Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine.
Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine.
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DOI：
10.1038/s41467-022-34980-5
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发表时间：
2022-11-24
影响因子：
16.6
通讯作者：
Rinehart, Jesse
中科院分区：
文献类型：
--
作者：
Moen, Jack M.;Mohler, Kyle;Rogulina, Svetlana;Shi, Xiaojian;Shen, Hongying;Rinehart, Jesse
蛋白质磷酸化是一种普遍存在的翻译后修饰，用于通过调节蛋白质-蛋白质相互作用来调节细胞过程和蛋白质组结构。通过蛋白质组学监测识别磷酸化事件已经大大超过了我们使用传统策略进行功能分配的能力，这通常需要先验了解上游激酶。磷酸氨基酸特异性正交翻译系统、进化上不同的氨酰基-tRNA合成酶和能够共翻译插入磷酸氨基酸的tRNA对的发展，通过提供不依赖于激酶的磷蛋白生产方法，迅速提高了我们评估磷酸化生理功能的能力。尽管有这种实用性，但广泛的部署受到技术限制和无法重建复杂的磷调节网络的阻碍。在这里，我们通过优化遗传编码的磷酸苏氨酸翻译来表征磷酸依赖性激酶激活机制来应对这些挑战，随后开发多级蛋白质相互作用平台，以磷酸位点水平分辨率直接评估激酶和磷酸结合蛋白底物网络的重叠。蛋白质磷酸化是一种普遍存在的翻译后修饰，用于通过调节蛋白质-蛋白质相互作用来调节细胞过程和蛋白质组结构。在这里，作者优化了基因编码的磷酸苏氨酸，以使用大规模 DNA 阵列来研究 CHK2 激酶的调节，该阵列使磷酸蛋白质组表达技术能够识别 CHK2 底物和 14-3-3 相互作用之间的位点特异性重叠。
Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution. Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. Here the authors optimize genetically encoded phosphothreonine to study the regulation of CHK2 kinase using large-scale DNA arrays that enable phosphoproteome expression techniques to identify sitespecific overlap between CHK2 substrates and 14-3-3 interactions.
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使用扩展的遗传密码将磷酸酪氨酸的特定于位点掺入。
影响因子：
14.8
作者：
Hoppmann C;Wong A;Yang B;Li S;Hunter T;Shokat KM;Wang L
通讯作者：
Wang L
影响因子：
4.8
作者：
Lee, CH;Chung, JH
通讯作者：
Chung, JH
影响因子：
14.9
作者：
Hornbeck PV;Kornhauser JM;Tkachev S;Zhang B;Skrzypek E;Murray B;Latham V;Sullivan M
通讯作者：
Sullivan M
影响因子：
7.7
作者：
Kuncha, Santosh Kumar;Suma, Katta;Sankaranarayanan, Rajan
通讯作者：
Sankaranarayanan, Rajan
影响因子：
56.9
作者：
MCCLAIN, WH;FOSS, K
通讯作者：
FOSS, K