A kinase subunit of the human mediator complex, CDK8, positively regulates transcriptional activation
A kinase subunit of the human mediator complex, CDK8, positively regulates transcriptional activation
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DOI：
10.1111/j.1365-2443.2007.01036.x
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发表时间：
2007-01-01
期刊：
影响因子：
2.1
通讯作者：
Ohkuma, Yoshiaki
中科院分区：
文献类型：
--
作者：
Furumoto, Tadashi;Tanaka, Aki;Ohkuma, Yoshiaki
人甲状腺激素受体相关蛋白（TRAP）/介体和相关复合物通过调节因子介导转录。为了进一步了解这些复合物的结构和功能多样性，我们建立了三个HeLa细胞系，每个细胞系表达三个表位标记的人TRAP/介体亚基MED 6，MED 7和CDK 8中的一个，并通过亲和和HPLC-凝胶过滤色谱分离包含这些亚基的复合物。来自每个细胞系的最大复合物的分子量为1.5 MDa，并且具有几乎相同的亚基组成;我们将这些复合物命名为TRAP/介体样复合物1（TMLC 1）。另外观察到两种潜在的亚复合物：CDK 8细胞系（TMLC 2）的1-MDa复合物和MED 6细胞系（TMLC 3）的600-kDa复合物。这三种复合物都在体外调节转录; TMLC 1和TMLC 3增强转录激活，而TMLC 2抑制转录激活。TMLC 1和TMLC 2磷酸化RNA聚合酶II（Pol II），但TMLC 3没有。此外，TMLC 1主要与一般转录因子TFIIE、TFIIF和TFIIH相互作用，这些转录因子在转录起始和向延伸过渡期间发挥作用。在最后一个实验中，使用RNA干扰敲低CDK 8阻止了在内切酶测定中Gal 4-VP 16的转录激活。这与TMLC 1在体外对转录的影响一起表明CDK 8在转录激活中起积极作用。
The human thyroid hormone receptor-associated proteins (TRAP)/Mediator and related complexes mediate transcription through regulatory factors. To further understand the structural and functional diversity of these complexes we established three HeLa cell lines each expressing one of three epitope-tagged human TRAP/Mediator subunits, MED6, MED7, and CDK8 and isolated the complexes in which these subunits were contained by affinity and HPLC-gel filtration chromatography. The largest complexes from each cell line had a molecular mass of 1.5 MDa and possessed almost identical subunit compositions; we designated these complexes TRAP/Mediator-like complex 1 (TMLC1). Two potential subcomplexes were additionally observed: a 1-MDa complex from the CDK8-cell line (TMLC2) and a 600-kDa complex from the MED6-cell line (TMLC3). All three complexes regulated transcription in vitro; TMLC1 and TMLC3 augmented transcriptional activation, whereas TMLC2 repressed it. TMLC1 and TMLC2 phosphorylated RNA polymerase II (Pol II), but TMLC3 did not. Furthermore, TMLC1 predominantly interacted with the general transcription factors TFIIE, TFIIF, and TFIIH, which function during transcription initiation and the transition to elongation. In a final experiment, knockdown of CDK8 using RNA interference prevented transcriptional activation by Gal4-VP16 in a luciferase-assay. This, together with the effect of TMLC1 on transcription in vitro, suggests that CDK8 play positive roles in transcriptional activation.