Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta.
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, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdelta C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within approximately 10 A of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.
Das
Joydip
J
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA 02115, USA.
Addona
George H
GH
Sandberg
Warren S
WS
Husain
S Shaukat
SS
Stehle
Thilo
T
Miller
Keith W
KW
D000595:Amino Acid Sequence; D018681:Anesthetics, General / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004075:Diglycerides / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D018681:Anesthetics, General; D004075:Diglycerides; D011994:Recombinant Proteins; D014443:Tyrosine; C497259:Prkcd protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate