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ISRN Oncology
Volume 2013 (2013), Article ID 329063, 9 pages
http://dx.doi.org/10.1155/2013/329063
Research Article

PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

1Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA
2Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
3Center for Live-Cell Imaging, Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA
4Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48108, USA
5Van Andel Research Institute, Grand Rapids, MI 49503, USA
6Southwest Michigan Innovation Center, Kalamazoo, MI 49008, USA

Received 9 July 2013; Accepted 18 August 2013

Academic Editors: Y.-y. Liu and Y. Yu

Copyright © 2013 Tizhi Su et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.