Quantitation of protein kinase C by immunoblot-expression in different cell lines and response to phorbol esters

Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE‐GEYYNVPIPE). 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 leveis of immunoreactive polypeptide and extractable calcium‐ and phospholipid‐dependent kinase activity for various cell lines. Treatment of murine, 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.