Berghard A, Gradin K, Pongratz I, Whitelaw M, Poellinger L
Center for Biotechnology, Karolinska Institute, Huddinge, Sweden.
Mol Cell Biol. 1993 Jan;13(1):677-89. doi: 10.1128/mcb.13.1.677-689.1993.
Signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) is mediated by the intracellular dioxin receptor which, in its dioxin-activated state, regulates transcription of target genes encoding drug-metabolizing enzymes, such as cytochrome P-450IA1 and glutathione S-transferase Ya. Exposure of the dioxin receptor to dioxin leads to an apparent translocation of the receptor to the nucleus in vivo and to a rapid conversion of the receptor from a latent, non-DNA-binding form to a species that binds to dioxin-responsive positive control elements in vitro. This DNA-binding form of receptor appears to be a heterodimeric complex with the helix-loop-helix factor Arnt. In this study, we show that activation of the cytochrome P-450IA1 gene and minimal dioxin-responsive reporter constructs by the dioxin receptor was inhibited following prolonged treatment of human keratinocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Inhibition of the receptor-mediated activation response was also achieved by treatment of the cells with a number of protein kinase inhibitors, one of which, calphostin C, shows selectivity for protein kinase C. Taken together, these data suggest that protein kinase C-dependent phosphorylation may play an essential role in the dioxin signaling pathway. This hypothesis is supported by the observation that pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate inhibited the DNA-binding activity of the dioxin receptor in vivo. In vivo, the dioxin receptor was found to be a phosphoprotein. In vitro, dephosphorylation of the ligand-activated, heteromeric dioxin receptor form or dephosphorylation of the individual ligand-binding and Arnt receptor subunits inhibited the xenobiotic response element-binding activity. Moreover, dephosphorylation experiments with the individual receptor subunits prior to assembly of the xenobiotic response element-binding receptor form indicated that phosphorylation seemed to be important for the DNA-binding activity per se of the receptor, whereas Arnt appeared to require phosphorylation to interact with the receptor. Finally, a protein kinase C inhibitor-sensitive cytosolic catalytic activity that could restore the DNA-binding activity of the dephosphorylated dioxin receptor form was identified.
二噁英（2,3,7,8-四氯二苯并对二噁英）的信号转导由细胞内二噁英受体介导，该受体在其被二噁英激活的状态下，调节编码药物代谢酶的靶基因的转录，如细胞色素P-450IA1和谷胱甘肽S-转移酶Ya。二噁英受体暴露于二噁英会导致该受体在体内明显易位至细胞核，并使其从潜在的、不与DNA结合的形式迅速转变为在体外能与二噁英反应性阳性对照元件结合的形式。这种受体的DNA结合形式似乎是与螺旋-环-螺旋因子Arnt形成的异二聚体复合物。在本研究中，我们发现，在用佛波酯12-O-十四酰佛波醇-13-乙酸酯长时间处理人角质形成细胞后，二噁英受体对细胞色素P-450IA1基因和最小二噁英反应性报告基因构建体的激活受到抑制。用多种蛋白激酶抑制剂处理细胞也能抑制受体介导的激活反应，其中一种抑制剂钙泊三醇对蛋白激酶C具有选择性。综上所述，这些数据表明蛋白激酶C依赖性磷酸化可能在二噁英信号通路中起关键作用。这一假设得到以下观察结果的支持：用12-O-十四酰佛波醇-13-乙酸酯预处理细胞会抑制二噁英受体在体内的DNA结合活性。在体内，发现二噁英受体是一种磷蛋白。在体外，配体激活的异源二聚体二噁英受体形式的去磷酸化或单个配体结合和Arnt受体亚基的去磷酸化会抑制外源性反应元件结合活性。此外，在组装外源性反应元件结合受体形式之前对单个受体亚基进行去磷酸化实验表明，磷酸化似乎对受体本身的DNA结合活性很重要，而Arnt似乎需要磷酸化才能与受体相互作用。最后，鉴定出一种对蛋白激酶C抑制剂敏感的胞质催化活性，它可以恢复去磷酸化的二噁英受体形式的DNA结合活性。
