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

Citation
Jb. Schachter et al., 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, Biochimica et biophysica acta (G). General subjects, 1291(2), 1996, pp. 167-176
Citations number
60
Categorie Soggetti
Biology,Biophysics
ISSN journal
03044165
Volume
1291
Issue
2
Year of publication
1996
Pages
167 - 176
Database
ISI
SICI code
0304-4165(1996)1291:2<167:SAOPBA>2.0.ZU;2-R
Abstract
The present study examines the synergistic activation of PKC by arachi donic acid and diacylglycerols in phospholipid vesicles and demonstrat es that this combination of activators leads to the formation of a con stitutively 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 an d free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reve rsible. However, addition of arachidonic acid to diacylglycerol-contai ning vesicles resulted in irreversible PKC binding in the absence of c alcium. Immunoblot analysis indicated that the calcium-independent bin ding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly ca lcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the sub sequent activity was almost entirely resistant to calcium chelation. T his vesicle-associated form of the enzyme, when detergent extracted an d recombined with phospholipid vesicles, maintained significant 'const itutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a m odel of the physiological regulation of PKC in which the combined acti ons of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC.