Regulation of Protein Kinase C Theta by Phosphorylation

University Of California, San Diego, La Jolla CA

Investigators

Abstract

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.