Abstract 295: PKCε phosphorylates and modulates the cell membrane translocation of RhoA

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Abstract Accumulating evidence has clearly defined the importance of PKCε and small Rho GTPases in tumor metastasis. Previous studies in our laboratory have demonstrated that PKCε signals through RhoA to modulate tumor cell invasion and motility in head and neck squamous cell carcinoma. In this study, we further delineated the link between PKCε and RhoA. Recombinant PKCε directly phosphorylated recombinant RhoA using an in vitro kinase assay. Phosphopeptide mapping with LC-MSE identified T127 and S188 as confident phosphorylation sites and T19 and S26 as tentative phosphorylation sites on RhoA. Interestingly, recombinant PKCε was able to bind to recombinant RhoA in the absence of PKC activators and ATP. This observation demonstrates that the binding interaction between PKCε and RhoA does not require an active PKCε conformation and thus, the interaction between these proteins may be more complex than a typical substrate-kinase event. N-terminus fluorophore-tagged PKCε (CherryFP or CFP) and RhoA (GFP or YFP) were co-expressed in HEK293 cells to examine the interaction between these two proteins in live-cells. Live-cell fluorescence microscopy followed by FRET stoichiometric analysis confirmed the interaction between PKCε and RhoA in the cytoplasm of non-stimulated cells. Stimulation of the cells with PMA to activate PKCε resulted in the translocation of PKCε and RhoA from the cytoplasm to the cell membrane and increased the binding of PKCε to RhoA at the cell membrane by three-fold as measured by FRET. Taken together, our results demonstrate, for the first time, that PKCε phosphorylates, co-localizes, and regulates the cell membrane translocation of RhoA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 295.