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.

Figure 1

Experimental workflow for the analysis of the in vivo phosphoproteome of rat skeletal muscle.

Figure 2

Representative MS/MS spectrum for a novel phosphorylated peptide ₃₅₅GTGGVDTAAVADVpYDISNIDR₃₇₅ of sarcomeric mitochondrial creatine kinase. The ions corresponding to b₉, b₁₀, b₁₂ and y₅, y₆, y₇ are consistent with phosphorylation on Tyr368.

Figure 3

Subcellular location of identified phosphoproteins in rat skeletal muscle based on gene ontology annotation and literature search.

Figure 4

Tyrosine phosphorylation of enzymes involved in glycolysis and glycogen metabolism. Isoforms and/or subunits of enzymes involved in (A) glycolysis, (B) glycogen metabolism, and (C) regulation of muscle glycogen synthase (GYS1). Tyrosine phosphorylated proteins are shown in yellow boxes, and the number of identified tyrosine phosphorylation sites (pY) are indicated by red circles. Number of serine (Ser) residues on GYS1 indicates known Ser phosphorylation sites with their classical number of site given below the bar. AMPK, AMP-activated kinase; CaM-KII, calcium-calmodulin-dependent kinase II; CK1, casein kinase 1; CK2, casein kinase 2; DYRK1A, dual specificity tyrosine-phosphorylation-regulated kinase 1A; GSK3A, glycogen synthase kinase a; PHK, phosphorylase kinase; PKA, protein kinase A; PKC, protein kinase C.

Figure 4

Tyrosine phosphorylation of enzymes involved in glycolysis and glycogen metabolism. Isoforms and/or subunits of enzymes involved in (A) glycolysis, (B) glycogen metabolism, and (C) regulation of muscle glycogen synthase (GYS1). Tyrosine phosphorylated proteins are shown in yellow boxes, and the number of identified tyrosine phosphorylation sites (pY) are indicated by red circles. Number of serine (Ser) residues on GYS1 indicates known Ser phosphorylation sites with their classical number of site given below the bar. AMPK, AMP-activated kinase; CaM-KII, calcium-calmodulin-dependent kinase II; CK1, casein kinase 1; CK2, casein kinase 2; DYRK1A, dual specificity tyrosine-phosphorylation-regulated kinase 1A; GSK3A, glycogen synthase kinase a; PHK, phosphorylase kinase; PKA, protein kinase A; PKC, protein kinase C.

Figure 4

Tyrosine phosphorylation of enzymes involved in glycolysis and glycogen metabolism. Isoforms and/or subunits of enzymes involved in (A) glycolysis, (B) glycogen metabolism, and (C) regulation of muscle glycogen synthase (GYS1). Tyrosine phosphorylated proteins are shown in yellow boxes, and the number of identified tyrosine phosphorylation sites (pY) are indicated by red circles. Number of serine (Ser) residues on GYS1 indicates known Ser phosphorylation sites with their classical number of site given below the bar. AMPK, AMP-activated kinase; CaM-KII, calcium-calmodulin-dependent kinase II; CK1, casein kinase 1; CK2, casein kinase 2; DYRK1A, dual specificity tyrosine-phosphorylation-regulated kinase 1A; GSK3A, glycogen synthase kinase a; PHK, phosphorylase kinase; PKA, protein kinase A; PKC, protein kinase C.

Figure 5

Number of distinct pY sites by potential kinase family for all phosphoproteins predicted by KinasePhos [33]. Insulin receptor (INSR), Epidermal growth factor receptor (EGFR), Tyrosine kinase Src (Src), Spleen Tyrosine kinase (Syk), Abelson murine leukemia virus oncoprotein (Ab1).