Probing Akt-Inhibitor Interaction by Chemical Cross-Linking and Mass Spectrometry
Probing Akt-Inhibitor Interaction by Chemical Cross-Linking and Mass Spectrometry
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DOI：
10.1016/j.jasms.2009.04.004
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发表时间：
2009-08-01
影响因子：
3.2
通讯作者：
Kim, Hee-Yong
中科院分区：
文献类型：
--
作者：
Huang, Bill X.;Kim, Hee-Yong
丝氨酸/苏氨酸激酶Akt是调节细胞存活的关键酶。由于Akt的高活性已被证明与多种人类恶性肿瘤的发病机制有关，抑制Akt的激活是一种很有前景的癌症治疗策略。我们之前已经证明，Akt-膜相互作用会导致Akt结构域间排列从封闭到开放的变化，这反过来又允许Akt磷酸化/活化。在本研究中，我们展示了一种基于Akt构象变化的化学交联和180标记质谱分析来识别Akt抑制机制的新策略。通过定量比较两种域间交联肽，我们发现Akt与抑制剂（PI类似物）的结合导致开放的域间构象，其中PH和调节结构域远离激酶结构域，甚至在与膜相互作用之前，随后阻止Akt易位到质膜。相比之下，与另一种抑制剂（肽TCL1）孵育后，结构域间构象保持不变。随后与单层囊泡的相互作用表明，TCL1破坏了PH结构域的开放，使T308暴露在质膜上进行磷酸化。这种基于构象分子相互作用机制的新方法可能对特定Akt抑制剂或抗肿瘤药物的药物发现工作有用。[J] .质谱学报，2009,20,1504-1513 .
The serine/threonine kinase Akt is a critical enzyme that regulates cell survival. As high Akt activity has been shown to contribute to the pathogenesis of various human malignancies, inhibition of Akt activation is a promising therapeutic strategy for cancers. We have previously demonstrated that changes in Akt interdomain arrangements from a closed to open conformation occur upon Akt-membrane interaction, which in turn allows Akt phosphorylation/activation. In the present Study, we demonstrate a novel strategy to discern mechanisms for Akt inhibition based on Akt conformational changes using chemical cross-linking and 180 labeling mass spectrometry. By quantitative comparison of two interdomain cross-linked peptides, which represent the proximity of the domains involved, we found that the binding of Akt to an inhibitor (PI analog) caused the open interdomain conformation where the PH and regulatory domains moved away from the kinase domain, even before interacting with membranes, subsequently preventing translocation of Akt to the plasma membrane. In contrast, the interdomain conformation remained unchanged after incubating with another type of inhibitor (peptide TCL1). Subsequent interaction with unilamellar vesicles suggested that TCL1 impaired particularly the opening of the PH domain for exposing T308 for phosphorylation at the plasma membrane. This novel approach based on the conformation-based molecular interaction mechanism should be potentially useful for drug discovery efforts for specific Akt inhibitors or anti-tumor agents. (J Am Soc Mass Spectrom 2009, 20,1504-1513) (C) 2009 American Society for Mass Spectrometry