Multi-phosphorylation of the Intrinsically Disordered Unique Domain of c-Src Studied by In-Cell and Real-Time NMR Spectroscopy
Multi-phosphorylation of the Intrinsically Disordered Unique Domain of c-Src Studied by In-Cell and Real-Time NMR Spectroscopy
复制标题
DOI：
10.1002/cbic.201300139
复制
发表时间：
2013-09-23
期刊：
影响因子：
3.2
通讯作者：
Pons, Miquel
中科院分区：
文献类型：
--
作者：
Amata, Irene;Maffei, Mariano;Pons, Miquel
内含子无序区（IDR）是调节蛋白质功能所必需的翻译后修饰的优选位点。增强的局部迁移率的IDR有利于他们的观察，通过NMR光谱在体内。磷酸化事件可以发生在多个位点，并动态响应激酶-磷酸酶网络的变化。在这里，我们使用实时核磁共振光谱研究的影响激酶和磷酸酶存在于非洲爪蟾卵母细胞和卵提取物的磷酸化状态的“独特的域”的c-Src。我们随后的磷酸化S17在卵母细胞中，和S17，S69，和S75在卵提取物中的NMR光谱，MS和蛋白质印迹。添加特异性激酶抑制剂表明，S75和S69被不同于Cdk 1的CDK（细胞周期蛋白依赖性激酶）磷酸化。此外，尽管PKA（cAMP依赖性蛋白激酶）可以在体外磷酸化S17，但这并不是鸡蛋提取物中主要的S17激酶。PKA活性的变化影响CDK依赖性位点的磷酸化水平，从而表明激酶-磷酸酶网络的间接影响。这项研究提供了一个概念验证的使用实时在体内NMR光谱表征激酶/磷酸酶的影响，内在无序的监管结构域。
Intrinsically disordered regions (IDRs) are preferred sites for post-translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase-phosphatase networks. Here we used real-time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the "unique domain" of c-Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin-dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP-dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK-dependent sites, thus suggesting indirect effects of kinase-phosphatase networks. This study provides a proof-of-concept of the use of real-time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.