Activation of purified human protein kinase C alpha and beta I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate.
The increasing number of eukaryotic protein kinase C (PKC) isoenzymes which have been described has raised great interest in potential differences in the cellular expression, the mode of activation and the substrate specificity of these isoenzymes. The last two aspects have mostly been studied with isoenzymes purified from rat or bovine brain or from recombinant-baculovirus-infected insect cells. In this study, we have expressed the human PKC isoenzymes alpha and beta I in recombinant-baculovirus-infected insect cells. The isoenzymes were purified to homogeneity by a four-step procedure which included a reversible Ca(2+)-dependent association/dissociation to and from the endogenous membranes of the lysed insect cells. Characterization of the purified enzymes with respect to ATP requirement and substrate specificity, using the epidermal-growth-factor receptor peptide and histone III-S respectively, revealed no isoenzyme-specific differences. Activation by trypsin or Ca2+ and a variety of different phospholipids and phosphoinositides (in a mixed-micellar assay) gave the following results. Proteolytic cleavage of the PKC isoenzymes by trypsin generated fully activated phospholipid-independent PKC beta I, whereas PKC alpha reached only 50% of the activity obtained in the presence of phospholipids. PKC alpha and beta I showed no difference in their dependence on Ca2+, diacylglycerol (DAG) and phosphatidylserine (PS). Replacement of either DAG or PS by phosphatidylglycerol, cardiolipin, phosphatidylcholine and several phosphoinositides revealed that PtdIns(4,5)P2 can act as a PKC activator similar to DAG, whereas PtdIns can substitute for PS as a cofactor of activation. Thus, at least for the PKC isoenzymes alpha and beta I, a combination of PtdIns and PtdIns(4,5)P2 can fully replace PS and DAG in vitro as the classical activators of PKC.
University of Freiburg, Institute of Molecular Cell Biology, Germany.
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Kochs
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Hummel
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Fiebich
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Sarre
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Marmé
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Hug
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009036:Moths; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D004075:Diglycerides; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D014357:Trypsin; D002118:Calcium