Medical Faculty, Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany; German Center for Diabetes Research (DZD), Partner Düsseldorf, München-Neuherberg, Germany.
Medical Faculty, Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany.
J Biol Chem. 2021 Jan-Jun;296:100637. doi: 10.1016/j.jbc.2021.100637. Epub 2021 Apr 16.
TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis-Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[O]-ATP. These data revealed that Ser (K ∼6 μM) and Thr (K ∼25 μM) were preferential sites for phosphorylation by AKT, whereas Ser, Ser, Ser (K ∼10 μM), Ser (K ∼79 μM), and Ser were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.
TBC1D4 是一种 160 kDa 的多结构域 Rab GTPase 激活蛋白（RabGAP），是胰岛素和收缩激活的激酶 AKT 和 AMPK 的下游靶标。TBC1D4 的磷酸化与 GLUT4 从储存小泡（GSV）向细胞表面的易位有关。然而，其对酶活性的影响尚不清楚，因为之前的研究大多研究了缺乏已知磷酸化位点的截短 GAP 结构域。在本研究中，我们使用杆状病毒系统表达和纯化了重组全长 TBC1D4。尺寸排阻色谱和共免疫沉淀实验表明，全长 TBC1D4 形成约 600 kDa 的寡聚体。与截短的 GAP 结构域相比，全长 TBC1D4 显示出相似的底物特异性，但对 Rab10 的特异性 GAP 活性明显更高。使用高分辨率质谱，我们在 TBC1D4 中定位了 19 个 Ser/Thr 磷酸化位点。我们使用纯化激酶和稳定同位素标记的 γ-[O]-ATP 通过体外磷酸化测定来确定米氏动力学。这些数据表明，Ser（K ∼6 μM）和 Thr（K ∼25 μM）是 AKT 磷酸化的优先位点，而 Ser、Ser、Ser（K ∼10 μM）、Ser（K ∼79 μM）和 Ser 是 AMPK 的优先靶标。AKT 或 AMPK 对 TBC1D4 的磷酸化不会改变内在的 RabGAP 活性，但会破坏与胰岛素调节的氨肽酶（IRAP）的相互作用，IRAP 是参与 GLUT4 转运的 GSV 中的一种常驻蛋白。这些发现提供了证据，表明胰岛素和收缩可能主要通过破坏 RabGAP 向 GLUT4 囊泡的募集来调节 TBC1D4 的功能。
