![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
SM Dimitrijevic, WJ Ryves, PJ Parker and FJ Evans
Department of Pharmacognosy, School of Pharmacy, University of London, UK.
A mixed micellar assay was used to study the in vitro binding of [3H]phorbol-12, 13-dibutyrate ([3H]PDBu) to pure recombinant protein kinase C (PKC)-alpha, -beta 1, -beta 2, -gamma, -delta, -epsilon, and - zeta isotypes expressed in the baculovirus/insect cell system. Scatchard analysis revealed that all isotypes except PKC-zeta were able to specifically bind PDBu, with Kd values ranging from 1.6 to 18 nM in the presence of calcium. In the absence of calcium PKC-alpha, -beta 1, - beta 2, and -delta were observed to have a 2-3-fold drop in affinity, although Bmax values remained unchanged, at a stoichiometry of 1.4-2.8 mol of PDBu/mol of enzyme. Competition with specific [3H]PDBu binding was assessed for the phorbol esters PDBu, 12-tetradecanoylphorbol-13-O- acetate, 12-deoxyphorbol-13-O-phenylacetate, 12-deoxyphorbol-13-O- phenylacetate-20-acetate, thymeleatoxin, resiniferatoxin, and sapintoxin A. Resiniferatoxin and 12-deoxyphorbol-13-O-phenylacetate-20- acetate were found to compete effectively only with PDBu bound to the PKC-beta 1 and -beta 2 isotypes and were the least potent of the phorbol esters tested (IC50, > 5 microM). The phorbol esters sapintoxin A, 12-deoxyphorbol-13-O-phenylacetate, 12-tetradecanoylphorbol-13-O- acetate, and PDBu (in order of potency) competed for binding to all isotypes (IC50 values ranging from 2 to 70 nM), with unchanged or slightly decreased potency when calcium was replaced by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thymeleatoxin, which was similar in other respects to these potent phorbol esters, was found to be less able to compete with binding to PKC-alpha and -epsilon isotypes (IC50, 3-5 microM). It appears that, whereas the binding of phorbol esters to PKC depends primarily on the C20 substituent, other areas of the molecule have an influence on this interaction and the PKC isotypes themselves display heterogeneity in their phorbol ester- binding characteristics.
This article has been cited by other articles:
|
|
 |
|
  R. Kubitz, N. Saha, T. Kuhlkamp, S. Dutta, S. vom Dahl, M. Wettstein, and D. Haussinger Ca2+-dependent Protein Kinase C Isoforms Induce Cholestasis in Rat Liver J. Biol. Chem., March 12, 2004; 279(11): 10323 - 10330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Lenz, H. P. Reusch, N. Albrecht, G. Schultz, and M. Schaefer Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells J. Cell Biol., October 28, 2002; 159(2): 291 - 302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. A. Shen, O. I. Chertihin, R. L. Biltonen, and J. J. Sando Lipid-dependent Activation of Protein Kinase C-alpha by Normal Alcohols J. Biol. Chem., November 26, 1999; 274(48): 34036 - 34044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Dwivedi and G. N. Pandey Effects of Treatment with Haloperidol, Chlorpromazine, and Clozapine on Protein Kinase C (PKC) and Phosphoinositide-Specific Phospholipase C (PI-PLC) Activity and on mRNA and Protein Expression of PKC and PLC Isozymes in Rat Brain J. Pharmacol. Exp. Ther., November 1, 1999; 291(2): 688 - 704. [Abstract] [Full Text]
|
|
|
|
|
|
M. Medkova and W. Cho Interplay of C1 and C2 Domains of Protein Kinase C-alpha in Its Membrane Binding and Activation J. Biol. Chem., July 9, 1999; 274(28): 19852 - 19861. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Szallasi and P. M. Blumberg Vanilloid (Capsaicin) Receptors and Mechanisms Pharmacol. Rev., June 1, 1999; 51(2): 159 - 212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Slater, F. J. Taddeo, A. Mazurek, B. A. Stagliano, S. K. Milano, M. B. Kelly, C. Ho, and C. D. Stubbs Inhibition of Membrane Lipid-independent Protein Kinase Calpha Activity by Phorbol Esters, Diacylglycerols, and Bryostatin-1 J. Biol. Chem., September 4, 1998; 273(36): 23160 - 23168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Ansonoff and A. M. Etgen Estradiol Elevates Protein Kinase C Catalytic Activity in the Preoptic Area of Female Rats Endocrinology, July 1, 1998; 139(7): 3050 - 3056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fang, S. Gibson, M. Flowers, T. Furui, R. C. Bast Jr., and G. B. Mills Lysophosphatidylcholine Stimulates Activator Protein 1 and the c-Jun N-terminal Kinase Activity J. Biol. Chem., May 23, 1997; 272(21): 13683 - 13689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Slater, M. B. Kelly, J. D. Larkin, C. Ho, A. Mazurek, F. J. Taddeo, M. D. Yeager, and C. D. Stubbs Interaction of Alcohols and Anesthetics with Protein Kinase Calpha J. Biol. Chem., March 7, 1997; 272(10): 6167 - 6173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Slater, C. Ho, M. B. Kelly, J. D. Larkin, F. J. Taddeo, M. D. Yeager, and C. D. Stubbs Protein Kinase Calpha Contains Two Activator Binding Sites That Bind Phorbol Esters and Diacylglycerols with Opposite Affinities J. Biol. Chem., March 1, 1996; 271(9): 4627 - 4631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Nakajima, K. Yamauchi, S. Shigematsu, S. Ikeo, M. Komatsu, T. Aizawa, and K. Hashizume Selective Attenuation of Metabolic Branch of Insulin Receptor Down-signaling by High Glucose in a Hepatoma Cell Line, HepG2 Cells J. Biol. Chem., June 30, 2000; 275(27): 20880 - 20886. [Abstract] [Full Text] [PDF]
|
|
 |
 |
 |
|
 |
 |
 |
