Mechanisms and biological consequences of the nuclear receptor CAR activation

CAR激活机制：这个系统的独特之处在于，外源生物不会直接与CAR结合来激活它。我们先前确定内源性CAR的苏氨酸48在小鼠原代肝细胞中被磷酸化，苯巴比妥治疗使该苏氨酸去磷酸化，激活CAR并将其移位到细胞核中。我们确定蛋白磷酸酶2A是使CAR的苏氨酸48去磷酸化的酶。此外，激活的C-激酶受体1(RACK1)被认为是激活PP2A核心酶使CAR的苏氨酸48去磷酸化的重要调节亚基。苯巴比妥通过使苏氨酸38去磷酸化间接激活核受体CAR。我们现在已经确定了苯巴比妥激活CAR的潜在机制。苯巴比妥与EGFR的结合启动细胞信号，使RACK1的酪氨酸52去磷酸化。非磷酸化的RACK1作为调节亚基，刺激蛋白磷酸酶2A去磷酸化CAR中的苏氨酸38，使其激活。CAR含有分子内多肽XRS，可以调节苯巴比妥启动的RACK1信号，从而使苏氨酸38去磷酸化，从而激活它。在没有XRS的情况下，RACK1不能与CAR结合，并通过PP2Ac刺激去磷酸化。我们的研究确定苯巴比妥-EGFR-RACK1/PP2Ac-XRS是CAR激活的主要机制。 CAR中的苏氨酸38在大多数人类核受体如雌激素受体中作为磷酸化基序保守。我们检测了人类雌激素受体的丝氨酸212(小鼠ER中的丝氨酸216)，发现这种丝氨酸残基在中性粒细胞、嗜酸性粒细胞和单核细胞等白细胞的ER中被磷酸化。丝氨酸212的突变分析显示，磷酸化的ER调控着一组独特的基因。利用磷酸化的S216多肽抗体，现已证实在丝氨酸216处磷酸化的小鼠ER在中性粒细胞中表达，并调节子宫的迁移和渗透。因此，这种磷酸化基序可能赋予特定的核受体以不同的功能，远远超出CAR的功能。 异源生物信号串扰机制：在被外源生物激活后，CAR对基因的调控是不同的，赋予了CAR调控的基因表达的特异性。CAR通过与细胞信号的串扰获得这种特异性。我们已经确定了多种内源性细胞信号是CAR激活和功能的基本调节因子：p38MAPK、SGK2和GADD45(生长停滞和DNA损伤诱导的45)。鉴于这些发现，我们正在研究这些信号调节CAR激活和功能的分子机制。 CAR介导的疾病：慢性药物治疗，如苯巴比妥，已知会激活CAR并导致啮齿类动物的肝细胞癌(HCC)。我们现在已经将KCNK1和GADD45确定为苯巴比妥促进肝癌发展的CAR靶点：CAR与GADD45蛋白相互作用，这种相互作用抑制JNK1的磷酸化，从而抑制细胞凋亡，并可能促进肿瘤的发生。KCNK1是在男性肝脏中特异性诱导的，可以减轻肝脏增生。多溴联苯醚PBDE-47(2，2，4，4-四溴二苯基醚)是一种持久性和生物累积性溴化阻燃剂，几乎在所有美国人中都有发现。它与人类的多种健康影响(例如，内分泌干扰、生殖影响、发育神经毒性)有关，现在被发现是一种强大的人类汽车激活剂。他汀类药物现在被发现可以激活PXR-SGK2信号，诱导肝脏糖异生。

CAR activation mechanism: What is unique about this system is the fact that xenobiotics do not directly bind to CAR to activate it. We previously determined that threonine 48 of endogenous CAR is phosphorylated in mouse primary hepatocytes and that phenobarbital treatment de-phosphorylates this threonine, activating CAR and translocating it into the nucleus. We identified protein phosphatase 2A as the enzyme that de-phosphorylates threonine 48 of CAR. Moreover, receptor for activated C-kinase1 (RACK1) was characterized as the essential regulatory subunit that activates the PP2A core enzyme to de-phosphorylate threonine 48 of CAR. Phenobarbital indirectly activates nuclear receptor CAR by de-phosphorylating threonine 38. Here we have now determined the underlying mechanism through which phenobarbital activates CAR. Phenobarbital binding to EGFR initiates cell signaling to de-phosphorylate tyrosine 52 of RACK1. Non-phosphorylated RACK1, acting as the regulatory subunit, stimulates protein phosphatase 2A to de-phosphorylate threonine 38 of CAR for its activation. CAR contains the intra-molecular peptide XRS to regulates phenobarbital-initiated RACK1signaling, thereby de-phosphorylating threonine 38 for its activation. In the absence of the XRS, RACK1 is unable to bind to CAR and stimulates de-phosphorylation by PP2Ac. Our investigations have defined the phenobarbital-EGFR-RACK1/PP2Ac-XRS as the principle mechanism for CAR activation. Threonine 38 of CAR is conserved as a phosphorylation motif in the majority of human nuclear receptors such as estrogen receptors. We examined serine 212 of human estrogen receptor (serine 216 in mouse ER) and found that this serine residue is phosphorylated in ER in leukocytes such as neutrophils, eosinophils and monocytes. Mutation analysis of serine 212 revealed that phosphorylated ER regulates a unique set of the genes. Utilizing phosphor-S216 peptide antibody, it is now confirmed that mouse ER phosphorylated at serine 216 is expressed in neutrophils and regulates migration and infiltration into the uterus.Therefore, this phosphorylation motif may confer distinct functions to a given nuclear receptor far beyond that of CAR. Xenobiotic-signal crosstalk mechanism: Upon activation by xenobiotics, CAR regulates genes differently from one another, conferring specificity to CAR-regulated gene expression. CAR acquires this specificity via crosstalk with cell signaling. We have identified various endogenous cell signals as the essential regulator of CAR activation and function: p38 MAPK, SGK2 and the GADD45 (growth arrest and DNA-damage inducible 45). Given these findings, we are investigating the molecular mechanisms by which these signaling regulate CAR activation and functions. CAR-mediated diseases: Chronic treatment with drugs, such as phenobarbital, is known to activate CAR and cause hepatocellular carcinoma (HCC) in rodents. We have now characterized KCNK1 and GADD45 as a CAR target for phenobarbital promotion of HCC development: CAR interacts with GADD45 protein and this interaction inhibits phosphorylation of JNK1, thus repressing apoptosis and possibly promoting tumor genesis. KCNK1 is specifically induced in male livers and attenuates hepatic hyperplasia. Polybrominated diphenyl ether PBDE-47 (2, 2, 4, 4-tetrabromodipheyl ether), a persistent and bioaccumulative brominated flame retardant, is found in nearly all Americans. It has been associated with a wide variety of health effects in people (e.g., endocrine disruption, reproductive effects, developmental neurotoxicity)is now found to be a strong human CAR activator. Statins is now found to activate the PXR-SGK2 signaling to induce hepatic gluconeogenesis.