ATP-sensitive binding of melittin to the catalytic domain of protein kinase C

CA O'Brian and NE Ward

Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase that appears to play a critical role in the regulation of cell growth. Melittin was previously shown to inhibit Ca2+- and phosphatidylserine (PS)-dependent PKC activity with an inhibitory potency that was reduced as the PS concentration was elevated. In this report, we found that melittin could inhibit activation of PKC by Ca2+ and PS, with an IC50 of 3 microM. When the enzyme activity was released from regulation by Ca2+ and PS by the generation of an active catalytic fragment of PKC through limited proteolysis, melittin inhibited the enzyme activity with an IC50 of 25 microM. Through inhibitor binding studies and enzyme kinetics, we established that melittin binds directly to the catalytic domain of PKC and that the substrate MgATP can release bound melittin from PKC. Melittin bound to PKC in the absence of PKC cofactors, and MgATP completely disrupted the binding of melittin to PKC, whereas phosphoacceptor substrates did not. The catalytic fragment of PKC, which contains two potential ATP-binding sites according to sequence analysis of PKC-encoding cDNAs, also bound melittin. The kinetics of inhibition of the catalytic fragment were consistent with a noncompetitive inhibition with respect to the substrate ATP, providing evidence that the antagonism of the binding of melittin to PKC by MgATP is not due to a direct competition between MgATP and melittin at the active site of PKC.

Volume 36, Issue 3, pp. 355-359, 09/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

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