THE CATALYTIC DOMAIN OF PROTEIN-KINASE-C CHIMERAS MODULATES THE AFFINITY AND TARGETING OF PHORBOL ESTER-INDUCED TRANSLOCATION

Emerging evidence suggests important differences among protein kinase C (PKC) isozymes in terms of their regulation and biological functions , PKC is regulated by multiple interdependent mechanisms, including en zymatic activation, translocation of the enzyme in response to activat ion, phosphorylation, and proteolysis. As part of our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic and regulatory domains of PKC alpha, -delta, and -epsilon. These chimeras, which preserve the ov erall structure of the native PKC enzymes, were stably expressed in NI H 3T3 fibroblasts. Their intracellular distribution was similar to tha t of the endogenous enzymes, and they responded with translocation upo n treatment with phorbol 12-myristate 13-acetate (PMA). We found that the potency of PMA for translocation of the PKC alpha/x chimeras from the soluble fraction was influenced by the catalytic domain. The ED50 for translocation of PKC alpha/alpha was 26 nM, in marked contrast to the ED50 of 0.9 nM in the case of the PKC alpha/epsilon chimera. In ad dition to this increase in potency, the site of translocation was also changed; the PKC alpha/epsilon chimera translocated mainly into the c ytoskeletal fraction. PKCx/epsilon chimeras displayed twin isoforms wi th different mobilities on Western blots. PMA treatment increased the proportion of the higher mobility isoform. The two PKCx/epsilon isofor ms differed in their localization; moreover, their localization patter n depended on the regulatory domain, Our results emphasize the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.