Role of substrate in imparting calcium and phospholipid requirements to protein kinase C activation.
The role of substrate in influencing the cofactor requirements of the phospholipid- and Ca2+-dependent protein kinase C (PKC) was investigated by using several substrates. All of the substrates tested, including histone, troponin I, myosin light chain, protamine, poly(arginine, serine) (PAS), poly(lysine, serine) (PLS), and myelin basic protein (MBP), were found to interact with and aggregate phospholipid vesicles as well as phosphatidylserine (PS)-Triton mixed micelles. Phosphorylation of these different substrates by PKC indicated the presence of three distinct substrate categories: substrates such as protamine requiring no cofactors; substrates such as PLS, PAS, and MBP requiring only the presence of phospholipid; and substrates such as histone, myosin light chain, and troponin I requiring the presence of Ca2+ and phospholipid. Diacylglycerol was a major cofactor only with category C substrates. These different requirements correlated with the interaction of the substrate with phospholipid and/or enzyme. The substrates in category A interacted strongly with and aggregated PKC in a binary mixture. In the absence of Ca2+, PKC bound to substrates of category B directly but not to substrates in category C. Thus, substrate-enzyme binding eliminated the Ca2+ requirement of phosphorylation, and aggregation of substrate-enzyme complex eliminated the phospholipid requirements as well. Substrate-phospholipid interaction and substrate phosphorylation were inhibited by increasing salt concentrations, but the amount needed depended upon the substrate. Loss of PKC activity appeared to coincide with loss of substrate-PS aggregation while dissociation of PKC from the membranes required much higher salt concentrations. Poly(L-lysine) and poly(L-arginine), two potent inhibitors of PKC, also showed substrate-dependent inhibition characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D004735:Energy Transfer; D004789:Enzyme Activation; D007700:Kinetics; D009994:Osmolar Concentration; D010455:Peptides / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D011107:Polylysine / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D010455:Peptides; D010743:Phospholipids; D011107:Polylysine; C015462:polyarginine; D011493:Protein Kinase C; D002118:Calcium