Mechanisms and biological consequences of the nuclear receptor CAR activation

CAR激活机制：该系统的独特之处在于外源药物不直接与CAR结合来激活它。我们之前确定内源性CAR的苏氨酸48在小鼠原代肝细胞中被磷酸化，苯巴比妥治疗使这种苏氨酸去磷酸化，激活CAR并将其转运到细胞核中。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，活化c -激酶1受体（RACK1）被认为是激活PP2A核心酶使CAR的苏氨酸48去磷酸化的重要调控亚基。苯巴比妥通过去磷酸化苏氨酸38间接激活核受体CAR。现在我们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，使RACK1的酪氨酸52去磷酸化。非磷酸化的RACK1作为调控亚基，刺激蛋白磷酸酶2A去磷酸化CAR的苏氨酸38，使其活化。CAR含有分子内肽XRS来调节苯巴比妥引发的rack1信号，从而使苏氨酸38去磷酸化以激活其。在缺少XRS的情况下，RACK1无法与CAR结合并刺激PP2Ac的去磷酸化。我们的研究已经确定了苯巴比妥- egfr - rack1 /PP2Ac-XRS是CAR激活的主要机制。

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.