Dedigama-Arachchige Pavithra M, Acharige Nuwan P N, Pflum Mary Kay H
Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
Mol Omics. 2018 Apr 16;14(2):121-133. doi: 10.1039/c7mo00064b.
Phosphorylation is a key post-translational modification in cell signaling, which is regulated by the equilibrium activities of kinases and phosphatases. The biological significance of many phosphorylation events remains poorly characterized due to the scarcity of tools to discover phosphatases substrates. In prior work, we established kinase-catalyzed biotinylation where kinases accept the γ-modified ATP analog, ATP-biotin, to label phosphoproteins. Here, we developed a novel method to study substrates of phosphatases using kinase-catalyzed biotinylation termed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In a proof-of-concept experiment, K-BIPS was initially used to explore the substrates of phosphatases inhibited by okadaic acid. Many known phosphatase substrates were observed, confirming K-BIPS as a valid phosphatase substrate identification tool. Then, as a further application, K-BIPS was used to discover the substrates of the PP1-Gadd34 phosphatase complex in the context of unfolded protein response (UPR). In addition to the known substrate eIF2α, K-BIPS revealed several novel substrates, suggesting a more prominent role for the PP1-Gadd34 complex in UPR than previously appreciated. Overall, the two studies establish K-BIPS as a powerful tool to discover the cellular substrates of phosphatases.
磷酸化是细胞信号传导中一种关键的翻译后修饰，它由激酶和磷酸酶的平衡活性所调控。由于发现磷酸酶底物的工具匮乏，许多磷酸化事件的生物学意义仍未得到充分表征。在先前的工作中，我们建立了激酶催化的生物素化方法，即激酶接受γ-修饰的ATP类似物ATP-生物素以标记磷酸化蛋白。在此，我们开发了一种利用激酶催化的生物素化来研究磷酸酶底物的新方法，称为K-BIPS（激酶催化生物素化鉴定磷酸酶底物）。在一个概念验证实验中，K-BIPS最初被用于探索被冈田酸抑制的磷酸酶的底物。观察到了许多已知的磷酸酶底物，证实了K-BIPS是一种有效的磷酸酶底物鉴定工具。然后，作为进一步的应用，K-BIPS被用于在未折叠蛋白反应（UPR）的背景下发现PP1-Gadd34磷酸酶复合物的底物。除了已知的底物eIF2α外，K-BIPS还揭示了几种新底物，这表明PP1-Gadd34复合物在UPR中的作用比之前所认识到的更为突出。总体而言，这两项研究将K-BIPS确立为发现磷酸酶细胞底物的强大工具。
