ABSTRACT The importance of PKC in TCR signals controlling T cell activation and differentiation is well established. The selective requirement of PKC in allergic and autoimmune diseases, but not in antiviral responses, highlights it as a promising drug target. Realization of this potential requires a clear understanding of the mechanism(s) that regulate the unique, non-redundant functions and highly selective immunological synapse (IS) localization of PKC in T cells, which remain unknown. Our work during the last funding period revealed that within the large PKC family, PKC represents a unique paradigm with respect to the mechanisms that regulate these events and, ultimately, lead to the activation of critical transcription factors, which determine T cell fate. We propose to apply a multi-disciplinary approach in order to address the following unresolved questions: Aim 1) Positive regulation of PKC activation and localization. Using biochemical, genetic, imaging and biophysical approaches, we will address the hypothesis that the localization and activation of PKC in T cells are positively regulated by specialized mechanisms and plasma membrane interactions owing to the unique properties of its regulatory C1 and V3 domains. We will conduct a detailed structure- function analysis of the C1A, C1B and V3 domains of PKC, their phosphorylation, and their interaction with binding partners. Aim 2) Autoinhibition by the PKC C2 domain. Our studies indicate that the PKC N- terminal C2 domain negatively regulates its function, and that autoinhibition is relieved by TCR-induced phosphorylation of Tyr-90, and, furthermore, that C2 itself is a pTyr- and PIP2-binding domain. We will investigate the mechanisms through which autoinhibition is effected, and characterize C2-binding ligands and their functional significance. We will pursue Aims 1 and 2 as a collaborative consortium with Dr. Wonhwa Cho (U. Illinois), who will provide his expertise in biophysical and two-photon imaging approaches to further elucidate the specialized mechanisms that regulate PKC. Aim 3) Regulation of Ca2+/NFAT signaling by PKC. PKC-/- T cells display a Ca2+/NFAT signaling defect, but the mechanism linking PKC to Ca2+ signaling is unknown. Based on our recent findings, and given the important role of Ca2+ signaling in determining the balance between productive T cell activation and anergy/apoptosis, we will characterize this pathway. Specifically, we will address the hypothesis that PKC mediates a positive feedback loop that promotes PLC1 activation and, hence, Ca2+/NFAT signaling, via its functional and/or physical association with Tec family tyrosine kinase(s). Our studies will elucidate novel mechanisms that control the unique membrane localization and function of PKC in T cells. The new information will likely provide a rational basis for using PKC as a drug target in autoimmune and allergic diseases, and in T cell malignancies.