We demonstrate here a non-enzymatic method for in vitro activation of p38a kinase via chemical modification. Using a �tag and modify� strategy to install phosphocysteine (pCys), p38a was selectively phosphorylated at two distinct sites. One site was native to phosphorylation (180), the other not (172). A single phosphoform was generated in each case. Evaluation of enzymatic activity of all the p38a variants revealed that the kinase was only active when phosphorylated at 180 (as p38a-pC180), mimicking p38a-pT180. p38a-pC180 was able to process a natural protein substrate ATF2, thus demonstrating biological recognition. More detailed analysis of enzymatic action of p38a-pC180 showed that phosphorylation at 180 favoured substrate monophosphorylation. p38a-pC180 activity was further investigated with both Type I and II kinase inhibitors, where it was found that activity was modulated by both classes of inhibitor. Differences in cooperativity suggested that this could be used as an additional discriminator for inhibitor type. Given the high conversion to the single phosphoform of p38a-pC180, the use of protein chemical modification allows us to solely attribute these kinetic and binding effects to this phosphoform.

Giving p38a kinase a jump-start: p38a Activation induced by chemical modification

247th National Meeting (2014)

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