Tumor promoter 12-O-tetradecanoylphorbol-13-acetate and sn-1,2-dioctanoylglycerol increase the phosphorylation of protein kinase C in cells.
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 Mr 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 10T1/2 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or sn-1,2-dioctanoylglycerol. Exposure of cells to 100 nM 12-O-tetradecanoylphorbol-13-acetate for 15 min increased the phosphorylation of PKC by 5-fold in the particulate fraction, while treatment with 100 microM 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.
Molina
C A
CA
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Ashendel
C L
CL
D000818:Animals; D001921:Brain / Q000201:enzymology; D002461:Cell Line, Transformed; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D008970:Molecular Weight; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004075:Diglycerides; C044295:1,2-dioctanoylglycerol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate