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

Abstract

Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that an 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 an 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 EC50 (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 EC50 values of 78 mM, 8 mM and 340 microM respectively, suggesting the presence of an allosteric alcohol-binding site in 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 His236, Tyr238 was the preferred site for azioctanol. Inspection of the model structure of PKC epsilon C1B reveals that these residues are 3.46 A (1 A=0.1 nm) apart from each other and form a groove where His236 is surface-exposed and Tyr238 is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding in terms of both photolabelling and alcohol-induced SAPD binding in the mutant H236A/Y238A. Whereas Tyr238 was labelled in mutant H236A, His236 was labelled in mutant Y238A. The present results provide direct evidence for the presence of an allosteric alcohol-binding site on protein kinase C epsilon and underscore the role of His236 and Tyr238 residues in alcohol binding.

The role of protein kinase C epsilon in neural signal transduction and neurogenic diseases

“…Correlation with the increased GABA transmission in the animals was confirmed by the increased sensitivity to ethanol and the higher chloridion uptake upon stimulation [80]. Expression of the PKC ε in the brain controls ethanol-drinking behavior and it has an alcohol binding site in its second cysteine-rich regulatory domain [76,81]. In the studies of examining the role of PKC ε in this action of ethanol on ventral segmental area neurons, people find the results that the activation of PKC ε isoenzyme contributes to ethanol induced potentiation of functions [82], so drugs targeting PKC ε may be useful to curb excessive drinking and be a possible therapeutic target for development of anxiolytics [79].…”

ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells

“…Ethanol is a substrate of protein kinase C epsilon (PKCe), 35 and we used PMA as a pan-PKC substrate.…”

1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

“…A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα [1, 2], PKCε [3] and PKCδ [4, 5] contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC.…”

“…PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα [1, 2], PKCε [3] and PKCδ [4, 5] contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC.…”