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药物和外源药物不直接与CAR结合来激活它。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。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，刺激去磷酸化，从而激活CAR和核易位。蛋白磷酸酶2A （PP2A）利用活化c -激酶1受体（RACK1）作为调控亚基催化这种去磷酸化。在对苯巴比妥的反应中，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型二聚体介导的机制，通过该机制，CAR控制其磷酸化以响应EGFR信号和苯巴比妥。磷酸化的CAR形成其同型二聚体，将PP2A/RACK1结合位点埋在同型二聚体界面内，阻止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同型二聚体，允许PP2A/RACK1结合并使CAR去磷酸化以激活。

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.