The Scaffold Protein Shoc2/SUR-8 Accelerates the Interaction of Ras and Raf
The Scaffold Protein Shoc2/SUR-8 Accelerates the Interaction of Ras and Raf
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DOI：
10.1074/jbc.m109.053975
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发表时间：
2010-03-05
影响因子：
4.8
通讯作者：
Matsuda, Michiyuki
中科院分区：
文献类型：
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作者：
Matsunaga-Udagawa, Rie;Fujita, Yoshihisa;Matsuda, Michiyuki
Shoc2/SUR-8作为Ras和Raf的支架，正向调节Ras/ERK MAPK信号转导。在这里，我们使用基于荧光共振能量转移(FRET)的生物传感器，结合计算模型，研究了Shoc2在RAS的时空调节中的作用。在表皮生长因子刺激的HeLa细胞中，RNA介导的Shoc2基因敲除可使MEK和ERK的磷酸化水平降低一半，但不能激活RAS。为了实时监测RAS与Raf的结合，我们使用了一种FRET生物传感器，其中RAS和Raf的RAS结合域被连接在一起，并与受体和供体荧光蛋白夹在一起进行FRET测量。利用这种生物传感器，我们发现在表皮生长因子刺激下，Ras和Raf的快速相互作用需要Shoc2。为了解释这一动力学的分子机制，我们开发了两个计算模型，可以解释Shoc2在RAS-ERK信号转导中的作用。其中一个模型，Shoc2加速器模型，对实验观测提供了合理的解释。在这个Shoc2加速器模型中，Shoc2加速了RAS-Raf相互作用的结合和解离。我们认为Shoc2主要通过促进Ras-Raf的相互作用来调节Ras-ERK信号通路的时空模式。
Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.