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Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate and sn-1,2-Dioctanoylglycerol Increase the Phosphorylation of Protein Kinase C in Cells¹

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907

Phosphorylation of protein kinase C (PKC) may be an important mode of regulation of this enzyme that plays a key role in mouse skin tumor promotion and in mammalian cell signal transduction. To investigate this possibility, PKC was specifically immunoprecipitated from Abelson murine leukemia virus-transformed normal rat kidney cells that had been metabolically labeled with [³²P]orthophosphoric acid. The M_r 80,000 phosphoprotein that was specifically immunoprecipitated from Abelson murine leukemia virus-transformed normal rat kidney cells was found to be identical with purified rat brain PKC that had undergone cell-free autophosphorylation. This is based on comparisons of peptides generated by partial proteolysis with Staphylococcus aureus V₈ protease by one-dimensional polyacrylamide-sodium dodecyl sulfate gel electrophoresis and of tryptic peptides by reversed-phase high-pressure liquid chromatography. These data are consistent with phosphorylation of PKC in cells having occurred via autophosphorylation. The autophosphorylation of PKC was stimulated by treatment of C3H 10T cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or sn-1,2-dioctanoylglycerol. Exposure of cells to 100 nM 12-O-tetradecanoylphorbal-13-acetate for 15 min increased the phosphorylation of PKC by 5-fold in the particulate fraction, while treatment with 100 µM dioctanoylglycerol enhanced phosphorylation of PKC only by 2-fold. Phosphorylation of PKC in response to activation may have significance for altering the sensitivity of PKC to proteolytic down-regulation and/or to subsequent activation.

¹ This work was supported by Grants CA 36262, CA K04 01424, CA 09634 and GM F31 11691 from the NIH and Grant BC-636 from the American Cancer Society.

² To whom requests for reprints should be addressed.

Received 3/ 1/90. Accepted 6/24/91.

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