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

作者：Knight, James D. R.; Tian, Ruijun; Lee, Robin E. C.; Wang, Fangjun; Beauvais, Ariane; Zou, Hanfa; Megeney, Lynn A.; Gingras, Anne-Claude; Pawson, Tony; Figeys, Daniel; Kothary, Rashmi^*

来源：Skeletal Muscle, 2012, 2(1): 5.

DOI：10.1186/2044-5040-2-5

• differentiation
• FSBA
• kinase assay
• mitogen-activated protein kinase
• myoblast
• p38
• phosphorylation
• quantitative MS

Background: The p38 alpha 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 p38 alpha 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 p38 alpha 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 p38 alpha and beta isoforms.
Results: Applying the technique to p38 alpha 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 p38 beta revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.
Conclusion: Our results suggest p38 alpha has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38 beta 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.

• 出版日期2012
• 单位中国科学院; 中国科学院大连化学物理研究所