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，外源性物质不直接与CAR结合以激活它。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，苯巴比妥处理使该苏氨酸去磷酸化，激活CAR并将其易位到细胞核中。我们鉴定了蛋白磷酸酶2A作为使CAR的苏氨酸48去磷酸化的酶。此外，活化的C-激酶1受体（receptor for activated C-kinase 1，RACK 1）被表征为激活PP 2A核心酶以使CAR的苏氨酸48去磷酸化的必需调节亚基。苯巴比妥通过使苏氨酸38去磷酸化而间接激活核受体CAR。 在这里，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结合启动细胞信号传导以使RACK 1的酪氨酸52去磷酸化。非磷酸化的RACK 1作为调节亚基，刺激蛋白磷酸酶2A使CAR的苏氨酸38去磷酸化以使其活化。 CAR包含分子内肽XRS，以调节苯巴比妥引发的RACK 1信号传导，从而使苏氨酸38去磷酸化以使其活化。在不存在XRS的情况下，RACK 1不能与CAR结合并通过PP 2Ac刺激去磷酸化。 我们的研究已经将苯巴比妥-EGFR-RACK 1/PP 2Ac-XRS定义为CAR激活的主要机制。 CAR的苏氨酸38作为磷酸化基序在大多数人核受体如雌激素受体中是保守的。 我们检测了人雌激素受体的丝氨酸212（小鼠ER中的丝氨酸216），发现该丝氨酸残基在白细胞（如中性粒细胞、嗜酸性粒细胞和单核细胞）的ER中被磷酸化。 丝氨酸212的突变分析显示，磷酸化ER调节一组独特的基因。因此，这种磷酸化基序可以赋予给定的核受体远远超过CAR的独特功能。 外源性信号串扰机制：在被外源性物质激活后，CAR调节基因彼此不同，赋予CAR调节的基因表达特异性。CAR通过与细胞信号传导的串扰获得这种特异性。我们已经鉴定了多种内源性细胞信号作为CAR活化和功能的重要调节因子：p38 MAPK、SGK 2和GADD 45（生长停滞和DNA损伤诱导型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：已知用药物（如苯巴比妥）进行慢性治疗会激活CAR并在啮齿动物中引起肝细胞癌（HCC）。我们现在已经将KCNK 1和GADD 45表征为苯巴比妥促进HCC发展的CAR靶点：CAR与GADD 45蛋白相互作用，这种相互作用抑制JNK 1的磷酸化，从而抑制细胞凋亡并可能促进肿瘤发生。 KCNK 1在雄性肝脏中特异性诱导并减弱肝脏增生。已知3，5-二乙氧羰基-1，4-二氢可力丁（DDC）治疗可引起严重的肝损伤并使出生后肝祖卵圆细胞增殖。利用CAR KO小鼠，我们已经确定DDC激活CAR，并且这种激活对于肝损伤和卵圆细胞增殖的发展至关重要。

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. 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. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment is known to cause severe liver injury and proliferate postnatal hepatic progenitor oval cells. Utilizing CAR KO mice, we have determined that DDC activates CAR and this activation is essential for the developments of liver injury and oval cell proliferation.