DIOXIN-DEPENDENT ACTIVATION OF MURINE CYP1A-1 GENE-TRANSCRIPTION REQUIRES PROTEIN KINASE-C-DEPENDENT PHOSPHORYLATION
DIOXIN-DEPENDENT ACTIVATION OF MURINE CYP1A-1 GENE-TRANSCRIPTION REQUIRES PROTEIN KINASE-C-DEPENDENT PHOSPHORYLATION
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DOI：
10.1128/mcb.12.4.1856
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发表时间：
1992-04-01
影响因子：
5.3
通讯作者：
PUGA, A
中科院分区：
文献类型：
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作者：
CARRIER, F;OWENS, RA;PUGA, A
小鼠Cyp1a-1(细胞色素P(1)450)基因被2，3，7，8-四氯二苯并-对二恶英(TCDD)(二恶英)等诱导剂转录激活需要芳香烃受体，并与位于转录起始点上游约1kb的一个或多个芳香烃反应元件(AhRE)相互作用。我们发现，蛋白激酶活性的抑制剂2-氨基嘌呤处理小鼠肝癌HEPA-1细胞，可抑制TCDD诱导的细胞色素P1A1基因的表达，并抑制细胞色素P1A1酶活性的升高。凝胶迁移率改变分析表明，2-氨基嘌呤也能抑制AH受体与其AHRE靶标之间DNA-蛋白质复合体的形成。由于TCDD处理的HEPA-1细胞的核提取物在体外去磷酸化取消了ah受体与其同源AHRE序列形成特定复合体的能力，因此需要磷酸化来形成ah受体特异性复合体。为了确定几种已知的蛋白激酶中的任何一种是否参与了Cyp1a-1基因的转录调控，我们在TCDD诱导之前用其他九种蛋白激酶抑制剂处理HEPA-1细胞；分析了这些细胞的核提取物形成特定DNA-蛋白质复合体的能力。只有用蛋白激酶C抑制剂星形孢子素处理过的细胞的提取物才不能形成这些复合体。另外，星状孢子素可完全抑制TCDD诱导的细胞色素P1A1基因表达。用佛波酯延长处理时间使蛋白激酶C耗尽，可完全抑制TCDD诱导的细胞色素P1A1基因表达。我们的结论是：(I)磷酸化是形成转录复合体和转录激活Cyp1a-1基因所必需的；(Ii)磷酸化位点(S)存在于组成转录复合体的至少一个蛋白质上，可能是ah受体本身；(Iii)负责磷酸化的酶可能是蛋白激酶C。
Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYP1A1 mRNA induction by TCDD as well as the concomitant increase in CYP1A1 enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cyp1a-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYP1A1 mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYP1A1 mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cyp1a-1 gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the enzyme responsible for the phosphorylation is likely to be protein kinase C.