Phosphorylation of native and heme-modified CYP3A4 by protein kinase C: a mass spectrometric characterization of the phosphorylated peptides.
As an initial approach toward the characterization of the phosphorylation of cumene hydroperoxide (CuOOH)-inactivated cytochrome P450 (CYP3A4, the major human liver drug-metabolizing enzyme) and its role in the degradation of the inactivated protein, we have identified one of the major participating cytosolic kinase(s) as rat liver cytosolic protein kinase C (PKC) with the use of specific and general kinase inhibitors. Accordingly, we employed a model phosphorylation system consisting of purified PKC, gamma-S-[(32)P]ATP, and either native or CuOOH-inactivated purified recombinant His(6)-tagged CYP3A4. Lysylendoprotease (Lys)-C digestion of the phosphorylated CuOOH-inactivated CYP3A4(His)(6) followed by HPLC-peptide mapping and mass spectrometric (LC/MS/MS) analyses led to the isolation and the unambiguous identification of two PKC-phosphorylated CYP3A4 peptides: E(258)SRLEDT(p)QK(266) and F(414)LPERFS(p)K(421). Similar analyses of the PKC-phosphorylated native enzyme predominantly yielded E(258)SRLEDT(p)QK(266) as the phosphorylated peptide. Studies are currently in progress to determine whether phosphorylation of any or both of these peptides is required for the Ub-dependent 26S proteasomal degradation of CuOOH-inactivated CYP3A4.
Wang
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Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
Medzihradszky
K F
KF
Maltby
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D
Correia
M A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002853:Chromatography, Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010649:Phenylalanine; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine; D019311:Staurosporine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D019319:Okadaic Acid; D006418:Heme; D012694:Serine; D010649:Phenylalanine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D011493:Protein Kinase C; D019311:Staurosporine