title: Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS.
abstract: Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (<1% of total protein phosphorylation), only a few tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca(2+) homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states.
authors.affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy & Health Sciences, Wayne State University, Detroit, MI, USA.
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
authors.forename: Kurt
authors.initials: K
authors.lastname: Højlund
authors.forename: Moulun
authors.initials: M
authors.lastname: Luo
authors.forename: Christian
authors.initials: C
authors.lastname: Meyer
authors.forename: Thangiah
authors.initials: T
authors.lastname: Geetha
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Yi
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D008297:Male; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism*; D000097603:Dyrk Kinases
chemical_list: D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D009124:Muscle Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D011505:Protein-Tyrosine Kinases; D014443:Tyrosine; D000097603:Dyrk Kinases; C448021:glycogen synthase kinase 3 alpha