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.
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.
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人肝细胞色素 P450 3A4 的多位点磷酸化增强其 gp78 和 CHIP 介导的泛素化：其 Ser-478 残基在 gp78 催化反应中的关键作用。
DOI：
10.1074/mcp.m111.010132
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Correia,MariaAlmira
中科院分区：
文献类型：
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作者：
Wang,YongQiang;Guan,Shenheng;Acharya,Poulomi;Liu,Yi;Thirumaran,RanjitK;Brandman,Relly;Schuetz,ErinG;Burlingame,AlmaL;Correia,MariaAlmira
CYP 3A 4是一种整合的内质网（ER）锚定蛋白，是主要的人类肝脏细胞色素P450酶，负责超过50%的临床相关药物的处置。其蛋白质周转的改变可以影响药物代谢、药物间相互作用和化疗药物的生物利用度。这种CYP 3A 4转换通过经典的ER相关降解（ERAD）过程发生，该过程涉及UBC 7/gp 78和UbcH 5a/CHIP E2-E3复合物对26 S蛋白酶体靶向的泛素化。这些E3连接酶在CYP 3A 4 ERAD中顺序和协同作用，因为在培养的肝细胞中RNA干扰敲低每种酶导致功能活性酶的稳定。我们已经证明，UBC 7/gp 78介导的CYP 3A 4泛素化需要蛋白激酶（PK）A和PKC的蛋白磷酸化，并确定了三个残基（Ser-478，Thr-264和Ser-420），其磷酸化是细胞内CYP 3A 4 ERAD所必需的。我们在此记录了其中，Ser-478在UBC 7/gp 78介导的CYP 3A 4泛素化中起关键作用，其在其突变为磷酸化模拟物Asp残基时加速和增强，但在其Ala突变时减弱。有趣的是，CYP 3A 5是一种多态性表达的人肝CYP 3A 4同种型（含有Asp-478），在HepG 2细胞中被泛素化，但降解程度低于CYP 3A 4。这表明虽然Ser-478磷酸化对于UBC 7/gp 78介导的CYP 3A 4泛素化是必需的，但对于其ERAD是不够的。此外，我们现在报告，CYP 3A 4蛋白磷酸化PKA和/或PKC在其他网站以外的Ser-478，Thr-264，和Ser-420也增强UbcH 5a/CHIP介导的泛素化。通过蛋白质组学分析，我们确定了（i）12个可能参与CHIP-CYP 3A 4相互作用的额外磷酸化位点和（ii）8个先前未识别的CYP 3A 4泛素化位点，这些位点位于空间相关的Asp/Glu和可磷酸化的Ser/Thr残基簇内，可能用于接合每个E2-E3复合物。总的来说，我们的研究结果强调了ERAD中蛋白磷酸化和泛素化之间的相互作用，据我们所知，提供了通过蛋白磷酸化识别gp 78底物的第一个例子。
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.