Protein engineering of p38 MAP kinase for substrate identification

Protein kinases play a central role in signal transduction pathways. Many studies have reported that MAPK pathways are implicated in growth, differentiation, apoptosis and immunity. p38 MAPK is one of the major groups within the MAPK superfamily. Recent studies indicate that the p38 MAPK signalling pathway is one of the major intracellular signalling pathways regulating myogenesis.

Identification of kinase substrates is a major goal of kinase biology. In order to identify p38 MAPK substrates, we have generated mutations in human p38? to create a structural distinction between the ATP binding site of p38 MAPK and that of other kinases in the cell in order to identify its substrates using modified nucleotide analogs. Identification of the relevant substrates will help to clarify the molecular mechanism underlying the role of p38 MAPK. Here, it is shown that engineered kinases, are able to use synthesized ATP analogs, which are typically not recognized by natural kinases. In particular, this work demonstrates that Leu87Ala/Thr106Gly p38 MAPK could be useful in a proteomic approach for identifying novel p38 MAPK substrates.