NANOG is multiply phosphorylated and directly modified by ERK2 and CDK1 in vitro.
NANOG is multiply phosphorylated and directly modified by ERK2 and CDK1 in vitro.
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DOI：
10.1016/j.stemcr.2013.12.005
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发表时间：
2014-01-14
影响因子：
5.9
通讯作者：
Thomson, James A.
中科院分区：
文献类型：
--
作者：
Brumbaugh, Justin;Russell, Jason D.;Yu, Pengzhi;Westphall, Michael S.;Coon, Joshua J.;Thomson, James A.
NANOG是一种趋异同源框蛋白，是维持多能性和自我更新的转录回路的核心组成部分。虽然NANOG已经在转录水平上被广泛研究，但关于其翻译后调控知之甚少，可能是由于其低丰度和具有挑战性的物理性质。在这里，我们确定了11个磷酸化位点的内源性人类NANOG，其中9个映射到单个氨基酸。为了筛选赋予这些修饰的信号分子，我们开发了用于激酶特异性的多重测定（MAKS）。MAKS同时测试多达10种激酶的活性，同时直接鉴定底物和磷酸化的确切位点。使用MAKS，我们发现了ERK 2和CDK 1/CyclinA 2的位点特异性磷酸化，提供了关键信号通路和NANOG之间的推定联系。我们报告了内源性人NANOG上的11个磷酸化位点（9个定位）。我们引入了一种多重测定法来鉴定修饰给定底物的激酶。我们证明了NANOG在体外被ERK 2和CDK 1直接磷酸化。我们将细胞周期和生长因子介导的信号传导与NANOG联系起来。NANOG是一种核心多能性因子。在这里，Brumbaugh等人。表明NANOG在N末端被严重磷酸化，并且这些位点中的许多是脯氨酸指导的。为了将这些修饰置于信号通路的背景下，他们开发了激酶特异性的多重测定（MAKS），并表明NANOG在体外被ERK 2和CDK 1直接磷酸化。
NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS). MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG. We report 11 phosphorylation sites (9 localized) on endogenous, human NANOG We introduce a multiplexed assay to identify kinases that modify a given substrate We demonstrate that NANOG is directly phosphorylated by ERK2 and CDK1 in vitro We connect cell-cycle- and growth-factor-mediated signaling to NANOG NANOG is a core pluripotency factor. Here, Brumbaugh et al. show that NANOG is heavily phosphorylated on the N terminus and many of these sites are proline directed. To place these modifications in the context of signaling pathways, they developed the multiplexed assay for kinase specificity (MAKS) and show that NANOG is directly phosphorylated by ERK2 and CDK1 in vitro.
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