the CD3 signaling pathway with the CD4-associated pathway activates PLC even in the absence of CD45. The data demonstrate that the requirement for CD45 in coupling CD3/TcR to the PI-PLC activation cascade is not absolute, but rather substantiates a role for CD45 in modifying molecular interactions that control T cell activation.     

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells

MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.     

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.     

A critical role for p59(fyn) in CD2-based signal transduction

TCR- but not CD2-triggered IL-2 production is p56(lck) dependent. To test the hypothesis that p59(fyn), a second src-family protein tyrosine kinase (PTK) expressed in T lymphocytes, might be an essential upstream component of the CD2 signaling pathway, we generated human (h) CD2 transgenic (tg) fyn(+/+) and fyn(-/-) mice. Clustering of hCD2 molecules on resting peripheral T lymphocytes results in Ca(2+) mobilization, activation of MAPK and cellular proliferation. In contrast, in the absence of p59(fyn), these CD2-initiated activities are markedly reduced, while TCR-triggered proliferation is unaffected. Several CD2 pathway components regulated by p59(fyn) have been identified including phospholipase C-gamma1 (PLC-gamma1), Vav, protein kinase C-theta isoform (PKC-theta), docking protein (Dok), focal adhesion kinase (FAK) and Pyk2. Decreased inducible PKC-theta catalytic activity and Vav phosphorylation likely account for diminished p38 and JNK activation in hCD2tg fyn(-/-) mice. Moreover, deficiency in fyn-dependent PLC-gamma1 catalytic activity may contribute to reduced PKC-alpha-dependent ERK activation. Of note, CD2-dependent Dok but not linker from activated T cells (LAT) tyrosine phosphorylation requires p59(fyn). Furthermore, that FAK and Pyk2 are target substrates implies that p59(fyn) may be an important regulator of T cell adhesion as well. Collectively, these data identify p59(fyn) as a key PTK in CD2-mediated activation of mature T lymphocytes.     

Targets of p56(lck) activity in immature thymoblasts: stimulation of the Ras/Raf/MAPK pathway

Previous studies suggest that p56(lck) activity influences thymocyte development at a stage prior to TCR alphabeta expression. Transgenic mice that express high levels of p56(lck) activity during thymopoiesis develop thymic lymphomas consisting of cells with immature surface phenotypes. We have utilized cell lines derived from lck-induced thymic tumors to define biochemical pathways regulated by p56(lck) activity in immature thymocytes. Here we report that components of the Ras/Raf/MAPK pathway are constitutively activated in these lck-transformed immature thymoblasts. p56(lck) utilizes Shc and Grb2 adaptors to mediate activation of p21(ras) in the thymoblast lines by promoting tyrosine phosphorylation of the Shc protein and constitutive interaction between Shc and Grb2. The putative guanine nucleotide exchange factor p95(vav) is also maintained in constitutively tyrosine phosphorylated form as a result of elevated Lck activity. One target of activated Ras, the Raf-1 kinase, is hyperphosphorylated and downstream targets of activated Raf- 1, Erk1 and Erk2, are hyperphosphorylated and activated in Lck- transformed thymocytes. Forskolin treatment reverses Raf-1 hyperphosphorylation in the cells and inhibits proliferation by blocking G1/S transition. In contrast, conventional protein tyrosine kinase inhibitors block proliferation by arresting Lck thymoblasts at G2/M. Lck-mediated stimulation of the Ras/Raf/MAPK pathway is also required to maintain cell viability by preventing programmed cell death. In summary, p56(lck) activity stimulates G1/S transition in immature thymoblasts and maintains cell viability via transduction of constitutive activation signals downstream to components of the Ras/Raf/MAPK pathway.