PROJECT SUMMARY The overall vision of the proposed research is to gain a comprehensive understanding of how phosphorylation regulates the activity and function of a key regulator of immune signaling, the Ser/Thr protein kinase C (PKC) Theta (). This kinase is selectively expressed in hematopoietic cells where it transduces signals resulting in T cell and platelet activation.1,2 Its dysregulation is associated with a variety of pathophysiological conditions including blood cancers,3,4 inflammatory diseases,5 thrombosis,6 and hemostasis.7 Despite this, the regulation and function of PKC remains largely unknown and necessitates further investigation. Phosphorylation of PKC plays an essential role in regulating its maturation, catalytic activity, and subcellular localization,8 all of which are crucial for PKC function in T cells and platelets. This proposal aims to understand how phosphorylation at known conserved priming sites (activation loop, turn motif, hydrophobic motif),9 a bioinformatically-identified new potential priming site (Ser662), and an uncharacterized activation-induced site (Ser685), regulate the maturation, activity, and/or localization of PKC. Unbiased phosphoproteomics approaches have revealed that phosphorylation of Ser685 significantly increases in T cells10 and platelets11 in response to stimulation, however its function has not yet been determined due, in part, to a lack of available research tools. This site, and Ser662 are positioned on a key regulatory segment, the C-tail, and are evolutionarily conserved. The central hypothesis driving this proposal is that phosphorylation of S662 is involved in the maturation of PKC and that activation- induced phosphorylation of S685 promotes the re-autoinhibition of activated PKC to facilitate signal termination. To this end, I will investigate how nonphosphorylatable or phosphomimetic mutations at these residues impact PKC biochemical properties, cellular activity, subcellular localization, and downstream signaling (Aim 1). Additionally, I will examine the phosphoproteome of PKC in Jurkat cells and platelets and examine how phosphorylation at the agonist-induced site, Ser685, affects downstream signaling. I will also aim to identify the kinase(s) regulating PKC Ser685 phosphorylation using various phosphoproteomics approaches (Aim 2). These key studies will elucidate the functional impact of PKC phosphorylation at critical residues and how this influences downstream signaling. Moreover, this proposal will elucidate substrates and signaling networks regulated by PKC. Uncovering the regulation and function of PKC, a key regulator of T cells and platelets, is crucial to understanding T cell and platelet signaling in both normal and disease states.