A cDNA encoding protein kinase C identifies two species of mRNA in brain and GH3 cells.
Antiserum raised against purified protein kinase C (the Ca2+/phospholipid-dependent enzyme) (Ballester, R., and rosen, O. M. (1985) J. Biol. Chem. 260, 15194-15199) was used to screen a rat brain cDNA library in the prokaryotic expression vector lambda gt11. Three positive clones were isolated and shown to have overlapping restriction endonuclease maps. The positive recombinant phage with the longest cDNA insert (1.4 kilobases (kb)) was used for production of a beta-galactosidase fusion protein. Rabbit antiserum raised against the fusion protein recognized a single rat brain polypeptide of Mr 80,000 which was identified as protein kinase C by the following criteria: electrophoretic co-migration with purified protein kinase C, partial co-purification with protein kinase C, and disappearance from the cytosol of phorbol 12-myristate 13-acetate-treated GH3 cells. The nick-translated cDNA hybridized with two mRNAs, 8 kb and 3.5 kb, whose tissue distribution was in agreement with that reported for protein kinase C activity. Hybrid selection with immobilized cDNA identified mRNA encoding a protein of Mr 80,000 that could be precipitated by antibody to purified protein kinase C. Treatment of GH3 cells with phorbol 12-myristate 13-acetate, which promotes translocation and subsequent degradation of protein kinase C, did not alter the level of either message.
D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D005910:Glioma / Q000201:enzymology; D007106:Immune Sera; D007527:Isoenzymes / Q000235:genetics*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D011493:Protein Kinase C / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D011493:Protein Kinase C; D001616:beta-Galactosidase; D013755:Tetradecanoylphorbol Acetate