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

The Journal of Biological Chemistry
Joydip DasKeith W Miller

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 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 approximately 415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdelta C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdelta C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCdelta C1B was subjected to tryptic digest,...Continue Reading

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Citations

Dec 7, 2010·Biomolecular NMR Assignments·Brian P ZiembaJones N M David
Jan 1, 2008·Proceedings of the National Academy of Sciences of the United States of America·Enrique ArevaloKeith W Miller
Jul 13, 2012·The Journal of Biological Chemistry·Robert V StahelinWonhwa Cho
Nov 28, 2012·The Journal of Biological Chemistry·Brian P WeiserRoderic G Eckenhoff
Feb 25, 2014·Current Anesthesiology Reports·Brian P WeiserRoderic G Eckenhoff
May 13, 2009·The Biochemical Journal·Joydip DasSimon J Slater
May 5, 2005·Annual Review of Biophysics and Biomolecular Structure·Wonhwa Cho, Robert V Stahelin
Mar 25, 2015·Bioorganic & Medicinal Chemistry Letters·Souta MasudaYasumaru Hatanaka
Sep 1, 2006·Protein Science : a Publication of the Protein Society·Joydip DasKeith W Miller
May 2, 2008·Clinical Pharmacology and Therapeutics·Rg EckenhoffMb Kelz
Jun 11, 2009·Anesthesiology·Peter J WickleyDerek S Damron
May 23, 2013·Journal of Neurochemistry·Joydip DasGregg W Roman
Nov 15, 2019·Alcoholism, Clinical and Experimental Research·Joydip Das
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Feb 22, 2007·The Journal of Biological Chemistry·Damian P BrightNicholas P Franks
Sep 2, 2011·Bioorganic & Medicinal Chemistry·Joydip DasAnjoy Majhi

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