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Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4627-4631
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase
C Contains Two Activator Binding Sites That Bind Phorbol Esters
and Diacylglycerols with Opposite Affinities
(Received for publication, November 12,
1995)
Simon J.
Slater,
Cojen
Ho ,
Mary Beth
Kelly,
Jonathan D.
Larkin ,
Frank J.
Taddeo,
Mark D.
Yeager ,
Christopher D.
Stubbs
Based on marked differences in the enzymatic properties of
diacylglycerols compared with phorbol ester-activated protein kinase C
(PKC), we recently proposed that activation induced by these compounds
may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J.,
Rubin, E., and Stubbs, C. D.(1994) J. Biol. Chem. 269,
17160-17165). In the present study, direct evidence is provided
showing that phorbol esters and diacylglycerols bind simultaneously to PKC . Using a novel binding assay employing the fluorescent
phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and
low affinity was obtained. Thus, both binding and activation
dose-response curves for SAPD were double sigmoidal, which was also
observed for dose-dependent activation by the commonly used phorbol
ester, 4 -12-O-tetradecanoylphorbol-13-acetate (TPA). TPA
removed high affinity SAPD binding and also competed for the low
affinity site. By contrast with TPA, low affinity binding of SAPD was
inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to
the high affinity site was markedly enhanced. Again contrasting with
both TPA and DAG, the potent PKC activator, bryostatin-I (B-I),
inhibited SAPD binding to its high affinity site, while low affinity
binding was unaffected. Based on these findings, a model for PKC
activation is proposed in which binding of one activator to the low
affinity site allosterically promotes binding of a second activator to
the high affinity site, resulting in an enhanced level of activity.
Overall, the results provide direct evidence that PKC contains two
distinct binding sites, with affinities that differ for each activator
in the order: DAG > phorbol ester > B-I and B-I > phorbol
ester > DAG, respectively.

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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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