Expression, purification, and characterization of protein kinase C-epsilon.
Of the recently described members of the protein kinase C (PKC) family (-delta, -epsilon, -zeta), no detailed properties of the purified enzymes have been presented. Here we describe the expression of PKC-epsilon in insect cells using a baculovirus vector. The recombinant enzyme has been purified to homogeneity by sequential chromatography on DEAE-cellulose, serine-Sepharose, Mono Q, and Superose 12; the protein shows a molecular mass of 90 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PKC-epsilon is dependent upon phospholipid and diacylglycerol (or phorbol esters) for activity and displays a pattern of specificity for these effectors similar to other PKC isotypes. Similarly, inhibition of PKC-epsilon by staurosporine and H-7 parallels inhibition of other PKC isotypes. However, unlike PKC-alpha, -beta, and -gamma, PKC-epsilon shows no dependence upon Ca2+. Furthermore, the substrate specificity of PKC-epsilon is quite different from other characterized PKCs. The importance of functional diversity within the PKC family is discussed.
Schaap
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Ludwig Institute for Cancer Research, London, Great Britain.
Parker
P J
PJ
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D007304:Insect Viruses / Q000235:genetics; D007313:Insecta; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010957:Plasmids; D011493:Protein Kinase C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D004075:Diglycerides; D007527:Isoenzymes; D015240:Phorbol 12,13-Dibutyrate; D004247:DNA; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate