Resveratrol preferentially inhibits protein kinase C-catalyzed phosphorylation of a cofactor-independent, arginine-rich protein substrate by a novel mechanism
Jr. Stewart et al., Resveratrol preferentially inhibits protein kinase C-catalyzed phosphorylation of a cofactor-independent, arginine-rich protein substrate by a novel mechanism, BIOCHEM, 38(40), 1999, pp. 13244-13251
Resveratrol, a polyphenolic natural product abundantly present in grape ski
ns, is a candidate cancer chemopreventive agent that antagonizes each stage
of carcinogenesis and inhibits protein kinase C (PKC), a key mediator of t
umor promotion. While resveratrol has been shown to antagonize both isolate
d and cellular forms of PKC, the weak inhibitory potency observed against i
solated PKC cannot account for the reported efficacy of the polyphenol agai
nst PKC in cells. In this report, we analyze the mechanism of PKC inhibitio
n by resveratrol. Our results indicate that resveratrol has a broad range o
f inhibitory potencies against purified PKC that depend on the nature of th
e substrate and the cofactor dependence of the phosphotransferase reaction.
Resveratrol weakly inhibited the Ca2+/phosphalidylserine-stimulated activi
ty of a purified rat brain PKC isozyme mixture (IC50 = 90 mu M) by competit
ion with ATP (K-i = 55 mu M). Consistent with the kinetic evidence for a ca
talytic domain-directed mechanism, resveratrol inhibited the lipid-dependen
t activity of PKC isozymes with divergent regulatory domains similarly, and
it was even more effective in inhibiting a cofactor-independent catalytic
domain fragment (CDF) of PKC generated by limited proteolysis. This suggest
ed that regulatory features of PKC might impede resveratrol inhibition of t
he enzyme. To explore this, we examined the effects of resveratrol on PKC-c
atalyzed phosphorylation of the cofactor-independent substrate protamine su
lfate, which is a polybasic protein that activates PKC by a novel mechanism
. Resveratrol potently inhibited protamine sulfate phosphorylation (IC50 =
10 mu M) by a mechanism that entailed antagonism of the activation of PKC b
y protamine sulfate and did not involve competition with either substrate.
On the basis of the presence of PKC isozymes at subcellular sites rich in p
olybasic proteins, it has been proposed that certain endogenous polybasic P
KC substrates may activate PKC in cells by the same mechanism as protamine
sulfate. Our results suggest that antagonism by resveratrol of the phosphor
ylation of cellular PKC substrates that resemble protamine sulfate in their
interactions with PKC may contribute to the efficacy of resveratrol agains
t PKC in cells.