Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

The novel protein kinase C (PKC) isoform, PKC theta, is selectively expressed in T lymphocytes and is a sine qua non for T cell antigen receptor (TCR)-triggered activation of mature T cells. Productive engagement of T cells by antigen-presenting cells (APCs) results in recruitment of PKC theta to the T cell-APC contact area--the immunological synapse--where it interacts with several signaling molecules to induce activation signals essential for productive T cell activation and IL-2 production. The transcription factors NF-kappa B and AP-1 are the primary physiological targets of PKC theta, and efficient activation of these transcription factors by PKC theta requires integration of TCR and CD28 costimulatory signals. PKC theta cooperates with the protein Ser/Thr phosphatase, calcineurin, in transducing signals leading to activation of JNK, NFAT, and the IL-2 gene. PKC theta also promotes T cell cycle progression and regulates programmed T cell death. The exact mode of regulation and immediate downstream substrates of PKC theta are still largely unknown. Identification of these molecules and determination of their mode of operation with respect to the function of PKC theta will provide essential information on the mechanism of T cell activation. The selective expression of PKC theta in T cells and its essential role in mature T cell activation establish it as an attractive drug target for immunosuppression in transplantation and autoimmune diseases.

Molecular cloning and biochemical studies identified protein kinase C (PKC) enzymes as members of a distinct family of serine/threonine protein kinases, playing critical roles in the regulation of cellular differentiation and proliferation of diverse cell types (reviewed in reference 36). In an attempt to find PKC isoforms that are involved in growth control and/or activation of T lymphocytes, we have identified PKC-(5), whose human gene locus was recently mapped to chromosome 10p15 (15). PKC-is characterized by a unique tissue distribution, i.e., in skeletal muscle, lymphoid organs, and hematopoietic cell lines, particularly T cells (4,5,10,34,39,53), and by isoenzyme-specific activation requirements and substrate preferences in vitro (4). PKC-undergoes cytosol-to-membrane translocation in T cells stimulated with phorbol esters (4), implying that this isoform is likely to be involved in T-cell activation pathways. The unique expression and functional properties of PKC-suggest that it may play a specialized role in T-cell signaling pathways.T-cell activation results in the expression of interleukin-2 (IL-2), an autocrine growth factor that is a critical stimulus for the growth and differentiation of B and T lymphocytes. Pharmacological and biochemical studies indicate that activation of two major signaling pathways, one of which can be triggered by phorbol esters (such as phorbol 12-myristate 13-acetate [PMA]) and the other of which can be triggered by Ca 2ϩ ionophores, is required for induction of IL-2 (reviewed in reference 51). A substantial amount of work over the past several years has shown the requirement of cooperative interactions of several transcription factors, including AP-1, NF-B, NF-AT, and NF-IL2A (Oct-1), with the minimal inducible promoter/enhancer region of the IL-2 gene (11). Several lines of evidence point to AP-1 as a critical transcription factor for IL-2 regulation. AP-1 is a dimer of different members of the Fos (c-Fos, FosB, Fra-1, Fra-2, and FosB2) and Jun (c-Jun, JunB, and JunD) family of proteins (1). AP-1 thereby interacts with the IL-2 regulatory region directly (25,26,33,47) and also indirectly as a component of NF-AT and NF-IL2 (37, 50). AP-1 activity is regulated by de novo synthesis of Jun and Fos proteins, as well as by posttranslational modifications such as phosphorylation and dephosphorylation (1,8,9,30,43,48). Two potential AP-1-binding sites have been identified in the mouse and human IL-2 enhancer region at Ϫ150 bp (proximal AP-1) and Ϫ180 bp (distal AP-1). These elements show sequence similarity to the consensus AP-1 enhancer sequence and have been studied by deletional, mutational, and gel shift analyses (14,18,25,40). Most of these data support an important role for AP-1 in IL-2 transcription, especially as a result of the interaction with the proximal enhancer site (25).PKC has been implicated in the activation of AP-1 in T lymphocytes, as demonstrated by studies involving PKC-specific pharmacological inhibitors (24, 28) or PKC down-regulation by chronic phorbol este...

Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

Vav and PKCtheta play an early and important role in the TCR/CD28-induced stimulation of MAP kinases and activation of the IL-2 gene. Vav is also essential for actin cytoskeleton reorganization and TCR capping. Here, we report that PKCtheta function was selectively required in a Vav signaling pathway that mediates the TCR/CD28-induced activation of JNK and the IL-2 gene and the upregulation of CD69 expression. Vav also promoted PKCtheta translocation from the cytosol to the membrane and cytoskeleton and induced its enzymatic activation in a CD3/CD28-initiated pathway that was dependent on Rac and on actin cytoskeleton reorganization. These findings reveal that the Vav/Rac pathway promotes the recruitment of PKCtheta to the T cell synapse and its activation, essential processes for T cell activation and IL-2 production.

T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp561ck that is encoded by the kk protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it -plays an important role in T-cell development and activation. We have studied the regulation of pp56kk and found that this kinase can be rapidly activated by an endogenous mechanism present in T-lymphocyte membranes. This activation was sensitive to sodium orthovanadate and O-phosphotyrosine, consistent with the involvement of a phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphatase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) in pp56&k activation. Based on a recent report demonstrating that CD45, the leukocyte common antigen, is a membrane-bound PTPase, we analyzed its role in pp56Ick activation. CD45 was found to be the major (>90%) PTPase in membranes of the murine T-lymphoma line BW5147. Moreover, activation of pp56kk was undetectable in a mutant BW5147 line lacking CD45 expression (and the associated PTPase activity). In contrast, activation of pp56kk was readily detected in the wild-type lymphoma line. More important, when immunoprecipitated CD45 was added to pp56~kk, the TPK activity of the latter increased >2-fold within minutes. This effect of CD45 was completely blocked by sodium orthovanadate. These rmdings indicate an important role for the CD45 PTPase in pp56'k activation. This role could be mediated by direct dephosphorylation of a regulatory tyrosine residue in ppS6kk.

Protein kinase C-theta (PKC-theta) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide-major histocompatibility complex (MHC) induces translocation of PKC-theta to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-theta regulatory domain and required Lck but not ZAP-70. In addition, PKC-theta was associated with Lck in the rafts. An isolated PKC-straight theta catalytic fragment did not partition into rafts or activate the transcription factor NF-kappa B, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-theta to rafts, which localize to the T cell synapse; this PKC-theta translocation is important for its function.

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1 − /−) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.