Washington Kareem, Ammosova Tatyana, Beullens Monique, Jerebtsova Marina, Kumar Ajit, Bollen Mathieu, Nekhai Sergei
Center for Sickle Cell Disease, Department of Biochemistry and Molecular Biology, Howard University, 2121 Georgia Avenue, Washington, D. C. 20059, USA.
J Biol Chem. 2002 Oct 25;277(43):40442-8. doi: 10.1074/jbc.M205687200. Epub 2002 Aug 15.
Transcription by RNA polymerase-II (RNAPII) is controlled by multisite phosphorylation of the heptapeptide repeats in the C-terminal domain (CTD) of the largest subunit. Phosphorylation of CTD is mediated by the cyclin-dependent protein kinases Cdk7 and Cdk9, whereas protein serine/threonine phosphatase FCP1 dephosphorylates CTD. We have recently reported that human immunodeficiency virus-1 (HIV-1) transcription is positively regulated by protein phosphatase-1 (PP1) and that PP1 dephosphorylates recombinant CTD. Here, we provide further evidence that PP1 can dephosphorylate RNAPII CTD. In vitro, PP1 dephosphorylated recombinant CTD as well as purified RNAPII CTD. HeLa nuclear extracts were found to contain a species of PP1 that dephosphorylates both serine 2 and serine 5 of the heptapeptide repeats. In nuclear extracts, PP1 and FCP1 contributed roughly equally to the dephosphorylation of serine 2. PP1 co-purified with RNAPII by gel filtration and associated with RNAPII on immunoaffinity columns prepared with anti-CTD antibodies. In cultured cells treated with CTD kinase inhibitors, the dephosphorylation of RNAPII on serine 2 was inhibited by 45% by preincubation with okadaic acid, which inhibits phosphatases of PPP family, including PP1 but not FCP1. Our data demonstrate that RNAPII CTD is dephosphorylated by PP1 in vitro and by PPP-type phosphatase, distinct from FCP1, in vivo.
RNA聚合酶II（RNAPII）的转录受最大亚基C末端结构域（CTD）中七肽重复序列多位点磷酸化的控制。CTD的磷酸化由细胞周期蛋白依赖性蛋白激酶Cdk7和Cdk9介导，而蛋白丝氨酸/苏氨酸磷酸酶FCP1使CTD去磷酸化。我们最近报道，蛋白磷酸酶-1（PP1）对人类免疫缺陷病毒1型（HIV-1）转录起正调控作用，且PP1使重组CTD去磷酸化。在此，我们提供进一步证据表明PP1可使RNAPII CTD去磷酸化。在体外，PP1使重组CTD以及纯化的RNAPII CTD去磷酸化。发现HeLa细胞核提取物中含有一种PP1，它可使七肽重复序列的丝氨酸2和丝氨酸5去磷酸化。在细胞核提取物中，PP1和FCP1对丝氨酸2去磷酸化的贡献大致相同。PP1通过凝胶过滤与RNAPII共纯化，并在用抗CTD抗体制备的免疫亲和柱上与RNAPII结合。在用CTD激酶抑制剂处理的培养细胞中，用冈田酸预孵育可使RNAPII丝氨酸2的去磷酸化受到45%的抑制，冈田酸可抑制包括PP1但不包括FCP1的PPP家族磷酸酶。我们的数据表明，RNAPII CTD在体外由PP1去磷酸化，在体内由不同于FCP1的PPP型磷酸酶去磷酸化。
