We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKCca with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, APKCIB, lacking the coding region for amino acid positions 6 to 159 of PKCD was also constructed. These mutant kinase genes expressed under the control of the SRa promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKCa at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. APKCI3, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.Various hormones, growth factors, and neurotransmitters are known to elicit inositol phospholipid breakdown to produce diacylglycerol (DG) and inositol 1,4,5-trisphosphate in the target tissues (1, 19). DG and inositol 1,4,5-trisphosphate serve as second messengers for activation of protein kinase C (PKC) and calcium mobilization, respectively (1, 19). Phorbol ester and membrane-permeable DG have been used to study the function of PKC because they selectively activate PKC without inducing calcium mobilization in intact cells (25). By use of these agents, it has been shown that PKC activation and calcium mobilization are essential elements of cellular responses such as release reaction (12, 15), cell proliferation (11,30), and gene expression (3, 13). Since the phorbol ester induces the down regulation of PKC (24) and membrane-permeable DG is easily converted to the corresponding phosphatidic acid by the action of DG kinase (25), the activation of PKC by these agents appears to be transient in most cells, and therefore, it is inconvenient to use it in certain studies.In early studies of PKC, it was shown that partial proteolysis of this enzyme by a calcium-dependent protease generated a 51-kilodalton component with catalytic activity but without the requirement for activators such as phospholipid, phorbol ester, and calcium ion (25). The other product of proteolysis has been shown to bind to a complex of phospholipid, phorbol ester, and calcium ion (7). These results suggest that PKC might be composed of separate regulatory and catalytic domains.Recently, based on amino acid sequences of PKC deduced from cDNAs cloned from various species (4,9,14,(20)(21)(22), several domains of the PKC molecule have been delineated. The...