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The Nature of the Hydrophobic n -Alkanol Binding Site within the C1 Domains of Protein Kinase Cα

2004; American Chemical Society; Volume: 43; Issue: 23 Linguagem: Inglês

10.1021/bi049755z

1943-295X

Simon J. Slater, Steve A. Malinowski, Christopher D. Stubbs,

Pharmacogenetics and Drug Metabolism

The activator-binding sites within the C1 domains of protein kinase C (PKC) are also able to bind alcohols and anesthetics. In this study, the nature of the interaction of these agents with the hydrophobic region within the C1 domains was investigated and a structure−activity relationship for the alcohol effects was obtained. The effects of a series of n-alkanols on PKCα activity, determined using an in vitro assay system that lacked lipids, were found to be a nonlinear function of the chain length. In the absence of phorbol ester or diacylglycerol, 1-octanol potently activated PKCα in a concentration-dependent manner, while 1-heptanol was completely without effect, despite differing by one methylene unit. The minimal structural requirement for the activating effect corresponded to R−CH(OH)−(CH2)n−CH3, where R = H or an alkyl group and n ≥ 6. Consistent with this, 2-octanol, for which n = 5, was without effect on the activity, even though this alcohol is only marginally less hydrophobic than 1-octanol, whereas 2-nonanol, for which n = 6, was able to produce activity. Importantly, it was found that PKCα was activated to a greater extent by R-2-nonanol than by the S enantiomer. The potentiation of phorbol ester-induced, membrane-associated PKCα activity by long-chain n-alkanols reported previously (Slater, S. J., Kelly, M. B., Larkin, J. D., Ho, C, Mazurek, A, Taddeo, F. J., Yeager, M. D., Stubbs, C. D. (1997) J. Biol. Chem. 272, 6167−6173), was also found here for nonmembrane associated PKC, indicating that this effect is an intrinsic property of the enzyme rather than a result of membrane perturbation. Overall, the results suggest that the alcohol-binding sites within the C1 domains of PKCα contain spatially distinct hydrophilic and hydrophobic regions that impose a high degree of structural specificity on the interactions of alcohols and other anesthetic compounds, as well as diacylglycerols and phorbol esters.