The nature of the hydrophobic n-alkanol binding site within the C1 domains of protein kinase Calpha.
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 PKCalpha 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 PKCalpha 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)-(CH(2))(n)-CH(3), where R = H or an alkyl group and n >or= 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 PKCalpha was activated to a greater extent by R-2-nonanol than by the S enantiomer. The potentiation of phorbol ester-induced, membrane-associated PKCalpha 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 PKCalpha 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.
Slater
Simon J
SJ
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Malinowski
Steve A
SA
Stubbs
Christopher D
CD
D000438:Alcohols / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D057927:Hydrophobic and Hydrophilic Interactions; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D013237:Stereoisomerism
D000438:Alcohols; D010703:Phorbol Esters; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha