Taylor Eric B, An Ding, Kramer Henning F, Yu Haiyan, Fujii Nobuharu L, Roeckl Katja S C, Bowles Nicole, Hirshman Michael F, Xie Jianxin, Feener Edward P, Goodyear Laurie J
The Joslin Diabetes Center Section on Metabolism and Proteomics Core and Harvard Medical School, One Joslin Place, Boston, MA 02215, USA.
J Biol Chem. 2008 Apr 11;283(15):9787-96. doi: 10.1074/jbc.M708839200. Epub 2008 Feb 13.
The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.
160 kDa的Akt底物（AS160）会在Akt底物（PAS）基序上发生磷酸化，以响应胰岛素和骨骼肌收缩，从而调节葡萄糖摄取。在此，我们发现比目鱼肌、胫骨前肌和趾长伸肌中AS160蛋白表达与明显的AS160 PAS磷酸化之间存在解离现象。胫骨前肌裂解物中AS160的免疫去除导致160 kDa处PAS条带的去除极少，这表明存在另一种PAS免疫反应性蛋白。通过免疫沉淀和质谱分析，我们将该蛋白鉴定为AS160旁系同源物TBC1D1，这是一个调节脂肪细胞中GLUT4易位的肥胖候选基因。与所有其他组织相比，TBC1D1在骨骼肌中的表达高出几倍，并且是抗PAS抗体在胫骨前肌和趾长伸肌而非比目鱼肌中检测到的160 kDa处的主要蛋白。胰岛素、收缩以及AMP激活的蛋白激酶（AMPK）激活剂AICAR在体内的刺激增加了TBC1D1 PAS磷酸化。对来自小鼠骨骼肌的TBC1D1进行质谱分析，我们在TBC1D1上鉴定出几个新的磷酸化位点，并发现大多数是AMPK的共有或接近共有位点。光谱的半定量分析表明，与胰岛素相比，AICAR导致TBC1D1位点的总体磷酸化程度更高。纯化的Akt和AMPK在体外使TBC1D1磷酸化，并且AMPK而非Akt降低了TBC1D1的电泳迁移率。TBC1D1是骨骼肌中主要的PAS免疫反应性蛋白，在体内会被胰岛素、AICAR和收缩磷酸化。Akt和AMPK均可使TBC1D1磷酸化，但AMPK可能是更强有力的调节因子。
