Pin1 modulates the dephosphorylation of the RNA polymerase IIC-terminal domain by yeast Fcp1
Pin1 modulates the dephosphorylation of the RNA polymerase IIC-terminal domain by yeast Fcp1
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DOI：
10.1016/s0014-5793(02)02288-3
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发表时间：
2002-02-27
期刊：
影响因子：
3.5
通讯作者：
Lu, KP
中科院分区：
文献类型：
--
作者：
Kops, O;Zhou, XZ;Lu, KP
丝氨酸和苏氨酸残基N端的可逆磷酸化(pSer/Thr-Pro)是细胞内重要的信号机制。PSer/Thr-Pro部分以两种不同的顺式和反式构象存在，其转化由PPIase Pin1催化。其中，Pin1与RNA聚合酶11最大亚基的磷酸化C末端结构域(CTD)结合，但这种相互作用的生化和功能相关性尚不清楚。在这里，我们证实了CTD磷酸酶Fcp1可以抑制酵母中的Pin1突变。此外，这种遗传相互作用需要Fcp1的磷酸酶结构域和BRCT结构域，这表明Fcp1的定位具有关键作用。基于这些观察，我们开发了一种新的体外分析方法来分析Fcp1对CTD去磷酸化的作用，该方法只使用重组蛋白并模拟体内情况。这一实验为我们提供了强有力的证据，证明Pin1在体外能够刺激Fcp1对CTD去磷酸化，并且这种刺激依赖于Pin1‘S PPIase的活性。最后，Pin1显著增加了酵母中CtD在Ser(5)-Pro基序上的去磷酸化，而不是在Ser(2)-Pro上的去磷酸化，这可以用Pin1‘S底物特异性来解释。综上所述，我们的结果表明Pin1在调节CTD磷酸化中发挥了新的作用，并为依赖Pro异构化的蛋白质去磷酸化提供了进一步的例子。(C)2002年欧洲生化学会联合会。爱思唯尔科学公司出版。版权所有。
The reversible phosphorylation of serine and threonine residues N-terminal to proline (pSer/Thr-Pro) is an important signaling mechanism in the cell. The pSer/Thr-Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl-prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of the RNA polymerase 11, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1's PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser(5)-Pro motif, but not on Ser(2)-Pro in yeast, which can be explained with Pin1's substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization-dependent protein dephosphorylation. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.