Phosphorylation of Native and Heme-Modified CYP3A4 by Protein Kinase C: A Mass Spectrometric Characterization of the Phosphorylated Peptides
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- 作者：Xiangyang Wang ; Katalin F. Medzihradszky ; David Maltby ; and Maria Almira Correia
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：September 25, 2001
- 年：2001
- 卷：40
- 期：38
- 页码：11318 - 11326
- 全文大小：220K
- 年卷期：v.40,no.38(September 25, 2001)
- ISSN：1520-4995
文摘
As an initial approach toward the characterization of the phosphorylation of cumenehydroperoxide (CuOOH)-inactivated cytochrome P450 (CYP3A4, the major human liver drug-metabolizingenzyme) and its role in the degradation of the inactivated protein, we have identified one of the majorparticipating cytosolic kinase(s) as rat liver cytosolic protein kinase C (PKC) with the use of specific andgeneral kinase inhibitors. Accordingly, we employed a model phosphorylation system consisting of purifiedPKC, 
-S-[32P]ATP, and either native or CuOOH-inactivated purified recombinant His6-tagged CYP3A4.Lysylendoprotease (Lys)-C digestion of the phosphorylated CuOOH-inactivated CYP3A4(His)6 followedby HPLC-peptide mapping and mass spectrometric (LC/MS/MS) analyses led to the isolation and theunambiguous identification of two PKC-phosphorylated CYP3A4 peptides: E258SRLEDT(p)QK266 andF414LPERFS(p)K421. Similar analyses of the PKC-phosphorylated native enzyme predominantly yieldedE258SRLEDT(p)QK266 as the phosphorylated peptide. Studies are currently in progress to determine whetherphosphorylation of any or both of these peptides is required for the Ub-dependent 26S proteasomaldegradation of CuOOH-inactivated CYP3A4.
-S-[32P]ATP, and either native or CuOOH-inactivated purified recombinant His6-tagged CYP3A4.Lysylendoprotease (Lys)-C digestion of the phosphorylated CuOOH-inactivated CYP3A4(His)6 followedby HPLC-peptide mapping and mass spectrometric (LC/MS/MS) analyses led to the isolation and theunambiguous identification of two PKC-phosphorylated CYP3A4 peptides: E258SRLEDT(p)QK266 andF414LPERFS(p)K421. Similar analyses of the PKC-phosphorylated native enzyme predominantly yieldedE258SRLEDT(p)QK266 as the phosphorylated peptide. Studies are currently in progress to determine whetherphosphorylation of any or both of these peptides is required for the Ub-dependent 26S proteasomaldegradation of CuOOH-inactivated CYP3A4.