Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity.
The present study examines the synergistic activation of PKC by arachidonic acid and diacylglycerols in phospholipid vesicles and demonstrates that this combination of activators leads to the formation of a constitutively active, phospholipid-bound form of the enzyme. Activation of PKC was almost entirely calcium-dependent with vesicles containing dioleoylglycerol alone. In contrast, considerable calcium-independent activity was observed when vesicles contained both a diacylglycerol and free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reversible. However, addition of arachidonic acid to diacylglycerol-containing vesicles resulted in irreversible PKC binding in the absence of calcium. Immunoblot analysis indicated that the calcium-independent binding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly calcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the subsequent activity was almost entirely resistant to calcium chelation. This vesicle-associated form of the enzyme, when detergent extracted and recombined with phospholipid vesicles, maintained significant 'constitutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a model of the physiological regulation of PKC in which the combined actions of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC.
Laboratory of Adaptive Systems, National Institute for Neurological Disorders and Stroke, Bethesda, MD 20892, USA.
J B
JB
Schachter
D S
DS
Lester
D L
DL
Alkon
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology*; D004357:Drug Synergism; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D002614:Chelating Agents; D004075:Diglycerides; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D016718:Arachidonic Acid; D004533:Egtazic Acid; D011493:Protein Kinase C; D002118:Calcium