Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase C δ

Protein kinase C (PKC) is an important signal transduction protein whose cysteine-rich regulatory domain C1 has been proposed to interact with general anesthetics in both of its diacylglycerol/ phorbol ester -binding subdomains, the tandem repeats C1A and C1B. Previously, we identified an allosteric binding site on one of the two cysteine-rich domains, PKC delta C1B. To test the hypothesis that there is an additional anesthetic site on the other cysteine-rich subdomain, C1A, we subcloned, expressed in Escherichia coli, purified, and characterized mouse PKCd C1A. Octanol and butanol both quenched the intrinsic fluorescence of PKCd C1A in a saturable manner, suggesting the presence of a binding site. To locate this site, PKC delta C1A was photolabeled with three diazirine-containing alkanols, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry revealed that at low concentrations all three photoincorporated into PKC delta C1A with a stoichiometry of 1: 1 in the labeled fraction, but higher stoichiometries occurred at higher concentrations, particularly with azibutanol. Photocomplexes of PKCd C1A with azioctanols were separated from the unlabeled protein by HPLC, reduced, alkylated, digested with trypsin, and sequenced by mass spectrometry. All the azioctanols photolabeled PKCd C1A at residue Tyr-29, corresponding to Tyr-187 of the full-length PKC delta, and at a neighboring residue, Lys-40, suggesting there is an alcohol site in this vicinity. In addition, Glu-2 was photolabeled more efficiently by 3-azibutanol than by the azioctanols, suggesting the existence of a second, smaller site.