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
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
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.