Background: The p38a mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation,\r\nand does so in part through the phosphorylation and regulation of several transcription factors and chromatin\r\nremodelling proteins. However, whether p38a is involved in processes other than gene regulation during\r\nmyogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.\r\nMethods: To further characterise the involvement of p38a during myoblast differentiation, we developed and\r\napplied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In\r\naddition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in\r\nthe same experiment, and we made use of this to study the substrate specificities of the p38a and b isoforms.\r\nResults: Applying the technique to p38a resulted in the identification of seven in vivo phosphorylation sites on six\r\nproteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison\r\nwith p38b revealed that substrate specificity does not discriminate these two isoforms, but rather that their\r\ndistinguishing characteristic appears to be cellular localisation.\r\nConclusion: Our results suggest p38a has a novel cytoplasmic role during myogenesis and that its unique cellular\r\nlocalisation may be why p38b and other isoforms cannot compensate for its absence. The substrate-finding\r\napproach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and\r\nfor uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.
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