Identification of multiple cyclin subunits of human P-TEFb
Identification of multiple cyclin subunits of human P-TEFb
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DOI：
10.1101/gad.12.5.755
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发表时间：
1998-03-01
影响因子：
10.5
通讯作者：
Price, DH
中科院分区：
文献类型：
--
作者：
Peng, JM;Zhu, YR;Price, DH
从无效延伸到有效延伸的转变被认为是由正转录延伸因子 b (P-TEEb) 通过 RNA 聚合酶 II 最大亚基的羧基末端结构域 (CTD) 磷酸化来控制的。果蝇 P-TEFb 最近被鉴定为与细胞周期蛋白亚基（细胞周期蛋白 T）配对的细胞周期蛋白依赖性激酶 (CDK9)。我们在此展示了人 P-TEFb（T1 和 T2）的多个细胞周期蛋白亚基的克隆。由于选择性剪接，细胞周期蛋白 T2 有两种形式（T2a 和 T2b）。细胞周期蛋白 T1 和 T2 均普遍表达。对 HeLa 核提取物 (HNE) 进行的免疫沉淀和免疫耗竭实验表明，细胞周期蛋白 T1 和 T2 以相互排斥的方式与 CDK9 相关，并且几乎所有 CDK9 都与细胞周期蛋白 T1 或 T2 相关。 Sf9 细胞中产生的重组 CDK9/cyclin T1、CDK9/cyclin T2a 和 CDK9/cyclin T2b 具有 DRB 敏感激酶活性，并在体外转录延伸中发挥作用。激活 CDK9 需要细胞周期蛋白 T1 或 T2，并且细胞周期蛋白羧基末端的截短会降低但不会消除 P-TEFb 活性。共转染实验表明，所有三种 CDK9/细胞周期蛋白组合均显着激活 CMV 启动子。
The transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEEb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). We demonstrate here the cloning of multiple cyclin subunits of human P-TEFb (T1 and T2). Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicated that cyclin T1 and T2 were associated with CDK9 in a mutually exclusive manner and that almost all CDK9 was associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possessed DRB-sensitive kinase activity and functioned in transcription elongation in vitro. Either cyclin T1 or T2 was required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduced, but did not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter.