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首页> 外文期刊>Cellular Signalling >Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle
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Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle

机译：细胞周期M期细胞周期蛋白依赖性蛋白激酶使磷酸酶抑制剂2中的Pro-X-Thr-Pro位点磷酸化

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Protein phosphorylation serves as a primary mechanism for triggering events during mitosis and depends on coordinated regulation of kinases and phosphatases. Protein Ser-Thr phosphatase-1 (PP1) activity is essential for the metaphase to anaphase transition and the most ancient regulator of PP1 conserved from yeast to human is inhibitor-2 (I-2), an unstructured heat-stable protein. A unique sequence motif in I-2 from various species surrounds a phosphorylation site PXTP that can be phosphorylated in biochemical assays by GSK3, MAPK and CDK kinases. Here we used a phosphosite specific antibody to investigate the phosphorylation of I-2. We fractioned extracts from HeLa cells arrested with nocodazole and assayed for PXTP kinases using recombinant I-2. One major and two minor peaks of kinase activity were identified and the major peak contained both active MAPK and cdk1::cyclinB1, confirmed by immumoblotting. Cells released from a double thymidine block synchronously progressed through mitosis and immunoblotting revealed transient phosphorylation of endogenous I-2 in cells only during mitosis, and corresponding phosphorylation of histone H3 (Ser10) and PP1 (Thr320). Activation of cdk1::cyclinB1 was coincident with I-2 phosphorylation, but neither MAPK nor GSK3 were phosphorylated at this time, so we concluded that in living cells only cdk1::cyclinB1 phosphorylated the PXTP site in 1-2. Immunofluorescent staining of cells with the PXTP phosphosite antibody revealed highly specific staining of mitotic cells prior to anaphase, at which point the staining disappeared. Thus, phosphorylation of I-2 is catalyzed by cdk1::cyclinB1 and staining with a specific antibody should prove useful as a selective marker of cells in the early stages of mitosis. (c) 2005 Published by Elsevier Inc.
机译：蛋白质磷酸化是有丝分裂期间触发事件的主要机制，并且取决于激酶和磷酸酶的协调调节。蛋白质Ser-Thr磷酸酶1（PP1）的活性对于从中期到后期的过渡至关重要，从酵母到人保守的最古老的PP1调节剂是抑制剂2（I-2），这是一种非结构化的热稳定蛋白。来自各种物种的I-2中独特的序列基序围绕着一个磷酸化位点PXTP，该位点在生物化学分析中可以被GSK3，MAPK和CDK激酶磷酸化。在这里，我们使用了磷酸酶特异性抗体来研究I-2的磷酸化。我们分级分离了被Nocodazole阻滞的HeLa细胞的提取物，并使用重组I-2分析了PXTP激酶。鉴定了一个激酶活性的主要峰和两个次要峰，并且该主要峰同时包含活性MAPK和cdk1 :: cyclinB1，并通过免疫印迹证实。从双胸腺嘧啶核苷释放的细胞通过有丝分裂而同步进行，免疫印迹显示仅在有丝分裂期间细胞内源性I-2的瞬时磷酸化，以及组蛋白H3（Ser10）和PP1（Thr320）的相应磷酸化。 cdk1 :: cyclinB1的激活与I-2磷酸化同时发生，但此时MAPK和GSK3均未被磷酸化，因此我们得出结论，在活细胞中只有cdk1 :: cyclinB1在1-2中使PXTP位点磷酸化。用PXTP磷酸位点抗体对细胞进行的免疫荧光染色显示，后期有丝分裂细胞具有高度特异性的染色，此时染色消失了。因此，cdk1 :: cyclinB1催化I-2的磷酸化，用特异性抗体染色应证明在有丝分裂的早期阶段可用作细胞的选择性标记。 （c）2005年由Elsevier Inc.发布。

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