Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry
Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry
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DOI：
10.1007/s13361-015-1286-8
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发表时间：
2016-02-01
影响因子：
3.2
通讯作者：
Ge, Ying
中科院分区：
文献类型：
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作者：
Yu, Deyang;Peng, Ying;Ge, Ying
AMP 激活蛋白激酶 (AMPK) 是一种丝氨酸/苏氨酸蛋白激酶，对于调节所有真核细胞的能量代谢至关重要。它是一种异三聚体蛋白质复合物，由一个催化亚基（α）和两个调节亚基（β和γ）组成。 AMPK α 在残基 312 处的 C 端截短产生了一种蛋白质，在缺乏 β 和 γ 亚基的情况下，该蛋白质在 Thr172 磷酸化时具有活性，该蛋白质被称为 AMPK 催化结构域，通常用于在体外激酶测定中替代 AMPK 异三聚体复合物。然而，缺乏对 AMPK 催化结构域的全面表征。在此，我们在大肠杆菌中表达了带有His标签的人AMPK催化域（记为AMPK（Delta）），使用自上而下的质谱（MS）全面表征了AMPK（Delta）的基础状态和体外磷酸化后的特征，并评估了AMPK（Delta）的磷酸化如何影响其活性。出乎意料的是，我们发现细菌表达的AMPK(Delta)被基础磷酸化，并且磷酸化位点定位于His标签。我们发现 AMPK(Delta) 具有明显的基础活性，能够磷酸化自身及其底物，而不激活 Thr172 的磷酸化。此外，我们的数据表明 Thr172 是唯一被其上游激酶（肝激酶 B1）磷酸化的位点，并且这种磷酸化显着增加了 AMPK(Delta) 的激酶活性。重要的是，我们证明自上而下的 MS 与体外磷酸化测定相结合是监测磷酸化反应和确定激酶底物系统中磷酸化事件顺序的有效方法。
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). C-terminal truncation of AMPK alpha at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of beta and gamma subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPK(Delta)) in E. coli, comprehensively characterized AMPK(Delta) in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPK(Delta) affects its activity. Unexpectedly, we found that bacterially-expressed AMPK(Delta) was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPK(Delta) had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPK(Delta). Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.