PKCε has an alcohol-binding site in its second cysteine-rich regulatory domain

Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that ail alcohol-binding site is present in PKC alpha in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKC epsilon-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present Study was to investigate whether ail alcohol-binding site could be present in PKC epsilon. Here we show that ethanol inhibited PKC epsilon activity in a concentration-dependent manner with an EC(50) (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKC epsilon C1B in a concentration-dependent manner with EC(50) values of 78 mM, 8 mM and 340 mu M respectively, suggesting the presence of ail allosteric alcohol-binding site ill this subdomain. To identify this site, PKC epsilon C1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His(236), Tyr(238) was the preferred site for azioctanol. Inspection of the model structure of PKC epsilon C1B reveals that these residues are 3.46 angstrom (1 angstrom = 0.1 nm) apart from each other and form a groove where His(236) is surface-exposed and Tyr(238) is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding ill terms of both photolabelling and alcohol-induced SAPID binding in the mutant H236A/Y238A. Whereas Tyr(228) was labelled in mutant H236A, His(236) was labelled in mutant Y238A. The present results provide direct evidence for the presence of,ill allosteric alcohol-binding site oil protein kinase C epsilon and underscore the role of His(236) and Tyr(238) residues in alcohol binding.