Visualization of Ras-PI3K interaction in the endosome using BiFC
Visualization of Ras-PI3K interaction in the endosome using BiFC
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DOI：
10.1016/j.cellsig.2009.07.004
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发表时间：
2009-11-01
影响因子：
4.8
通讯作者：
Ohba, Yusuke
中科院分区：
文献类型：
--
作者：
Tsutsumi, Kaori;Fujioka, Yoichiro;Ohba, Yusuke
最近的研究表明，时空调控的重要性，在细胞内信号的多样性和特异性。在这里，我们表明，Ras-PI 3 K信号在磷脂酰肌醇代谢的内体通过活细胞成像的局部调节中起着重要的作用，通过使用双分子荧光互补技术，其中分子相互作用是由荧光发射指示。使用Ras和Ras结合结构域的几种可能的组合，我们确定了一组最佳的探针分子，其在活性和非活性形式的Ras之间产生最显著的荧光强度增加。这种组合揭示，在所测试的Ras效应物中，磷脂酰-肌醇3-激酶（PI 3 K）特别涉及内体中的信号传导。我们还发现全长PI 3 K以EGF和Ras依赖的方式被募集到内体，这似乎是PI 3 K在该隔室中激活所必需的。综上所述，这些发现表明Ras-PI 3 K信号传导的时空调节可能决定了PI 3 K的激活和随后的内体下游信号传导。(C)2009 Elsevier Inc. All rights reserved.
Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidy-linositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome. (C) 2009 Elsevier Inc. All rights reserved.