title: Synthetic phosphorylation of p38α recapitulates protein kinase activity.
abstract: 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.
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford , Mansfield Road, Oxford OX1 3TA, U.K.
authors.forename: K Phin
authors.initials: KP
authors.lastname: Chooi
authors.forename: Sébastien R G
authors.initials: SR
authors.lastname: Galan
authors.forename: Ritu
authors.initials: R
authors.lastname: Raj
authors.forename: James
authors.initials: J
authors.lastname: McCullagh
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.forename: Lyn H
authors.initials: LH
authors.lastname: Jones
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Davis
mesh_terms: D051698:Activating Transcription Factor 2 / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004789:Enzyme Activation; D007700:Kinetics; D020935:MAP Kinase Signaling System; D048308:Mitogen-Activated Protein Kinase 14 / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D051698:Activating Transcription Factor 2; D048308:Mitogen-Activated Protein Kinase 14; D003545:Cysteine