Mechanisms and biological consequences of the nuclear receptor CAR activation

核受体CAR激活机制：该系统的独特之处在于，治疗剂和外源性物质并不直接结合CAR来激活它，我们之前确定CAR的苏氨酸38通过其磷酸化和去磷酸化Sci来调节这种间接激活。信号了2013年）。 Thr 38的磷酸化使CAR失活并将其保留在细胞质中。药物如苯巴比妥抑制表皮生长因子受体（EGFR）信号传导以刺激CAR活化和核转位的去磷酸化。 蛋白磷酸酶2A（PP 2A）利用活化C激酶1受体（RACK 1）作为调节亚基催化这种去磷酸化。 响应于苯巴比妥，磷酸化CAR募集PP 2Ac/RACK 1进行去磷酸化和活化。在这里，我们现在已经确定了同源二聚体介导的机制，CAR通过该机制控制其对EGFR信号传导和苯巴比妥的磷酸化反应。 磷酸化的CAR形成其同源二聚体，其将PP 2A/RACK 1结合位点埋在同源二聚体界面内，防止CAR去磷酸化并保持CAR失活。 苯巴比妥间接和CAR配体直接解离CAR同二聚体，允许PP 2A/RACK 1结合并使CAR去磷酸化以活化。 CAR需要p38 MAPK来结合并激活其靶基因。一旦CAR结合启动子作为p38复合物，其在苏氨酸38处被p38磷酸化，从而失活。因此，p38连接细胞核中的CAR激活和失活。相反，CAR减弱小鼠肝脏中p38的磷酸化，这可能是肝细胞癌的细胞信号传导。 CAR的苏氨酸38作为磷酸化基序在大多数人核受体中是保守的。 我们检测了雌激素受体α的丝氨酸216，其在小鼠免疫细胞如中性粒细胞和脑小胶质细胞中被磷酸化。随后，我们产生了敲入小鼠（Esr 1 S216 A）携带非磷酸模拟Ser 216 Ala突变，并发现磷酸化ER α是小胶质细胞中的抗炎剂。我们对11种不同的核受体的研究将保守基序的磷酸化定义为核受体的蛋白质降解信号。发现FXR利用这种磷酸化来连接细胞核中的配体活化、失活和降解。因此，DNA结合结构域中保守的磷酸化基序为CAR以外的核受体调控提供了分子基础。 核受体PXR，功能和疾病： PXR被他汀类药物等药物激活。我们以前证明，他汀类药物激活的PXR招募蛋白磷酸2C去磷酸化丝氨酸/苏氨酸激酶2（SGK 2）。磷酸化的SGK 2，利用非磷酸化的SGK 2作为共调节子来激活致突变基因（Sci. Rep，2014）。 这种PXR-SGK 2信号传导可能导致他汀类药物治疗引起的副作用，增加血糖水平和发展2型糖尿病的风险。 我们现在已经确定PXR是葡萄糖调节的信号转导因子，其调节肝脏的新生。在禁食期间，小鼠肝脏中PXR在Ser 350处磷酸化。 这种磷酸化是由葡萄糖调节的。 牛痘病毒相关激酶1（VRK 1）可直接磷酸化人肝细胞中的Ser 350。与他汀类药物激活的PXR类似，磷酸化的PXR支架PP 2C使SGK 2去磷酸化，从而激活致肿瘤基因。因此，PXR最初似乎是作为一种信号传导器在禁食或饥饿期间维持葡萄糖稳态。

Nuclear receptor CAR activation mechanism: What is unique about this system is the fact that therapeutics and xenobiotics do not directly bind to CAR to activate it. We previously determined that threonine 38 of CAR regulates this indirect activation through its phosphorylation and dephosphorylation Sci. Signal. 2013). Phosphorylation of Thr38 inactivates CAR and retains it in the cytoplasm. Drugs such as phenobarbital repress epidermal growth factor receptor (EGFR) signaling to stimulate dephosphorylation for CAR activation and nuclear translocation. Protein phosphatase 2A (PP2A) catalyzes this dephosphorylation utilizing the receptor for activated C-kinase1 (RACK1) as the regulatory subunit. In response to phenobarbital, phosphorylated CAR recruits PP2Ac/RACK1 for dephosphorylation and activation. Here we have now determined the homodimer-mediated mechanism by which CAR controls its phosphorylation in response to EGFR signaling and to phenobarbital. Phosphorylated CAR forms its homodimer that buries the PP2A/RACK1 binding site within the homodimer interface, preventing CAR from dephosphorylation and keeping CAR being inactivated. Phenobarbital indirectly and CAR ligands directly dissociate CAR homodimer, allowing PP2A/RACK1 to bind and dephosphorylate CAR for activation. CAR is found to require p38MAPK to bind to and activate its target genes. Once CAR binds and a promoter as a p38 complex, it is phosphorylated at threonine 38 by p38, thereby inactivated. Thus, p38 links CAR activation and inactivation in the nucleus. Conversely, CAR attenuates phosphorylation of p38 in mouse liver, which can be a cell signaling for hepatocellular carcinoma. Threonine 38 of CAR is conserved as a phosphorylation motif in the majority of human nuclear receptors. We examined serine 216 of estrogen receptor alpha that is phosphorylated in mouse immune cells such as neutrophils and brain microglia. Subsequently, we generated Knock-In mice (Esr1S216A) bearing non-phosphomimetic Ser216Ala mutation and found that phosphorylated ERalpha is an anti-inflammatory in microglia. Our study with 11 different nuclear receptors defined phosphorylation of the conserved motif as protein degradation signal of nuclear receptors. FXR was found to utilize this phosphorylation to link ligand activation, inactivation and degradation in the nucleus. Thus, the conserved phosphorylation motif within the DNA binding domain has provided a molecular basis for nuclear receptor regulations beyond CAR. Nuclear receptor PXR, functions and diseases: PXR is activated by drugs such as statins. We previously demonstrated that statin-activated PXR recruits protein phosphate 2C to dephosphorylate serine/threonine kinase 2 (SGK2). Phosphorylated SGK2, utilizing non-phosphorylated SGK2 as a co-regulator to activate gluconeogenic genes (Sci. Rep, 2014). This PXR-SGK2 signaling may contribute to side-effects caused by statin therapy, increasing blood glucose levels and risk to develop type 2 diabetes. We have now characterized that PXR is glucose-regulated signal transduction factor that regulate hepatic gluconeogenesis. PXR is phosphorylated at Ser350 in mouse liver during fasting. This phosphorylation is regulated by glucose. Vaccinia virus-related kinase 1 (VRK1) directly phosphorylates Ser350 in human liver cells cultured in low glucose media. Similar to statin-activated PXR, phosphorylated PXR scaffolds PP2C to dephosphorylate SGK2, activating gluconeogenic genes. Thus, it appears that PXR has originally eveloved as a signal transducer to maintain glucose homeostasis during fasting or starvation.