Kim J B, Sharp P A
Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 2001 Apr 13;276(15):12317-23. doi: 10.1074/jbc.M010908200. Epub 2001 Jan 5.
The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol II in vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.
细胞周期蛋白依赖性激酶9（CDK9）-细胞周期蛋白T激酶复合物，即正性转录延伸因子b（P-TEFb），在人类免疫缺陷病毒转录过程中刺激RNA聚合酶（Pol）II的延伸过程。P-TEFb与人免疫缺陷病毒Tat蛋白及反式激活应答元件结合形成特异性复合物，从而介导有效的延伸。在此，我们表明，在体外，与RNA Pol II的羧基末端结构域相比，P-TEFb优先磷酸化hSPT5。P-TEFb对hSPT5的磷酸化发生在其羧基末端重复结构域的苏氨酸和丝氨酸残基上。此外，我们提供了多条证据表明P-TEFb是一种不依赖细胞周期蛋白依赖性激酶激活激酶（CAK）的激酶。例如，CDK9不会被CAK磷酸化，而CDK2-细胞周期蛋白A激酶活性会被CAK显著增强。因此，P-TEFb很可能以一种不依赖CAK的方式，通过磷酸化其底物hSPT5和RNA Pol II的CTD来参与RNA Pol II延伸的调控。
