Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

Abstract

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-β) and determine protein abundance of ATPsyn-β 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-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results: Seven phosphorylation sites were identified on ATPsyn-β 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 ∼30% higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50% in lean and healthy, but not insulin-resistant, individuals. Conclusions/interpretation: These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes. © 2009 Springer-Verlag.