Molecular Physiology Group, Department of Exercise and Sport Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark.
J Physiol. 2010 Nov 15;588(Pt 22):4539-48. doi: 10.1113/jphysiol.2010.194811. Epub 2010 Sep 13.
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 5AMP-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.
TBC1D1 是一种 Rab-GTPase 激活蛋白，参与调节骨骼肌中的 GLUT4 易位。我们在此评估了运动引起的人骨骼肌中 TBC1D1 Ser237 磷酸化和 14-3-3 蛋白结合能力的调节。在单独的实验中，健康男性进行了持续 30s、2min 或 20min 的全力自行车运动。在所有运动方案后，TBC1D1 Ser237 磷酸化增加（∼70-230%，P < 0.005），在 20min 骑行后观察到最大反应。有趣的是，TBC1D1 与 14-3-3 蛋白结合的能力显示出相似的调节模式，增加了 60-250%（P < 0.001）。此外，当在体外评估时，重组 5AMP 激活的蛋白激酶（AMPK）诱导人 TBC1D1 的 Ser237 磷酸化和 14-3-3 结合特性。为了进一步表征 AMPK 作为调节 TBC1D1 的上游激酶的作用，从全身 α1 或 α2 AMPK 敲除和野生型小鼠的伸趾长肌（EDL）中刺激体外收缩。在野生型和 α1 敲除小鼠中，收缩导致 Ser237 磷酸化增加约 100%（P < 0.001）。有趣的是，α2 敲除小鼠的肌肉表现为 TBC1D1 蛋白含量降低（∼50%，P < 0.001）以及基础和收缩刺激（∼60%，P < 0.001）的 Ser237 磷酸化降低，即使在考虑到 TBC1D1 蛋白含量降低后也是如此。这项研究表明，TBC1D1 在人骨骼肌中对运动的反应是 Ser237 磷酸化和 14-3-3 蛋白结合能力增加。此外，我们表明，催化 α2 AMPK 亚基是调节小鼠 EDL 肌肉中 TBC1D1 Ser237 磷酸化的 AMPK 活性的主要（但可能不是唯一）供体。
