Raft Protein Clustering Alters N-Ras Membrane Interactions and Activation Pattern
Raft Protein Clustering Alters N-Ras Membrane Interactions and Activation Pattern
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DOI：
10.1128/mcb.05570-11
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发表时间：
2011-10-01
影响因子：
5.3
通讯作者：
Henis, Yoav I.
中科院分区：
文献类型：
--
作者：
Eisenberg, Sharon;Beckett, Alison J.;Henis, Yoav I.
Ras蛋白是至关重要的致癌介质，其运输、膜定位和脂质筏关联决定了它们的同型特异性生物学反应。因此，它们的时空动态受到严格调节。虽然对H-和K-Ras进行了广泛的研究，但关于N-Ras（一种病因性致癌因子）的信息有限。在这里，我们报告了一种调节N-Ras的激活依赖时空组织的新机制，其受生物相关刺激的调节，以及对信号传导的异构体特异性作用。我们将另一种膜蛋白的贴片/固定化与光漂白后的荧光恢复（patch-FRAP）和FRAP光束大小分析相结合，研究N-Ras膜的相互作用。筏相关蛋白的聚类，无论是糖基磷脂酰肌醇锚定的流感病毒血凝素（HA-GPI）还是纤维连接蛋白受体，都以胆固醇依赖的方式选择性地增强了N-Ras-GTP（优先与筏结构域相关）的质膜-细胞质交换。电镜（EM）分析显示，N-Ras-GTP定位在胆固醇敏感簇中，当HA-GPI交联时，N-Ras-GTP优先脱离。HA-GPI聚集增强了表皮生长因子（EGF）刺激的N-Ras-GTP的高尔基室（GC）积累和信号传导。值得注意的是，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N-Ras的激活模式（如EGF）被筏蛋白聚集改变，这增强了N-Ras- gtp筏定位和去棕榈酰化，导致其在再棕榈酰化后的交换和GC积累。这一机制证明了Ras亚型与raft纳米结构域的激活依赖性差异关联的功能信号作用。
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.