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-actvating 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, AMPK alpha 2 inactive transgenic mice (AMPK alpha 2i 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-ammoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231. 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 AMPK alpha 2i 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