Protein kinase C-epsilon (PKC-⑀) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-⑀ and actin. Full-length recombinant PKC-⑀, but not PKC-␤II, -␦, -, or -, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-⑀ amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-⑀ or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-⑀ colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-⑀ phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-⑀ sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-⑀ in a catalytically active conformation.

Protein kinase C-epsilon coordinately regulates changes in cell growth and shape. Cells overproducing protein kinase C-epsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions that are reminiscent of the morphology observed in ras-transformed fibroblasts. Here we report that the regulatory C1 domain contains an actin binding hexapeptide motif that is essential for the morphogenic effects of protein kinase C-epsilon in cultured NIH3T3 murine fibroblasts. The extension of elongate processes by protein kinase C-epsilon transformed fibroblasts appeared to be driven by a kinase-independent mechanism that required organized networks of both actin and microtubules. Flow cytometry of phalloidin-stained cells demonstrated that protein kinase C-epsilon significantly increased the cellular content of polymerized actin in NIH3T3 cells. Studies with a cell-free system suggest that protein kinase C-epsilon inhibits the in vitro disassembly of actin filaments, is capable of desequestering actin monomers from physiologically relevant concentrations of thymosin beta4, and increases the rate of actin filament elongation by decreasing the critical concentration of actin. Based on these and other observations, it is proposed that protein kinase C-epsilon may function as a terminal downstream effector in at least one of the signaling pathways that mitogens engage to initiate outgrowth of cellular protrusions.