Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Mol Cell Biol. 2011 Oct;31(19):3938-52. doi: 10.1128/MCB.05570-11. Epub 2011 Aug 1.
The trafficking, membrane localization, and lipid raft association of Ras proteins, which are crucial oncogenic mediators, dictate their isoform-specific biological responses. Accordingly, their spatiotemporal dynamics are tightly regulated. While extensively studied for H- and K-Ras, such information on N-Ras, an etiological oncogenic factor, is limited. Here, we report a novel mechanism regulating the activation-dependent spatiotemporal organization of N-Ras, its modulation by biologically relevant stimuli, and isoform-specific effects on signaling. We combined patching/immobilization of another membrane protein with fluorescence recovery after photobleaching (patch-FRAP) and FRAP beam size analysis to investigate N-Ras membrane interactions. Clustering of raft-associated proteins, either glycosylphosphatidylinositol-anchored influenza virus hemagglutinin (HA-GPI) or fibronectin receptors, selectively enhanced the plasma membrane-cytoplasm exchange of N-Ras-GTP (preferentially associated with raft domains) in a cholesterol-dependent manner. Electron microscopy (EM) analysis showed N-Ras-GTP localization in cholesterol-sensitive clusters, from which it preferentially detached upon HA-GPI cross-linking. HA-GPI clustering enhanced the Golgi compartment (GC) accumulation and signaling of epidermal growth factor (EGF)-stimulated N-Ras-GTP. Notably, the cross-linking-mediated enhancement of N-Ras-GTP exchange and GC accumulation depended strictly on depalmitoylation. We propose that the N-Ras activation pattern (e.g., by EGF) is altered by raft protein clustering, which enhances N-Ras-GTP raft localization and depalmitoylation, entailing its exchange and GC accumulation following repalmitoylation. This mechanism demonstrates a functional signaling role for the activation-dependent differential association of Ras isoforms with raft nanodomains.
Ras 蛋白是关键的致癌介质，其转运、膜定位和脂筏缔合决定了它们的亚型特异性生物学反应。因此，它们的时空动力学受到严格调控。尽管 H-和 K-Ras 受到了广泛研究，但对于致癌因素 N-Ras 的信息则有限。在这里，我们报告了一种调节 N-Ras 激活依赖性时空组织的新机制，以及其对生物相关刺激的调制和对信号的亚型特异性影响。我们结合了另一种膜蛋白的贴片/固定化与光漂白后荧光恢复（patch-FRAP）和 FRAP 光束大小分析，以研究 N-Ras 与膜的相互作用。糖基磷脂酰肌醇锚定的流感病毒血凝素（HA-GPI）或纤连蛋白受体等筏相关蛋白的聚类选择性地增强了 N-Ras-GTP（优先与筏域相关）在胆固醇依赖性的质膜-细胞质交换。电子显微镜（EM）分析表明，N-Ras-GTP 定位于胆固醇敏感的簇中，在 HA-GPI 交联时，它优先从这些簇中脱离。HA-GPI 聚类增强了表皮生长因子（EGF）刺激的 N-Ras-GTP 的高尔基体（GC）积累和信号转导。值得注意的是，HA-GPI 交联介导的 N-Ras-GTP 交换和 GC 积累的增强严格依赖于去棕榈酰化。我们提出，Ras 蛋白聚类改变了 N-Ras 的激活模式（例如，通过 EGF），增强了 N-Ras-GTP 与脂筏纳米域的激活依赖性关联，从而在重新棕榈酰化后促进其交换和 GC 积累。这种机制证明了 Ras 亚型与脂筏纳米域的激活依赖性差异缔合在功能信号中的作用。
