Synthetic Phosphorylation of p38α Recapitulates Protein Kinase Activity
| Autor: | James S. O. McCullagh, Sébastien R. G. Galan, Lyn H. Jones, Benjamin G. Davis, Ritu Raj, Shabaz Mohammed, K. Phin Chooi |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular MAP Kinase Signaling System Protein Conformation Mitogen-activated protein kinase kinase Biochemistry Catalysis Substrate Specificity MAP2K7 Mitogen-Activated Protein Kinase 14 Colloid and Surface Chemistry Protein phosphorylation ASK1 Cysteine Phosphorylation Protein kinase A Binding Sites Activating Transcription Factor 2 biology MAP kinase kinase kinase Chemistry Communication Cyclin-dependent kinase 2 General Chemistry Enzyme Activation Kinetics biology.protein Cyclin-dependent kinase 9 |
| Zdroj: | Journal of the American Chemical Society |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/ja4095318 |
| Popis: | Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38α. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38α 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 p38α 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 p38α and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II. |
| Databáze: | OpenAIRE |
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