UC San Diego

Post-translational modifications govern almost every aspect of cell biology. The addition and removal of functional groups on proteins lead to complex signal transduction pathways. This dissertation focuses on elucidating regulatory mechanisms of two enzymes whose activity and signaling are interlocked in the serine/threonine phosphorylation realm: Protein Kinase C (PKC) and Protein Phosphatase 2A (PP2A). The conventional class of PKC isozymes (cPKC) is transiently activated by Ca2+ and diacylglycerol (DG) to allow phosphorylation of downstream substrates. Importantly, in the absence of these second messengers, PKC adopts an autoinhibited conformation where it has low activity and is quite stable in the cell. We first address the need for a structure of autoinhibited PKC by building a model of this conformation to generate testable hypotheses as to important residues that are involved in interdomain contacts. The second part of this dissertation utilizes our model to assess the effect of two disease-associated mutations at the same position (R42H from cancer and R42P from spinocerebellar ataxia) that are both predicted to disrupt an interaction that contributes to autoinhibition. Mutations that disrupt autoinhibitory contacts typically result in quality-control degradation. We use biochemical, cellular, and structural approaches to demonstrate that these mutants have higher basal activity and are in an open conformation as our model predicts. Whereas histidine at this position allows canonical downregulation both basally and in response to activators, R42P bypasses the ubiquitination associated with activator-induced degradation. These studies highlight the fine-tuning of PKC levels in the cell and provide examples of how two diseases manipulate this regulation to exert opposing effects on PKC. Finally, we examined potential mechanisms by which PP2A regulates PKC dephosphorylation and levels. We show that a novel motif identified on PKC may contribute to its rate of dephosphorylation and that the PP2A regulatory subunit B56δ can regulate PKC expression through regions of B56δ that are not conserved within the B56 family. Together, this work illustrates multiple mechanisms of PKC regulation with the goal of understanding how to properly target PKC isozymes in disease.