Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the squence of the bovine brain enzyme (LLNQEE-CEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the levels of immunoreactive polypeptide and extractable calcium-and phospholipid-dependent kinase activity for various cell lines. Treatment of rnurine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13-dibutyrate protein kinase C was essentially undetectable by 40 hours; the half-life of this down-regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium-and phospholipid-dependent kinase activity precisely parallels the phorbol ester induced down-regulation of binding and responsiveness in Swiss 3T3 cells.
Protein kinase C is a calcium-and phospholipid-depen-Associated with this cellular desensitization, down-regdent kinase that is implicated in a variety of cellular ulation of phorbol ester binding has been documented in responses including long-term cell growth and tumour several cell types (Solanki et al., 1981; Collins and Rozpromotion (reviewed in Nishizuka, 1984a,b). Physiologi-engurt, 1982b;Phillips and Jaken, 1983; Collins and cally protein kinase C appears to be regulated by di-Rozengurt, 1984). The relationship between the phorbol acylglycerol, which is generated by phospholipid turn-ester receptor and protein kinase C has prompted invesover and activates the enzyme at submicromolar cal-tigations into the nature of this down-regulation with cium concentrations (see Berridge and Irvine, 1984; respect to protein kinase C. It has been shown that Nishizuka, 1986).
down-regulated cells show little or no measurable pro-The ability of diacylglycerols to activate protein ki-tein kinase C activity (Rodriguez-Pena and Rozengurt, nase C in vitro can be mimicked by biologically active 1984; Blackshear et al., 1985;Ballester and Rosen, 1985); phorbol esters (Castagna et al., 1982). These tumour furthermore there is evidence that the actual concentrapromoters and a number of structurally related and tion of protein kinase C polypeptide is severely diminunrelated promoters (Miyake et al., 1984; Fujiki et al., ished in down-regulated cells (Blackshear et al., 1985; 1984) can activate protein kinase C in vitro at concentra-Ballester and Rosen, 1985). It is not clear, however, tions comparable with those used to elicit biological whether the time course for the acquirement of cellular responses; this suggests that these tumour promoters refractoriness, reduction of phorbol ester binding, and may function through direct activation of protein kinase measurable protein kinase C (Rodriguez-Pena and Roz-C. Indeed, there is much evidence to suggest that the engurt, 1984) occur in parallel with the loss of cellular high affinity binding site for these promoters is polypeptide.