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

Abstract

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 Cδ, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintosin-D, bound to PKCδ C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, λmax, shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted λmax of the PKCδ C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to ∼415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCδ C1B was photolabeled with three photoactivable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCδ C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCδ C1B 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 PKCδ C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within ∼10 Å of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.