Exercise-induced TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle

TY - JOUR

T1 - Exercise-induced TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle

AU - Frøsig, Christian

AU - Pehmøller, Christian

AU - Birk, Jesper Bratz

AU - Richter, Erik

AU - Wojtaszewski, Jørgen

N1 - CURIS 2010 5200 105

PY - 2010/11

Y1 - 2010/11

N2 - TBC1D1 is a Rab-GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise-induced regulation of TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all-out cycle exercise lasting either 30 s, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (~70-230%, P < 0.005), with the greatest response observed after 20 min of cycling. Interestingly, capacity of TBC1D1 to bind 14-3-3 protein showed a similar pattern of regulation, increasing 60-250% (P < 0.001). Furthermore, recombinant 5'AMP-activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14-3-3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock-out and wild-type mice were stimulated to contract in vitro. In wild-type and α1 knock-out mice, contractions resulted in a similar ~100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock-out mice were characterized by reduced protein content of TBC1D1 (~50%, P < 0.001) as well as in basal and contraction-stimulated (~60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14-3-3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle. In response to exercise, glucose uptake in muscle is increased by translocation of glucose transport proteins (GLUT4) from the cell interior to the cell surface. TBC1D1 is a recently identified protein believed to be involved in regulation of GLUT4 translocation in skeletal muscle. We show here that high-intensity cycle exercise in humans leads to regulation of TBC1D1, including increased phosphorylation of serine 237 and increased capacity of TBC1D1 to bind regulatory 14-3-3 proteins. We also provide evidence supporting the idea that the enzyme responsible for this regulation is AMPK, a kinase activated in response to AMP accumulation as observed during high-intensity exercise. This is the first study to demonstrate that TBC1D1 is regulated in response to exercise in human skeletal muscle, thus supporting a physiological role of TBC1D1 in regulation of glucose uptake during exercise.

AB - TBC1D1 is a Rab-GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise-induced regulation of TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all-out cycle exercise lasting either 30 s, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (~70-230%, P < 0.005), with the greatest response observed after 20 min of cycling. Interestingly, capacity of TBC1D1 to bind 14-3-3 protein showed a similar pattern of regulation, increasing 60-250% (P < 0.001). Furthermore, recombinant 5'AMP-activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14-3-3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock-out and wild-type mice were stimulated to contract in vitro. In wild-type and α1 knock-out mice, contractions resulted in a similar ~100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock-out mice were characterized by reduced protein content of TBC1D1 (~50%, P < 0.001) as well as in basal and contraction-stimulated (~60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14-3-3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle. In response to exercise, glucose uptake in muscle is increased by translocation of glucose transport proteins (GLUT4) from the cell interior to the cell surface. TBC1D1 is a recently identified protein believed to be involved in regulation of GLUT4 translocation in skeletal muscle. We show here that high-intensity cycle exercise in humans leads to regulation of TBC1D1, including increased phosphorylation of serine 237 and increased capacity of TBC1D1 to bind regulatory 14-3-3 proteins. We also provide evidence supporting the idea that the enzyme responsible for this regulation is AMPK, a kinase activated in response to AMP accumulation as observed during high-intensity exercise. This is the first study to demonstrate that TBC1D1 is regulated in response to exercise in human skeletal muscle, thus supporting a physiological role of TBC1D1 in regulation of glucose uptake during exercise.

U2 - 10.1113/jphysiol.2010.194811

DO - 10.1113/jphysiol.2010.194811

M3 - Journal article

C2 - 20837646

SN - 0022-3751

VL - 588

SP - 4539

EP - 4548

JO - Journal of Physiology

JF - Journal of Physiology

IS - 22

ER -