Our efforts are directed at understanding the early events in the interaction of phorbol ester tumor promoters with cells and tissues. Particular attention is being devoted to the analysis of the major phorbol ester receptor, protein kinase C. Most of the evidence for the role of protein kinase C in phorbol ester action is indirect. Its involvement in the mitogenic response of Swiss 3T3 cells was demonstrated directly by microinjection, using Swiss 3T3 cells down-regulated by chronic phorbol ester treatment. Emerging evidence suggests prominent proteolytic processing of protein kinase C. The catalytically active fragment of the enzyme was prepared by tryptic digestion and was characterized. Phospholipids regulated its activity in a pH- and substrate-dependent fashion, distinct from the activation of the intact enzyme by the interaction of phospholipids at the regulatory domain. The regulatory domain, being devoid of enzymatic activity and thus a pseudo-receptor, was also generated and is now being characterized. Analysis of modulators of protein kinase C other than the phorbol esters afford unique insights into its function. Bryostatins, macrocylic lactones, activate protein kinase C and compete for phorbol ester binding. Paradoxically, they blocked phorbol ester action in two differentiating systems (HL-60 promyelocytic leukemia cells and Friend erythroleukemia cells) and blocked some but not other responses in keratinocytes. In HL-60 cells, the bryostatins induced a similar but distinct pattern of phosphorylation. Diglycerides are the postulated endogenous analogs of the phorbol esters. In support of this hypothesis, treatment of mouse keratinocytes with bromooctanoic acid, which blocks triglyceride formation and causes diglyceride accumulation, induces effects similar to the phorbol esters. Bromooctanoic acid may provide a tool for assessing the tumor promoting activity of the diglycerides. Modeling studies comparing different classes of protein kinase C activators indicated putative common features. Novel structures incorporating these features inhibited phorbol ester binding and induced typical phorbol ester responses such as inhibition of EGF binding or phosphorylation of a 40 kd platelet protein.
