title: Multisite phosphorylation of human liver cytochrome P450 3A4 enhances Its gp78- and CHIP-mediated ubiquitination: a pivotal role of its Ser-478 residue in the gp78-catalyzed reaction.
abstract: CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation.
authors.affiliation: Department of Cellular & Molecular Pharmacology, University of California, San Francisco, California 94158-2517, USA.
authors.forename: YongQiang
authors.initials: Y
authors.lastname: Wang
authors.forename: Shenheng
authors.initials: S
authors.lastname: Guan
authors.forename: Poulomi
authors.initials: P
authors.lastname: Acharya
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Ranjit K
authors.initials: RK
authors.lastname: Thirumaran
authors.forename: Relly
authors.initials: R
authors.lastname: Brandman
authors.forename: Erin G
authors.initials: EG
authors.lastname: Schuetz
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Maria Almira
authors.initials: MA
authors.lastname: Correia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D060746:Endoplasmic Reticulum-Associated Degradation; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D040901:Proteomics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D060285:Receptors, Autocrine Motility Factor / Q000378:metabolism*; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D065692:Cytochrome P-450 CYP3A Inhibitors; D034741:RNA, Small Interfering; D025801:Ubiquitin; D012694:Serine; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C
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