Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cδ

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/ phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1: 2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to similar to415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdeltaC1B was photolabeled with three photoactivable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdeltaC1B at a stoichiometry of 1: 1 in the labeled fraction. The photolabeled PKCdeltaC1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdeltaC1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within similar to10 Angstrom of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.