Our research focuses on the molecular basis of signal transduction cascades initiated following triggering of the antigen-specific T cell receptor (TCR)/CD3 complex and costimulatory receptors. T cells play important effector and regulatory functions in the immune system, and aberrations in their activation can lead to immunological diseases. Therefore, understanding of the biochemical events occurring during T cell activation is important in order to rationally devise pharmacological treatments designed to modulate the T cell response in favor of the host. Signaling cascades in T cells involve enzymes as well as adaptor proteins that mediate protein-protein interactions essential for the formation of multi-subunit signaling complexes. Some of these signaling elements are specifically expressed in T cells or in hematopoietic cells in general and, thus, constitute potential tissue-selective drug development targets. We have focused our research efforts on several hematopoietic (or T cell)-specific signaling proteins, including PKCθ, SLAT and LAT.

1. PKCθ. PKC enzymes play critical roles in the differentiation and proliferation of many cell types, including T cells, and in the response to diverse stimuli. Little is known, however, about the substrate specificity and role of individual PKC isoforms in distinct activation and developmental events in T cells. In 1993, we cloned and identified a novel PKC isoform, PKCθ. It is characterized by a unique tissue distribution, i.e., in skeletal muscle, lymphoid organs, and hematopoietic cell lines, in particular T cells. PKCθ plays an important role in T cell activation: It selectively activates the transcription factors AP-1 and NF-κB, and integrates TCR and CD28 signals, which lead to activation of the CD28 response element (RE) in the interleukin-2 (IL-2) gene promoter. PKCθ also colocalizes with the TCR to the T cell synapse that forms at the contact area between antigen-specific T cells and antigen-presenting cells. Consistent with its important role in T cell activation, mature T cells from PKCθ-deficient mice display severely reduced proliferation and IL-2 production, along with impaired activation of NF-κB, AP-1 and NFAT.

Other studies in the past year identified SPAK, a Ste20-related mitogen-activated protein kinase kinase kinase (MAP3K), as a PKCθ-interacting kinase. The COOH-terminal 99 amino acids of SPAK mediated this interaction. Recombinant SPAK was directly phosphorylated by PKCθ, and mutagenesis identified Ser-311 and Ser-325 in the catalytic domain of SPAK as the major phosphorylation sites. Like PKCθ, TCR/CD28 costimulation enhanced the catalytic activity of SPAK. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCθ-deficient T cells. Limiting amounts of transfected wild type SPAK synergized with a constitutively active PKCθ mutant to activate AP-1, but not NF-κB. In addition, a kinase-inactive mutant of SPAK suppressed the TCR/CD28-induced activation of AP-1, but not NF-κB. These results define SPAK as a direct substrate and immediate target of PKCθ in a TCR/CD28-induced signaling pathway leading selectively to AP-1 activation.

Studies in the past year also addressed the potential role of PKCθ as a T cell survival signal. CD8+ T cells are crucial for host defense against invading pathogens and malignancies. However, relatively little is known about the intracellular signaling events that control the genetic program of their activation and differentiation into effector and memory cells. We used CD8+ T cells from TCR-transgenic (Tg) mice crossed to PKCθ-deficient mice to study the role of PKCθ in these processes. We found that PKCθ is not required for antigen-induced CD8+ T cell proliferation, but is critical for T cell survival and differentiation into functional, cytokine-producing CTLs. Antigen-stimulated PKCθ-/- T cells underwent accelerated apoptosis, which was reversed by retroviral-mediated expression of Bcl-2 or Bcl-xL, and displayed markedly reduced activation of Erk and JNK kinases. Our findings indicate that PKCθ is a critical survival factor required for CD8+ T cell survival and CTL differentiation.

Current studies address several major questions: First, we are interested in elucidating the mechanism that selectively mediates the translocation of PKCθ to the IS and/or membrane lipid rafts, including the characterization of a putative scaffold protein that links PKCθ to the cytoskeleton and the synapse. Second, studies continue to identify intermediate components leading from PKCθ to activation of NF-κB, AP-1 and the CD28 RE. Lastly, we investigate how Lck-induced tyrosine phosphorylation of PKCθ in its regulatory domain modulates its localization and function.

2. SLAT, a novel Th2-expressed adapter protein. Th2-derived cytokines play a central role in the pathophysiology of allergy and asthma. However, very little is known about proximal, TCR-coupled signaling events that regulate the Th1/Th2 differentiation process. Therefore, we initiated studies to elucidate the TCR-proximal signaling pathways that operate in Th2 cells, based on the notion that Th2-specific signaling elements represent potential targets for development of drugs that will selectively interfere with Th2 function. We previously reported the isolation and initial characterization of a novel protein, termed SWAP-70-Like Adapter of T cells (SLAT), which is selectively expressed at high levels in thymocytes and in differentiated Th2 cells. TCR signaling induces association of SLAT with ZAP-70 tyrosine kinase in Th2 cells, and this results in inhibition of ZAP-70 recruitment to the TCR complex and its activation. We hypothesize that SLAT favors the development and expansion of Th2 cells by selectively inhibiting the function of ZAP-70 in T cells. In addition, we found that the C-terminal region of SLAT is related to Dbl-homology (DH) domains, and it activates Rac1 and Cdc42, but not RhoA. We are currently studying whether SLAT also possesses a regulated GEF activity in T cells. In addition, we continued the backcrossing of SLAT-deficient mice onto the B6 and Balb/c backgrounds and begun to characterize SLAT-/- mice on a mixed background. Preliminary analysis indicates that SLAT-/- T cells cultured under Th2-inducing conditions display a bias towards Th1 development. Stimulation of SLAT-/- purified T cells with cross-linked anti-CD3 plus -CD4 antibodies revealed an increase in the basal phosphotyrosine content of SLAT-/- T cells and a ~2-fold increase in the inducible tyrosine phosphorylation of ZAP-70 on Tyr-319, a marker of its catalytic activity. SLAT-/- splenic CD4+ or CD8+ T cells also display a marked increase in the proportion of CD69high, CD44high and CD62Llow cells. These changes suggest that the SLAT mutation leads to increased proportion of mature/activated/memory T cells in the periphery, and are generally consistent with our hypothesis that SLAT is a negative regulator of TCR/costimulatory receptor signaling.