Imaging of Ras-MAP kinase signal transduction cascade

基于绿色荧光蛋白和荧光共振能量转移(FRET)的技术的最新发展使我们能够监测信号分子在活细胞中发生的构象变化。我们已经成功地开发了监测Ras家族GTP酶活性变化的探针，并显示了活细胞中Ras活性的动态变化。一个关键的信号分子c-Raf位于RAS下游，丝裂原活化蛋白激酶(MAPK)的上游。在这里，我们用基于荧光共振能量转移(FRET)原理的探针研究了RAS信号转导到MEK的机制。与前人的模型一致，发现c-Raf具有两种构象：开放活性构象和闭合非活性构象。RAS结合诱导c-Raf从封闭构象向开放构象转变，使c-Raf与MEK结合。在胞质RAS突变体的存在下，c-Raf与细胞质中的MEK结合，但不能磷酸化。相反，胞质RAS突变体通过膜靶向c-Raf显著增强MEK的磷酸化。这些结果证实了RAS诱导的构象变化在c-Raf激活MEK中的重要作用。

The recent development of a technology based on GFP and fluorescence resonance energy transfer (FRET) has enabled us to monitor conformational changes in signaling molecules as they occur in living cells. We have successfully developed probes to monitor the activity change of Ras-family GTPases and shown the dynamic change of Ras activity in living cells. A key signaling molecule, c-Raf, is situated downstream from Ras and upstream from the mitogen-activated protein kinase (MAPK) kinase (MEK). Here, we studied the mechanism underlying the signal transduction from Ras to MEK by using probes based on the principle of fluorescence resonance energy transfer (FRET). In agreement with previous models, it was found that c-Raf adopted two conformations: open active and closed inactive. Ras binding induced the c-Raf transition from closed to open conformation, which enabled c-Raf to bind to MEK. In the presence of a cytosolic Ras mutant, c-Raf bound to, but failed to phosphorylate, MEK in the cytoplasm. In contrast, the cytosolic Ras mutant significantly enhanced MEK phosphorylation by a membrane-targeted c-Raf. These results demonstrated the essential role of Ras-induced conformational change in MEK activation by c-Raf.