We investigated the phosphorylation signatures of two Rab GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 sites that did not respond to either exercise or insulin (TBC1D4: S666), that responded to insulin only (TBC1D4: S318), that responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and that responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin stimulated leg, Akt phosphorylation on both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2β2γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2β2γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK was regulating phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species are difficult.
Journal of Physiology, 2014, Vol 592, Issue 2, p. 351-375
AMP-Activated Protein Kinases; Adult; Animals; Exercise; GTPase-Activating Proteins; Humans; Insulin; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Phosphorylation; Physical Exertion; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't