MOLECULAR ANALYSIS OF THE INTERACTIONS BETWEEN PROTEIN-KINASE C-EPSILON AND FILAMENTOUS ACTIN

Protein kinase C-epsilon (PKC-epsilon) contains a putative actin bindi ng motif that is unique to this individual member of the PKC gene fami ly. me have used deletion mutagenesis to determine whether this hexape ptide motif is required for the physical association of PKC-epsilon an d actin. Full-length recombinant PKC-epsilon, but not PKC-beta II, -de lta, -eta, or -zeta bound to filamentous actin in a phorbol ester-depe ndent manner. Deletion of PKC-epsilon amino acids 222-230, encompassin g a putative actin binding motif, completely abrogated this binding ac tivity. When NIH 3T3 cells overexpressing either PKC-epsilon off the d eletion mutant of this isozyme were treated with phorbol ester only wi ld-type PKC-epsilon colocalized with actin in tomes of cell adhesion. in binary reactions, it was possible to demonstrate that purified fila mentous actin is capable of directly stimulating PKC-epsilon phosphotr ansferase activity. These and other findings support the hypothesis th at a conformationally hidden actin binding motif in the PKC-epsilon se quence becomes exposed upon activation of this isozyme aad functions a s a dominant localization signal in NIH 3T3 fibroblasts. This protein- protein interaction is sufficient to maintain PKC-epsilon ire a cataly tically active conformation.