Mechanisms and biological consequences of the nuclear receptor CAR activation

核受体CAR激活机制：这个系统的独特之处在于，治疗药物和外源药物并不直接与CAR结合来激活它。我们先前确定，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。2013)。Thr38的磷酸化使CAR失活，并将其保留在细胞质中。苯巴比妥等药物抑制表皮生长因子受体(EGFR)信号，刺激CAR激活和核转位的去磷酸化。蛋白磷酸酶2A(PP2A)利用活化的C-激酶受体1(RACK1)作为调节亚单位来催化这种去磷酸化。作为对苯巴比妥的反应，磷酸化的CAR招募PP2Ac/RACK1进行去磷酸化和激活。在这里，我们现在已经确定了同源二聚体介导的CAR控制其磷酸化的机制，以响应EGFR信号和苯巴比妥。磷酸化的CAR形成其同源二聚体，将PP2A/RACK1结合位点埋入同源二聚体界面，防止CAR去磷酸化并保持CAR失活。苯巴比妥间接地和CAR配体直接解离CAR同源二聚体，使PP2A/RACK1结合并去磷酸化CAR以进行激活。 CAR被发现需要p38MAPK与其靶基因结合并激活。一旦CAR作为p38复合体与启动子结合，它就被p38在苏氨酸38处磷酸化，从而失活。因此，p38将细胞核内CAR的激活和失活联系在一起。相反，CAR减弱了小鼠肝脏中p38的磷酸化，这可能是肝细胞癌的一个细胞信号。 CAR中的苏氨酸38在大多数人类核受体中作为磷酸化基序保守。我们检测了雌激素受体α的丝氨酸216，它在小鼠免疫细胞中被磷酸化，如中性粒细胞和脑小胶质细胞。随后，我们产生了携带非仿磷Ser216Ala突变的敲入小鼠(Esr1S216A)，并发现磷酸化的ERpha在小胶质细胞中具有抗炎作用。我们对11种不同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。FXR被发现利用这种磷酸化将核内配体的激活、失活和降解联系在一起。因此，DNA结合域中保守的磷酸化基序为超越CAR的核受体调控提供了分子基础。 核受体PXR、功能与疾病： PXR被他汀类药物激活。我们先前证明了他汀类激活的PXR招募蛋白磷酸2C去磷酸化丝氨酸/苏氨酸激酶2(SGK2)。磷酸化的SGK2，利用非磷酸化的SGK2作为辅助调节因子来激活糖异生基因(Sci.代表，2014)。这种PXR-SGK2信号可能与他汀类药物治疗引起的副作用、升高血糖水平和发展成2型糖尿病的风险有关。 我们现在已经确定PXR是葡萄糖调节的信号转导因子，调节肝脏糖异生。在禁食期间，PXR在小鼠肝脏的Ser350处被磷酸化。这种磷酸化受葡萄糖的调节。痘苗病毒相关蛋白1(VRK1)直接磷酸化低糖培养的人肝细胞中的Ser350。与他汀类激活的PXR类似，磷酸化的PXR支架PP2C去磷酸化SGK2，激活糖异生基因。因此，PXR最初似乎被认为是一种信号转导，在禁食或饥饿期间维持血糖稳态。

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.