Activation of p38 mitogen activated protein kinase alpha during myocardial ischaemia results in myocardial damage. Identifying and targeting the relevant substrates downstream of p38alpha may potentially provide a therapeutic target to limit myocardial injury. We aim to identify the substrates of p38alpha particularly during myocardial ischaemia. We have adopted the analogue sensitive kinase strategy, as developed by the Shokat lab, of mutating the kinase to allow it to use expanded forms of ATP that wild type kinases are not capable of using. In addition, these ATP analogues are modified with a gamma thiophosphate instead of the gamma phosphate that is transferred during catalysis. This transferred thiophosphate acts as a label and allows for identification and isolation of the substrates of the kinase. We have characterised an analogue sensitive form of p38 and confirmed it to have a similar substrate specificity to wild type p38alpha. We have successfully optimized the conditions for labelling p38alpha substrates in mouse heart homogenates exposed to the analogue sensitive kinase and the expanded form of ATP. We isolated and used MS to identify the substrates and the sites of phosphorylation. 61 proteins with phosphorylation at p38 consensus sequences were identified and we are currently in the process of validating these results. In conclusion we have optimized the conditions for identification and isolation of substrates of p38 alpha in the mouse heart. In the future, we will use the identification of these substrates to elucidate the mechanism through which p38alpha mediates its effect during myocardial ischaemia.
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