Activity Of P56lck Research Articles

The Polycomb-group protein ENX-2 interacts with ZAP-70

Human ENX-2 is a homologue of Drosophila Enhancer of zeste, which is a member of Polycomb-group proteins regulating the expression of homeotic genes as chromatin-associated proteins. In this study, we demonstrate that ENX-2 plays an important role as a signaling molecule involved in T cell receptor-mediated signaling pathway. In immunoprecipitation experiments, ENX-2 and zeta associated protein-70 (ZAP-70) were co-precipitated from T cell lysate. When probed with an anti-phospho-tyrosine antibody, ENX-2 was found to be phosphorylated on tyrosine. On the other hand, ENX-2 was not phosphorylated on tyrosine in the mutant Jurkat cell, J.Cam1.6 lacking the activity of lymphocyte protein tyrosine kinase p56 lck. The interaction between ENX-2 and ZAP-70 was abolished in the mutant cell. Furthermore, in-vitro kinase assay using purified p56 lck demonstrated that ENX-2 became tyrosine phosphorylated by this kinase. These findings show that the phosphorylation of ENX-2 is responsible for the interaction between ENX-2 and ZAP-70.

Signaling through P2X7 Receptor in Human T Cells Involves p56 lck , MAP Kinases, and Transcription Factors AP-1 and NF-κB

ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate multiple downstream signaling events in a human T-lymphoblastoid cell line.

High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC.

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b). Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.

Constitutive endocytosis and degradation of the pre-T cell receptor.

The pre-T cell receptor (TCR) signals constitutively in the absence of putative ligands on thymic stroma and signal transduction correlates with translocation of the pre-TCR into glycolipid-enriched microdomains (rafts) in the plasma membrane. Here, we show that the pre-TCR is constitutively routed to lysosomes after reaching the cell surface. The cell-autonomous down-regulation of the pre-TCR requires activation of the src-like kinase p56lck, actin polymerization, and dynamin. Constitutive signaling and degradation represents a feature of the pre-TCR because the γδTCR expressed in the same cell line does not exhibit these features. This is also evident by the observation that the protein adaptor/ubiquitin ligase c-Cbl is phosphorylated and selectively translocated into rafts in pre-TCR– but not γδTCR-expressing cells. A role of c-Cbl–mediated ubiquitination in pre-TCR degradation is supported by the reduction of degradation through pharmacological inhibition of the proteasome and through a dominant-negative c-Cbl ubiquitin ligase as well as by increased pre-TCR surface expression on immature thymocytes in c-Cbl–deficient mice. The pre-TCR internalization contributes significantly to the low surface level of the receptor on developing T cells, and may in fact be a requirement for optimal pre-TCR function.

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.

A Non-class I MHC Intestinal Epithelial Surface Glycoprotein, gp180, Binds to CD8

The activation of CD8+ T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the modulation of mucosal immune responses due to the fact that these T cells appear to have regulatory rather than cytolytic activity. A 180-kDa glycoprotein (gp180) has been identified and shown to be important in CD8+ T cell activation by intestinal epithelial cells. In this study, we examine, in further detail, the role that the CD8 molecule plays in this interaction. It has been previously shown that monoclonal antibodies against gp180 inhibited the activation of CD8-associated p56lck in T cells. Although indirectly suggested by these data, there was no evidence that the activation of this protein tyrosine kinase was a direct result of gp180 interacting with the CD8 molecule. In this study, we document that soluble gp180 is able to bind to CD8–Fc fusion proteins and is absorbed by human CD8α but not CD4 transfected murine T cells and that this interaction is dependent upon carbohydrate on the gp180 molecule. Furthermore, the sites used for binding by gp180 are distinct from those used by the conventional CD8 ligand, class I MHC. Thus, gp180 appears to be a novel CD8 ligand that plays an important role in the activation of CD8-associated kinases and of CD8+ T cells.

INABILITY OF 50 HZ MAGNETIC FIELDS TO REGULATE PKC- AND CA2+-DEPENDENT GENE EXPRESSION IN JURKAT CELLS

We have previously reported that the T cell line Jurkat registers the exposure of a sinusoidal extremely low frequency magnetic field at the level of the plasma membrane, resulting in activation of the tyrosine kinase p56lck, increase in inositol-3-phosphate levels and increase in intracellular calcium concentration within minutes. To elucidate if these events associated with changes in intracellular calcium ion levels were biologically significant, transient transfections of Jurkat cells were performed with calcium-ion dependent reporter constructs. Three different enhancer/promoter constructs were studied coupled to the luciferase reporter gene. The luciferase activity of each construct was measured after treatment of transfected cells to EMF exposure alone, or in combination with ionomycin, phorbol ester or cross-linking anti-CD3 antibodies. There was no indication that the used EMFs could influence any of these reporter constructs.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells.

Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP-70 kinase and the TCR/CD3 gamma-chain mutants, but did not protect the src p56(lck)-deficient cells. Similarly, MHC-I ligation did not protect anisomycin-treated src p56(lck)-deficient cells against apoptosis. These data suggest that MHC-I-induced inhibition of apoptosis depends on intact src p56(lck) activity, but not on major secondary messenger molecules associated with TCR signaling. Overall the results support the idea that signal transduction by MHC-I molecules is involved in homeostatic processes of importance for T-cell survival and death.

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.

L-Selectin Tyrosine Phosphorylates Cbl and Induces Association of Tyrosine-Phosphorylated Cbl with CrkL and Grb2

L-Selectin-mediated rolling of leukocytes on endothelial cells is an important step for lymphocyte homing and an early event in the immune response to pathogens or inflammatory stimuli. We have previously elucidated intracellular signaling cascades upon L-selectin engagement resulting in activation of Ras, Rac and JNK as well as cytoskeletal changes, oxygen release, ceramide synthesis and receptor capping. Activation of the src-tyrosine kinase p56lck is followed by phosphorylation of the L-selectin molecule and MAP-K. Here we show a tyrosine kinase dependent phosphorylation of the Cbl adapter protein after L-selectin engagement in lymphocytes. Phosphorylation of Cbl was absent in Jurkat cells that are pharmacologically treated with tyrosine kinase inhibitors and in lck-deficient JCaM cells. There is an activation induced association of tyrosine phosphorylated Cbl with Grb2 and CrkL, respectively, but not CrkII. Therefore, the adapter protein Cbl plays a role in L-selectin signaling and might modulate immune function by the specific recruitment of signaling molecules to multiprotein complexes.

TGF-beta abrogates TCR-mediated signaling by upregulating tyrosine phosphatases in T cells.

TGF-beta is known to inhibit many of the immune cell functions including T cell proliferation and IL-2 production. The mechanism of such TGF-beta-mediated inhibition of T cell functions is poorly understood. The present study examined the effects of TGF-beta on the activation of protein tyrosine kinases (PTK) P56lck, P59fyn, and Zap-70, and protein tyrosine phosphatases (PTP) SHP-1 and SHP-2. A balance between the actions of PTK and PTP is critical for appropriate T cell activation. These studies were carried out using nylon wool-purified splenic T cells from healthy Sprague-Dawley rats. Results from these studies showed that incubation of T cells with TGF-beta inhibited the activation of P56lck, P59fyn and Zap-70. The decrease in these three protein tyrosine kinases was accompanied by an increase in the activation of the protein tyrosine phosphatase SHP-1. There was no change in the phosphorylation of SHP-2 with and without pretreatment of T cells with TGF-beta. The decrease in P56lck, P59fyn kinase activity, and Zap-70 phosphorylation was prevented when T cells were stimulated with anti-CD3 in the presence of pervanadate, an inhibitor of PTP. These results suggested that TGF-beta-mediated inhibition of P56lck, P59fyn, and Zap-70 is likely due to an up-regulation of protein tyrosine phosphatases such as SHP-1.

Ig-like transcript 2 (ILT2)/leukocyte Ig-like receptor 1 (LIR1) inhibits TCR signaling and actin cytoskeleton reorganization.

Ig-like transcript 2 (ILT2)/leukocyte Ig-like receptor 1 (LIR1) is a receptor, specific for MHC class I molecules, that inhibits lymphoid and myeloid cells. Here, we analyzed the molecular and cellular mechanisms by which ILT2 modulates T cell activation in primary CTLs and transfected T cell lines. We found that cross-linking with the TCR and the activity of Src tyrosine kinase p56(lck) were required for phosphorylation of ILT2 and subsequent recruitment of Src homology protein 1. In contrast, ILT2 triggering resulted in reduced phosphorylation of TCRzeta and linker for activation of T cells, which led to reduced TCRzeta-ZAP70 complex formation, as well as extracellular signal-related kinase 1 and 2 activation. Furthermore, ILT2 inhibited both superantigen and anti-TCR Ab-induced rearrangement of the actin cytoskeleton. The inhibitory effect mediated by ILT2 is probably concentrated at the APC-T cell interface because both TCR and ILT2 were strongly polarized toward the APC upon engagement by their specific ligands. Thus, ILT2 inhibits both signaling and cellular events involved in the activation of T cells.

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.

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.

Surfactant modulates intracellular signaling of the adhesion receptor L-selectin.

Intraalveolar leukocyte accumulation is one of the hallmarks during respiratory distress. In the intraalveolar space, leukocyte activation is mediated by pathogens, cytokines, and different ligands binding to adhesion molecules. Leukocyte stimulation via the adhesion molecule L-selectin is specifically induced by ligands expressed on leukocytes, platelets, endothelial cells, or lipopolysaccharide. Recently, we have demonstrated that leukocyte activation by L-selectin transmits several intracellular signaling cascades resulting in capping and cytoskeletal changes, the activation of kinases and neutral sphingomyelinase, the recruitment of adaptor proteins to the cell membrane, the activation of the small G-proteins Ras and Rac, and the release of oxygen. In the present study, we examined the effects of surfactant on L-selectin-induced signal transduction in leukocytes. Using fluorescence microscopy, we provide evidence that preincubation of leukocytes with surfactant significantly inhibits receptor capping; 28+/-7% of cells show capping after L-selectin stimulation versus 8+/-5% and 3+/-1% of cells after preincubation with Exosurf and Curosurf, respectively (p < 0.05). The activity of the neutral sphingomyelinase in cell lysates is also modulated by surfactant. In addition, we show that the activation of the tyrosine kinase p56lck is diminished by approximately 50% after surfactant treatment. This results in inhibition in tyrosine phosphorylation of certain intracellular proteins. The interaction of the L-selectin molecule with its antibody was not influenced by surfactant as shown by flow cytometry. Surfactant inhibits intracellular signaling events of the L-selectin receptor in leukocytes and might therefore contribute to the modulatory effects of surfactant on immune function.

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Transforming growth factor β-mediated inhibition of src kinases is regulated by SHP-1

Abstract Background Transforming growth factor (TGF) β has been shown to suppress T cell effector functions. Although the mechanisms of such T cell suppression are not known, a disuption of the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) has been implicated. The present study investigated the roles of the src-related PTKs, P59fyn and P56lck, and the PTPs SHP-1 and SHP-2 in TGF-β-mediated T cell suppression. Methods Splenic T cells from rats were pretreated with TGF-β 25 ng ml−1 and subsequently stimulated with either anti-CD3 or anti-CD3 plus pervanadate (a PTP inhibitor). After incubation with specific antibodies and immunoprecipitation, the kinase activity of the PTKs was assessed by in vitro kinase assay. Phosphorylation of the PTPs was assessed using antiphosphotyrosine antibodies and immunoblotting. Results Incubation of T cells with TGF-β resulted in decreased activity of P59fyn and P56lck. Although TGF-β treatment resulted in a concomitant increase in SHP-1 activity, no change in SHP-2 activity was observed. When T cells were stimulated with anti-CD3 in the presence of pervanadate, the TGF-β-mediated decrease in P59fyn and P56lck activity was completely prevented. Conclusion These findings show that TGF-β treatment of T cells results in suppression of src kinases, and suggests that such inhibition of kinase activity is mediated by upregulation of SHP-1.

Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases

Engagement of interleukin-2 (IL-2) mediates the heterodimeridation of the common beta chain (β c) and common gamma chain (γ c) of the IL-2 receptor (IL-2R). This is sufficient and necessary for receptor activation and signal transduction. It is generally held that the IL-2R is activated by the trans-activity of the protein tyrosine kinases (PTKs) Jak1 and Jak3 associated with β c and γ c respectively. Transduction of proliferative signals requires Jak3 activity. A Jak3 independent signalling pathway involving p56 lck , generating anti-apoptotic signals, can be observed and requires the PROX domain of γ c. p56 lck can be activated by dephosphorylation of an inhibitory carboxyl terminal phosphorylated tyrosine residue (Y505). We propose that this is mediated by a PROX domain associated protein tyrosine phosphatase (PTP). Activation of p56 lck alone is insufficient for transduction of proliferative signals and thus works in concert with Jak3 mediated receptor activation. This indicates that both γ c domains are vital for signal transduction.

Protein Tyrosine Kinase p56 lck Is Required for Ceramide-induced but Not Tumor Necrosis Factor-induced Activation of NF-κB, AP-1, JNK, and Apoptosis

Ceramide has been implicated as an intermediate in the signal transduction of several cytokines including tumor necrosis factor (TNF). Both ceramide and TNF activate a wide variety of cellular responses, including NF-kappaB, AP-1, JNK, and apoptosis. Whether ceramide 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 p56(lck) in ceramide- and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, isogeneic Lck-deficient T cells. Treatment with ceramide activated NF-kappaB, degraded IkappaBalpha, and induced NF-kappaB-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56(lck) kinase. These effects were specific to ceramide, as activation of NF-kappaB by phorbol 12-myristate 13-acetate, lipopolysaccharide, H(2)O(2), and TNF was minimally affected. p56(lck) was also found to be required for ceramide-induced but not TNF-induced AP-1 activation. Similarly, ceramide activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. Ceramide also induced cytotoxicity and activated caspases and reactive oxygen intermediates in Jurkat cells but not in JCaM1 cells. Ceramide activated p56(lck) activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56(lck) tyrosine kinase reversed the ceramide-induced NF-kappaB activation and cytotoxicity. Overall our results demonstrate that p56(lck) plays a critical role in the activation of NF-kappaB, AP-1, JNK, and apoptosis by ceramide but has minimal or no role in activation of these responses by TNF.