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.

Chemical Protein Modification through Cysteine

“…Also, because the p K a of the thiol proton varies from cysteine residue to cysteine residue, depending on its microenvironment, reaction at a lower pH can be possible. Over the last few years, the reaction has been applied to a range of proteins, followed by Michael‐type addition of thiol moieties, both by the Davis group and by others …”

Synthetic approaches to protein phosphorylation

“…It has been shown that that phosphoCys mimic can be recognized by a phospho-serine antibody with comparable efficiency to the natural PTM. [54] This method has been applied to the study of Aurora-A[55,57] and p38α[56] protein kinases and their regulation by phosphorylation. The semisynthetic phospho-Cys containing kinases showed similar catalytic activities to the enzymatically phosphorylated enzymes, confirming that the phospho-cysteine nicely recapitulates phospho-serine/phospho-threonine functionally.…”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…Several articles referring to pCys-containing proteins (Sun et al 2012;Chooi et al 2014;Bertran-Vicente et al 2016;Gulerez et al 2016;Zhang et al 2017) have reported evidence of S-linked phosphorylation survival throughout various proteomic workflows. The HCD spectra of the model mixture assembled in this study demonstrate that direct detection of pCys-peptides is feasible, especially if their spectra exhibit NL-triplets, 80 Da losses from fragment ions, or ideally fragment ions carrying the pCys-residue.…”