title: A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts.
abstract: BACKGROUND
  The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON, K1H 8L6, Canada. rkothary@ohri.ca.
authors.forename: James Dr
authors.initials: JD
authors.lastname: Knight
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
authors.forename: Robin Ec
authors.initials: RE
authors.lastname: Lee
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.forename: Ariane
authors.initials: A
authors.lastname: Beauvais
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Megeney
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kothary