Protein Kinase C-δ and Protein Kinase C-ε Cooperatively Enhance Epithelial Cell Spreading via Transactivation of Epidermal Growth Factor Receptor and Actin-Dependent Phosphorylation of Focal Adhesion-Associated Proteins

Regulation of cell spreading and migration is a critical process for epithelial wound healing and tissue morphogenesis. Formation of lamellipodia and disassembly of focal adhesion during cell migration are two important events that require coordinated actin rearrangement, yet the signaling pathways that lead to such modulation are not fully described. Tyrosine phosphorylation of the focal adhesionassociated proteins such as Src, focal adhesion kinase (FAK) and paxillin has been linked to increased cell spreading, though the underlying mechanism is not well understood. Previously we showed that protein kinase C (PKC) regulates actin remodeling events in T84 intestinal epithelial cells but little is known regarding the role of PKC during cellular spreading. In the current study, we used two models, 1) the subconfluent T84 cell spreading model and 2) in vitro wound healing model, to investigate the involvement of the PKC signaling pathway in regulation of epithelial cell motility. In subconfluent cells, phorbol 12-myristate 13-acetate (PMA), which activates the conventional cPKC isoform PKCα and the novel nPKC isoforms PKCδ and PKCε of T84 cells, dramatically accelerated T84 cell spreading with concurrent increase in tyrosine phosphorylation of Src, FAK and paxillin. These effects were completely abolished by the PKC inhibitor Gö6850 and PKCδspecific inhibitor rottlerin. In contrast, the cPKC-selective inhibitor Gö6976 had no effect. Furthermore, siRNA-mediated down-regulation of PKCδ and PKCε, but not PKCα, significantly attenuated cell spreading, indicating that two novel isoforms PKCδ and PKCε are the critical elements participating in regulation of epithelial cell spreading. Tyrosine phosphorylation of FAK and paxillin by PMA was mediated by ligand-dependent transactivation of epidermal growth factor receptor (EGFR) which was selectively phosphorylated at Tyrosine-992 by PMA stimulation. Incubation with an EGFR neutralizing antibody or EGFR kinase inhibitor tyrphostin AG1478 completely abolished PMA-induced phosphorylation of FAK and paxillin as well as PMA-induced cell spreading, indicating that EGFR transactivation plays a central role in mediating PMA-elicited signaling for cellular spreading. We also determined the translocation of PKCδ during PMA-induced cell spreading of T84 cells. PMA caused PKCδ to translocate to the basal compartment, yet pretreatment with Gö6850 or siRNA directed against PKCε dramatically redistributed PKCδ to the lateral membrane; Gö6976, rottlerin, or siRNAs against PKCα or PKCδ were ineffective in preventing basal translocation of PKCδ, indicating that activity of PKCε determines basal translocation of PKCδ in response to PMA. Since PKCε has been shown to regulate actin rearrangement, we hypothesized that PKCε translocates PKCδ to the basal compartment in an actin-dependent manner. To test this hypothesis, we observed PMA-induced translocation of PKCδ in the presence of the chemical manipulators of actin cytoskeleton. Indeed, when PMA-elicited actin remodeling was disrupted by jasplakinolide or cytochalasin D, PKCδ translocated to the lateral membrane, mimicking the effects of Gö6850 and PKCε-siRNA. Simultaneously, no increase in phosphorylation of Src, FAK, and paxillin was observed. Furthermore, the tet-Ser peptide inhibitor which was shown to inhibit PKCε-induced actin remodeling by blocking MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation redistributed PKCδ to the lateral membrane and abolished phosphorylation of the focal adhesion proteins. These data demonstrate that actin remodeling by PKCε is critical for basal translocation of PKCδ for phosphorylation of focal adhesion targets, emphasizing the cooperation between two PKC isoforms for regulation of epithelial cell spreading. To further examine the role of PKC during wound healing, we examined the translocation of the PKC isoforms in response to scraped wounding. Activation of PKCα, PKCδ and PKCε was evident upon wounding as determined by increased isoform association with the membrane fraction. Inhibition of all three isoforms with Gö6850 prior to wounding completely prevented formation of lamellipodia, a critical step necessary to initiate forward movement of the cells. Consistently, cell migration toward the wound area was inhibited in the presence of Gö6850. Inhibition of PKCα with Gö6976 and PKCδ with rottlerin, however, did not abolish lamellipodia formation. Stimulation of FAK and paxillin phosphorylation was also evident during wound healing. Taken together, the current study establishes a novel role played by the PKC isoforms, particularly PKCδ and PKCε, in regulation of epithelial cell motility.