%0 Journal Article
%T PKC¦Å Phosphorylates and Mediates the Cell Membrane Localization of RhoA
%A Tizhi Su
%A Samuel Straight
%A Liwei Bao
%A Xiujie Xie
%A Caryn L. Lehner
%A Greg S. Cavey
%A Theodoros N. Teknos
%A Quintin Pan
%J ISRN Oncology
%D 2013
%R 10.1155/2013/329063
%X Protein kinase C¦Å (PKC¦Å) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKC¦Å and RhoA was defined. Phosphopeptide mapping revealed that PKC¦Å phosphorylates RhoA at T127 and S188. Recombinant PKC¦Å bound to recombinant RhoA in the absence of ATP indicating that the association between PKC¦Å and RhoA does not require an active ATP-docked PKC¦Å conformation. Activation of PKC¦Å resulted in a dramatic coordinated translocation of PKC¦Å and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKC¦Å 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 PKC¦Å-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKC¦Å is able to recruit RhoA to the cell membrane indicating that the association between PKC¦Å and RhoA is proximal to the active catalytic site and perhaps independent of a PKC¦Å-RhoA phosphorylation event. This work demonstrates, for the first time, that PKC¦Å phosphorylates and modulates the cell membrane translocation of RhoA. 1. Introduction Numerous publications have clearly defined the role of PKC¦Å as transforming oncogene in fibroblasts and epithelial cells. overexpression of PKC¦Å 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 PKC¦Å overexpression were invasive and displayed a polarized morphology with extended long cellular membrane protrusions [3]. Epidermis-specific PKC¦Å transgenic mice developed highly malignant and metastatic squamous cell carcinomas in response to 12-O-tetradecanoylphorbol-13-acetate stimulation [4]. PKC¦Å was demonstrated to transform androgen-dependent LNCaP prostate cancer cells into an androgen-independent variant [5]. Moreover, transgenic mice with selective overexpression of PKC¦Å in the prostate epithelium developed prostate hyperplasia and prostate intraepithelial neoplasia [6]. Our laboratory demonstrated that inhibition of PKC¦Å in MDA-MB231 cells, a highly metastatic breast cancer cell line with elevated PKC¦Å levels, was sufficient to dramatically decrease in vivo tumor growth and reduce the incidence of lung metastasis [7].
%U http://www.hindawi.com/journals/isrn.oncology/2013/329063/