Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Mol Biol Cell. 2010 Mar 15;21(6):1088-96. doi: 10.1091/mbc.e09-06-0455. Epub 2010 Jan 13.
Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca(2+) in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca(2+) concentration or inhibition of calmodulin. Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.
Ras 的下游是一种关键的信号分子，即 Raf1。已经表明，钙离子浓度的增加可以调节 Ras 依赖性的 Raf1 激活；然而，这种效应的机制仍然难以捉摸。在这里，为了表征 Ca(2+) 在 Ras 信号转导至 Raf1 中的作用，我们使用了一种 Ras 的合成鸟嘌呤核苷酸交换因子 (GEF)，即 eGRF。在表达 eGRF 的 HeLa 细胞中，cAMP 类似物 007 能够像表皮生长因子 (EGF)一样有效地激活 Ras，而 007 对 Raf1、MEK 和 ERK 的激活程度约为 EGF 的一半。使用基于荧光共振能量转移的生物传感器，发现 Raf1 在质膜上的激活不仅需要 Ras 的激活，还需要钙离子浓度的增加或钙调蛋白的抑制。此外，发现 Shoc2 敲低会消除 Raf1 的 Ca(2+)依赖性激活，Shoc2 是一种结合 Ras 和 Raf1 的支架蛋白。这些观察结果表明，Shoc2 支架蛋白以 Ca(2+)和钙调蛋白依赖的方式调节 Ras 依赖性 Raf1 激活。
