Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry
Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry
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DOI：
10.1074/mcp.m600026-mcp200
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发表时间：
2006-06-01
影响因子：
7
通讯作者：
Kim, Hee-Yong
中科院分区：
文献类型：
--
作者：
Huang, Bill X.;Kim, Hee-Yong
AKT是一种丝氨酸/苏氨酸激酶，在细胞生存中起着关键作用。在生长因子受体的刺激下，胞浆Akt通过磷脂结合被募集到质膜上，并通过Thr(308)和Ser(473)的磷酸化被激活。虽然Akt的部分晶体结构已经被报道，但还没有证明整个分子的三维结构或激活过程中的连续构象变化。在这项研究中，我们通过化学交联和串联质谱仪探测全长Akt在溶液中的三维结构，证明了Akt在激活过程中经历了戏剧性的域间构象变化。交联结果不仅提供了新的结构信息，而且揭示了Akt分子中各个结构域在静止状态、膜相互作用状态、磷酸化状态和底物结合状态下的独特空间排列。我们的数据为Akt激活序列的逐步结构域间构象变化提供了一个新的模型，为进一步研究Akt-膜、Akt-蛋白质和/或Akt-药物在溶液中的相互作用奠定了基础，以了解参与细胞生存生理和病理生理过程的分子机制。
Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr(308) and Ser(473). Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival.