Chemical Tools To Decipher Regulation of Phosphatases by Proline Isomerization on Eukaryotic RNA Polymerase II.
Chemical Tools To Decipher Regulation of Phosphatases by Proline Isomerization on Eukaryotic RNA Polymerase II.
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DOI：
10.1021/acschembio.5b00296
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发表时间：
2015-10-16
影响因子：
4
通讯作者：
Zhang YJ
中科院分区：
文献类型：
--
作者：
Mayfield JE;Fan S;Wei S;Zhang M;Li B;Ellington AD;Etzkorn FA;Zhang YJ
脯氨酸异构化极大地影响生物信号，但在蛋白质中是微妙且难以检测的。我们的特点是这种知之甚少的RNA聚合酶II羧基末端结构域（CTD）磷酸化状态的调控机制，使用新的，直接的，定量的化学工具。我们确定了三个CTD磷酸酶的脯氨酸异构体偏好：Ssu 72作为顺式脯氨酸特异性，Scp 1和Fcp 1作为强烈的反式偏好。由于这种固有的特性，这些磷酸酶对催化脯氨酸异构化的酶（如PIN 1/Pin 1）的反应不同。我们证明，这种选择性调节RNA聚合酶II磷酸化状态存在于人类细胞内，与体外试验一致。这些结果支持了一种模型，其中脯氨酸异构酶选择性地增强了对顺式脯氨酸具有特异性的CTD调节因子的一个子集的活性，而不是下游酶活性的整体增强。这导致CTD在体外和细胞中的多样化磷酸化状态。我们提供了化学工具来研究脯氨酸异构化及其在转录和其他生物学环境中选择性增强信号传导的能力。
Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.