Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America.
PLoS One. 2012;7(5):e36469. doi: 10.1371/journal.pone.0036469. Epub 2012 May 14.
Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.
Shoc2 是一种假定的支架蛋白，可与 RAS 和 RAF 相互作用，并正向调节细胞外信号调节蛋白激酶 1 和 2（ERK1/2）的信号转导。为了阐明 Shoc2 调节表皮生长因子（EGF）受体（EGFR）激活 ERK1/2 的机制，我们研究了 Shoc2 的亚细胞定位。在 EGFR 激活后，内源性 Shoc2 和红色荧光蛋白标记的 Shoc2 从细胞质易位到包含 Rab7 的晚期内体的子集。Shoc2 的内体募集被 GDP 结合的 H-RAS（N17S）突变体的过表达和网格蛋白的 RNAi 敲低所阻断，表明需要 RAS 活性和网格蛋白依赖性内吞作用。Shoc2 的 RNAi 耗竭强烈抑制了低生理浓度 EGF 激活的 ERK1/2，而野生型重组 Shoc2 的表达可挽救该激活。相比之下，Shoc2（S2G）突变体（被豆蔻酰化并存在于类诺南综合征患者中）不能挽救 Shoc2 耗竭细胞中 ERK1/2 的激活。Shoc2（S2G）未定位在晚期内体中，而是存在于质膜和早期内体上。这些数据表明，Shoc2 靶向晚期内体可能有助于在 EGF 刺激细胞的生理条件下诱导 EGFR 诱导的 ERK 激活，因此可能参与了通过 RAS-RAF 模块的信号时空调节。
