Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription.
Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription.
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DOI：
10.1186/1742-4690-2-47
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发表时间：
2005-07-27
期刊：
影响因子：
3.3
通讯作者：
Nekhai S
中科院分区：
文献类型：
--
作者：
Ammosova T;Washington K;Debebe Z;Brady J;Nekhai S
HIV-1达特蛋白募集人正转录延伸因子P-TEFb（由CDK 9和细胞周期蛋白T1组成）至HIV-1反式激活应答（TAR）RNA。CDK 9通过TFIIH维持在去磷酸化状态，并在TFIIH解离时进行磷酸化。因此，CDK 9在与HIV-1前起始复合物结合之前的去磷酸化可能对HIV-1转录很重要。其他人和我们以前表明，蛋白磷酸酶-2A和蛋白磷酸酶-1调节HIV-1的转录。在本研究中，我们分析了PP 2A和PP 1在体外和体内对CDK 9去磷酸化和HIV-1转录的相对贡献。在体外，PP 2A而非PP 1使CDK 9自磷酸化去磷酸化，并减少P-TEFb、达特和TAR RNA之间的复合物形成。冈田酸抑制PP 2A抑制基础以及Tat诱导的HIV-1转录，而PP 1的重组核抑制剂（NIPP 1）抑制PP 1只抑制Tat诱导的体外转录。在培养的细胞中，低浓度的冈田酸，抑制PP 2A，只有轻度抑制Tat诱导的HIV-1转录。相反，Tat介导的HIV-1转录被NIPP 1的表达强烈抑制。冈田酸诱导内源性以及瞬时表达的CDK 9的磷酸化，但在表达NIPP 1的细胞中未观察到这种诱导。此外，冈田酸不诱导具有Thr 186突变或具有参与CDK 9自磷酸化的Ser-329、Thr-330、Thr-333、Ser-334、Ser-347、Thr-350、Ser-353和Thr-354残基突变的CDK 9的磷酸化。我们的研究结果表明，虽然PP 2A在体外去磷酸化自磷酸化的CDK 9，在培养的细胞中，PP 1可能去磷酸化CDK 9，并有助于调节激活的HIV-1转录。
HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo. In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9. Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription.
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