Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in
1Center for Metabolic Biology, School of Life Sciences, Arizona State University, PO Box 87370, Tempe, AZ 85287, USA. k.hojlund@dadlnet.dk
Aims/Hypothesis:
Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-beta) and determine protein abundance of ATPsyn-beta and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals.
Methods:
Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-beta using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps.
Results:
Seven phosphorylation sites were identified on ATPsyn-beta purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a approximately 30% higher phosphorylation of ATPsyn-beta at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-beta protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-beta by approximately 50% in lean and healthy, but not insulin-resistant, individuals.
Conclusions/Interpretation:
These data demonstrate that ATPsyn-beta is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-beta together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-beta may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.
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