Synthetic Phosphorylation of p38α Recapitulates Protein Kinase Activity
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- 作者：K. Phin Chooi ; Sébastien R. G. Galan ; Ritu Raj ; James McCullagh ; Shabaz Mohammed ; Lyn H. Jones ; Benjamin G. Davis
- 刊名：Journal of the American Chemical Society
- 出版年：2014
- 出版时间：February 5, 2014
- 年：2014
- 卷：136
- 期：5
- 页码：1698-1701
- 全文大小：360K
- ISSN：1520-5126
文摘
Through a 鈥渢ag-and-modify鈥?protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38伪. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38伪 bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38伪 and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38伪 and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.