Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser(231), Ser(660) and Ser(700)) and one predicted Akt phosphorylation site (Thr(590)) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser(231) and Ser(660), tended to increase Ser(700) phosphorylation, but had no effect on Thr(590). AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser(231), Ser(660) and Ser(700), but not Thr(590), whereas insulin only increased Thr(590) phosphorylation. Basal and contraction-stimulated TBC1D1 Ser(231), Ser(660) and Ser(700) phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr(590) phosphorylation. Contraction-stimulated TBC1D1 Ser(231) and Ser(660) phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.