A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor
A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor
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DOI：
10.1124/dmd.108.024695
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发表时间：
2009-04-01
影响因子：
3.9
通讯作者：
Chen, Taosheng
中科院分区：
文献类型：
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作者：
Pondugula, Satyanarayana R.;Brimer-Cline, Cynthia;Chen, Taosheng
孕烷X受体（PXR）在多种生理过程中发挥着至关重要的作用。然而，负责的信号传导机制还没有很好地定义;最有可能的是，PXR的多种功能是由其磷酸化调节的。因此，我们试图确定高度保守的Thr（57）突变是否影响人类PXR（hPXR）功能。进行定点诱变以产生磷酸化缺陷型（hPXR（T57 A））和磷酸化模拟型（hPXR（T57 D））突变体。基因报告，蛋白质印迹，免疫细胞化学，哺乳动物双杂交和电泳迁移率变动分析被用来研究细胞色素P450 3A 4（CYP 3A 4）启动子激活，蛋白水平，定位，辅因子相互作用，和CYP 3A 4启动子结合的hPXR突变体，分别。hPXR（T57 D）而不是hPXR（T57 A）失去了其转录活性。这两种突变都没有改变hPXR的蛋白水平和与类固醇受体共激活因子-1的相互作用。hPXR和hPXR（T57 A）表现出均匀的核分布，而hPXR（T57 D）表现出独特的点状核定位模式，类似于与类维生素A和甲状腺受体（SMRT）沉默介体共定位的功能受损的hPXR突变体，尽管SMRT的沉默并不能挽救hPXR（T57 D）改变的功能。然而，hPXR（T57 D），而不是hPXR（T57 A），削弱了hPXR结合CYP 3A 4启动子的能力，与突变体的反式激活功能一致。此外，70 kDa形式的核糖体蛋白S6激酶（p70 S6 K）在体外磷酸化hPXR并抑制其转录活性，而hPXR（T57 A）部分抵抗p70 S6 K的抑制作用。我们的研究确定了一个功能显著的磷酸化模拟突变体（hPXR（T57 D）），并显示p70 S6 K磷酸化和hPXR反式激活的调节，以支持磷酸化在调节hPXR功能中起重要作用的观点。
The pregnane X receptor (PXR) plays crucial roles in multiple physiological processes. However, the signaling mechanisms responsible are not well defined; it is most likely that multiple functions of PXR are modulated by its phosphorylation. Therefore, we sought to determine whether mutation at a highly conserved Thr(57) affects human PXR (hPXR) function. Site-directed mutagenesis was performed to generate phosphorylation-deficient (hPXR(T57A)) and phosphomimetic (hPXR(T57D)) mutants. Gene reporter, Western blotting, immunocytochemistry, mammalian two-hybrid, and electrophoretic mobility shift assays were used to study cytochrome P450 3A4 (CYP3A4) promoter activation, protein levels, localization, cofactor interaction, and CYP3A4 promoter binding of the hPXR mutants, respectively. hPXR(T57D), but not hPXR(T57A), lost its transcriptional activity. Neither mutation altered hPXR's protein levels and interaction with steroid receptor coactivator-1. hPXR and hPXR(T57A) exhibited a homogenous nuclear distribution, whereas hPXR(T57D) exhibited a distinctive punctate nuclear localization pattern similar to that of hPXR mutants with impaired function that colocalize with silencing mediator of retinoid and thyroid receptors (SMRT), although silencing of SMRT did not rescue the altered function of hPXR(T57D). However, hPXR(T57D), but not hPXR(T57A), impaired hPXR's ability to bind to the CYP3A4 promoter, consistent with the mutant's transactivation function. Furthermore, the 70-kDa form of ribosomal protein S6 kinase (p70 S6K) phosphorylated hPXR in vitro and inhibited its transcriptional activity, whereas hPXR(T57A) partially resisted the inhibitory effect of p70 S6K. Our studies identify a functionally significant phosphomimetic mutant (hPXR(T57D)) and show p70 S6K phosphorylation and regulation of hPXR transactivation to support the notion that phosphorylation plays important roles in regulating hPXR function.