Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA. 1. Introduction Numerous publications have clearly defined the role of PKCε as transforming oncogene in fibroblasts and epithelial cells. overexpression of PKCε in NIH3T3 fibroblasts and FRC/TEX CL D rat colonic epithelial cells was shown to increase cell proliferation, enhance anchorage-independent colony formation, and induce a highly tumorigenic in vivo phenotype with tumor incidence of 100% [1, 2]. In addition, NIH3T3 fibroblasts with PKCε overexpression were invasive and displayed a polarized morphology with extended long cellular membrane protrusions [3]. Epidermis-specific PKCε transgenic mice developed highly malignant and metastatic squamous cell carcinomas in response to 12-O-tetradecanoylphorbol-13-acetate stimulation [4]. PKCε was demonstrated to transform androgen-dependent LNCaP prostate cancer cells into an androgen-independent variant [5]. Moreover, transgenic mice with selective overexpression of PKCε in the prostate epithelium developed prostate hyperplasia and prostate intraepithelial neoplasia [6]. Our laboratory demonstrated that inhibition of PKCε in MDA-MB231 cells, a highly metastatic breast cancer cell line with elevated PKCε levels, was sufficient to dramatically decrease in vivo tumor growth and reduce the incidence of lung metastasis [7].
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