Protein Kinase C-binding partners & phosphorylation
Protein Kinase C (PKC) comprises a family of phospholipid-dependent Ser/Thr kinases, implicated in a broad array of cellular processes. PKC activity is subject to a complex network of regulatory inputs, including co-factor binding, phosphorylation and protein-protein interaction. In addition, the chronic activation of PKC frequently leads to its down-regulation this process may be intrinsic to the tumour promoting activity of the phorbol esters. The aim of this work was to investigate the regulation of the novel PKC-e isoform by binding partners and phosphorylation, with a particular focus on the process of agonist-induced degradation. A yeast 2-hybrid screen was performed using a PKC-e bait and two novel binding partners were identified, both with associations to the ubiquitin/proteasome system VHL Binding Protein 1 (VBP1) and F-box WD40 protein 7 (Fbw7). Interactions were verified in mammalian cells and mapped to the catalytic domain of PKC-e. Extensive studies revealed that neither partner influenced the process of PKC-e down-regulation. However, Fbw7a was demonstrated to represent an in vitro PKC substrate. The site of phosphorylation was mapped to Ser-18 and, using phospho-specific antibodies, was shown to be phosphorylated in the cell. PKC-e activity is required for its agonist-induced degradation. Studies were therefore undertaken to investigate autophosphorylation, which may be implicated in this process. Serine residues 234, 316 and 368 were identified as novel PKC-e autophosphorylation sites. Using phospho- specific antibodies, all three sites were shown to be occupied in response to PKC-e activation. Phosphorylation at these sites was found not to influence agonist-induced PKC-e down-regulation. However, a critical role was established for phosphorylated Ser-368, in the recruitment of the PKC-e binding partner, 14-3-3(3. Together these findings provide insight into the mechanisms controlling PKC-e activity and demonstrate a relationship between regulation through phosphorylation and protein-protein interaction.