Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

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.

ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-θ V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-θ-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-θ from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-θ, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases.

Triggering of the antigen-specific T cell receptor-CD3 complex (TCR-CD3) stimulates a rapid phospholipase C-mediated hydrolysis of inositol phospholipids, resulting in the production of second messengers and in T cell activation and proliferation. The role of tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited TPK activity and tyrosine phosphorylation of the TCR zeta subunit, but not phospholipase C activity, genistein prevented TCR-CD3-mediated phospholipase C activation, interleukin-2 receptor expression, and T cell proliferation. These findings indicate that tyrosine phosphorylation is an early and critical event that most likely precedes, and is a prerequisite for, inositol phospholipid breakdown during receptor-mediated T cell activation.

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Protein kinase C-(PKC ) is a Ca 2؉ -independent PKC isoform that is selectively expressed in T lymphocytes (and muscle), and is thought to play an important role in T cell receptor-induced activation. To gain a better understanding of the function and regulation of PKC , we have employed the yeast two-hybrid system to identify PKC -interacting proteins. We report the isolation and characterization of a cDNA encoding a novel 335-amino acid (37.5-kDa) PKC -interacting protein termed PICOT (for PKC-interacting cousin of thioredoxin). PICOT is expressed in various tissues, including in T cells, where it colocalizes with PKC . PICOT displays an N-terminal thioredoxin homology domain, which is required for the interaction with PKC. Comparison of the unique C-terminal region of PICOT with expressed sequence tag data bases revealed two tandem repeats of a novel domain that is highly conserved from plants to mammals. Transient overexpression of full-length PICOT (but not its N-or C-terminal fragments) in T cells inhibited the activation of c-Jun N-terminal kinase (but not extracellular signal-regulated kinase), and the transcription factors AP-1 or NF-B. These findings suggest that PICOT and its evolutionary conserved homologues may interact with PKC-related kinases in multiple organisms and, second, that it plays a role in regulating the function of the thioredoxin system.Members of the protein kinase C (PKC) 1 family of intracellular serine/threonine kinases play critical roles in the regulation of cellular differentiation and proliferation in many cell types and in response to diverse stimuli, including hormones, neurotransmitters, and growth factors (1-3). The PKC family consists of 11 known mammalian members that are expressed in a wide variety of tissues and cell types. Based on sequence similarities, domain structures, and cofactor requirements, PKC isoenzymes can be grouped into three subfamilies. 1) The Ca 2ϩ -dependent conventional enzymes, consisting of PKC-␣, -␤I, -␤II, and -␥, contain three conserved domains, namely the diacylglycerol/phorbol ester binding C1 domain, which contains two repeats of a cysteine-rich zinc finger, the phospholipid-and calcium-binding C2 domain, and the catalytic C3 and C4 domains.2) The Ca 2ϩ -independent enzymes (PKC-␦, -⑀, -, -and -) are termed novel PKCs. The C2-like N-terminal domain of these enzymes can bind acidic phospholipids but not Ca 2ϩ . 3) A third PKC subfamily, termed atypical PKCs, includes PKCand -/ that possess a single cysteine-rich domain, lacking the ability to bind phospholipids or phorbol esters. PKC activity is regulated by defined cofactors that interact with specific regions of the regulatory domain as well as transphosphorylation by serine/threonine kinases and autophosphorylation. The activation is accompanied by a conformational change that releases the basic pseudosubstrate region from the catalytic cleft of the kinase domain. In addition, interaction with specific proteins, termed receptors for activated PKC, that function as selective scaffolds for a...

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