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.