Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes
Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes
复制标题
DOI：
10.1074/jbc.m509344200
复制
发表时间：
2006-03-31
影响因子：
4.8
通讯作者：
Matsuda, M
中科院分区：
文献类型：
--
作者：
Fujioka, A;Terai, K;Matsuda, M
为了理解Ras/ERK MAPK级联，包括Ras、Raf、MEK和ERK，已经开发了几种动力学模拟模型。然而，对于这些模型的开发至关重要的大量参数仍然缺失，需要任意设置。在这里，我们的目标是使用荧光探针收集这些缺失的参数。首先，对信号分子的水平进行定量。其次，为了监测ERK的激活和核转位，我们开发了基于荧光共振能量转移原理的探针。第三，使用可以非常快速地突出显示的荧光标记来估计Ras. Raf、Raf. MEK和MEK. ERK复合物的解离常数。最后，使用相同的荧光标记来测量ERK和MEK的核质穿梭率。利用这些参数，我们建立了一个由Ras/ERKMAPK级联反应的最小必需成员组成的动力学模拟模型。这个简单的模型再现了所观察到的ERK激活和核转位的基本特征。在该模型中，Raf的浓度显著影响刺激后磷酸化MEK和磷酸化ERK的水平。通过使用小干扰RNA技术降低Raf水平，实验证实了这一预测。这一观察结果验证了本研究中收集的参数的有用性。
To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.