Abstract
Multiple kinase activities are required for skeletal muscle differentiation. However, the mechanisms by which these kinase pathways converge to coordinate the myogenic process are unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determined specific peptide sequences using LC-MS/MS. To place these phosphopeptides into a rational context, a bioinformatics approach was used. Phosphorylation sites were matched to known site-specific and to site non-specific kinase-substrate interactions, and then other substrates and upstream regulators of the implicated kinases were incorporated into a model network of protein-protein interactions. The model network implicated several kinases of known relevance to myogenesis including AKT, GSK3, CDK5, p38, DYRK, and MAPKAPK2 kinases. This combination of proteomics and bioinformatics technologies should offer great utility as the volume of protein-protein and kinase-substrate information continues to increase.
Highlights
Multiple kinase activities are required for skeletal muscle differentiation
There is evidence that p38 acts through multiple mechanisms including phosphorylation of the myogenic regulatory factors (MRFs) cofactor myocyte enhancer factor 2 (4 – 6), phosphorylation of the MyoD cofactor E47, and enhanced recruitment of the SWI/SNF chromatin-remodeling complex to MRF-targeted promoters [7]
JNK1 is normally inactive during myogenesis, and its activation leads to muscle pathology [10]
Summary
Cells and Sample Preparation—C2C12 myoblasts were cultured in growth medium (10% fetal bovine serum, Dulbecco’s modified Eagle’s medium), and whole cell lysates were collected either 0 or 24 h after a change to differentiation medium (2% horse serum, Dulbecco’s modified Eagle’s medium) as described previously [13]. Preparation of Samples for IMAC—Approximately 15 g of phosphoprotein-enriched protein extract was suspended in 50 mM NH4HCO3 containing Promega modified trypsin (10 ng/l). IMAC Enrichment Using GELoader Tips—30 l of prepared IMAC resin was added to the peptide sample in binding buffer (100 mM NaCl in 1% acetic acid) and agitated for 15 min before loading into a GELoader tip. Resin was rinsed once by 20 l of a solution of 100 mM NaCl in 30% acetonitrile and 1% acetic acid and eluted in two steps by 250 mM ammonium phosphate, pH 9. Using 40 l of a solution of 5% acetic acid as binding buffer, the tryptic peptides were transferred to a Pierce column, left for 15 min at room temperature, and centrifuged for 1 min at 3000 ϫ g.
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