期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：7996-8001
DOI：10.1073/pnas.1509123112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceRat sarcoma (Ras) proteins play central roles in both normal and oncogenic signaling. Mechanisms of how Ras interacts with its effectors on the cell membrane, however, are still poorly understood, significantly hampering efforts to target this molecule in human cancer. Here we have used quantitative superresolution fluorescence microscopy in combination with carefully engineered biological systems to show that Ras dimers drive oncogenic signaling through the Raf-MAPK pathway. Our study suggests a new, dimer model of Ras-Raf signaling and the potential value of Ras dimers as a therapeutic target. Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.
关键词：Ras dimer ; MAPK signaling ; cancer ; single molecule imaging ; superresolution microscopy
