Synergistic Activation of Protein Kinase C, -I, a
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- 作者：Simon J. Slater ; Shawn K. Milano ; Brigid A. Stagliano ; Kevin J. Gergich ; Cojen Ho ; Anthony Mazurek ; Frank J. Taddeo ; Mary Beth Kelly ; Mark D. Yeager ; and Christopher D. Stubbs
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：March 23, 1999
- 年：1999
- 卷：38
- 期：12
- 页码：3804 - 3815
- 全文大小：134K
- 年卷期：v.38,no.12(March 23, 1999)
- ISSN：1520-4995
文摘
Protein kinase C
(PKC) has been shown to contain two discrete activator sites with differingbinding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiatedlevel of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., andStubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancementof activity was examined with respect to the Ca2+ dependences of membrane association and accompanyingconformational changes that lead to activation. The association of PKC with membranes containing12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioleoylglycerol (DAG), determined from tryptophanto dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+levels (
1 
M). However, PKC was found to be inactive even though membrane association was completeat these Ca2+ levels and further titration of Ca2+ to a concentration of ~100 M was required for activation.This increase in Ca2+ concentration also led to a further increase in RET, which was due to a Ca2+-induced activating conformational change, as verified by an accompanying increase in the PKC tryptophanfluorescence anisotropy. Coaddition of DAG and TPA resulted in a reduction in the Ca2+ levels requiredfor both the conformational change and enzyme activation. Also, it was found that incubation of theenzyme with TPA alone resulted in a time-dependent increase in the Ca2+-independent PKC activity,the rate and extent of which was further enhanced upon coaddition with DAG. 
he results suggest thatthe enhanced level of activity induced by coaddition of DAG and TPA involves both Ca2+-dependent andCa2+-independent activating conformational changes which result in active conformers of PKC distinctfrom those formed by interaction with either activator separately.
(PKC) has been shown to contain two discrete activator sites with differingbinding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiatedlevel of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., andStubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancementof activity was examined with respect to the Ca2+ dependences of membrane association and accompanyingconformational changes that lead to activation. The association of PKC with membranes containing12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioleoylglycerol (DAG), determined from tryptophanto dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+levels (1 M). However, PKC was found to be inactive even though membrane association was completeat these Ca2+ levels and further titration of Ca2+ to a concentration of ~100 M was required for activation.This increase in Ca2+ concentration also led to a further increase in RET, which was due to a Ca2+-induced activating conformational change, as verified by an accompanying increase in the PKC tryptophanfluorescence anisotropy. Coaddition of DAG and TPA resulted in a reduction in the Ca2+ levels requiredfor both the conformational change and enzyme activation. Also, it was found that incubation of theenzyme with TPA alone resulted in a time-dependent increase in the Ca2+-independent PKC activity,the rate and extent of which was further enhanced upon coaddition with DAG. he results suggest thatthe enhanced level of activity induced by coaddition of DAG and TPA involves both Ca2+-dependent andCa2+-independent activating conformational changes which result in active conformers of PKC distinctfrom those formed by interaction with either activator separately.