A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms.Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.Protein kinases are well-known regulators of cell signalling and cellular behaviour that execute their function through the covalent attachment of an ATP-derived phosphate to protein substrates. To understand the function of any protein kinase on a large and cell-wide scale first requires the development of a substrate screening technique that allows for the proteins phosphorylated by a kinase of interest to be comprehensively identified, ideally in a single experiment. Although substrate-finding techniques exist, they are hindered by problems that prevent them from being easily or readily employed [1-4] and are generally limited to providing in vitro substrate identifications that may or may not be relevant in vivo. In vivo approaches currently avail