title: Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway.
abstract: 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 KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) 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.
authors.affiliation: California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine (OCSSB), Oregon Health and Science University, Portland, OR 97239; nan@ohsu.edu grayjo@ohsu.edu mccormic@cc.ucsf.edu schu@stanford.edu.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Nan
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158;
authors.forename: Tanja M
authors.initials: TM
authors.lastname: Tamgüney
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158;
authors.forename: Eric A
authors.initials: EA
authors.lastname: Collisson
authors.affiliation: Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine (OCSSB), Oregon Health and Science University, Portland, OR 97239;
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Lin
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158;
authors.forename: Cameron
authors.initials: C
authors.lastname: Pitt
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158;
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Galeas
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
authors.forename: Sophia
authors.initials: S
authors.lastname: Lewis
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine (OCSSB), Oregon Health and Science University, Portland, OR 97239; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158; nan@ohsu.edu grayjo@ohsu.edu mccormic@cc.ucsf.edu schu@stanford.edu.
authors.forename: Joe W
authors.initials: JW
authors.lastname: Gray
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158; nan@ohsu.edu grayjo@ohsu.edu mccormic@cc.ucsf.edu schu@stanford.edu.
authors.forename: Frank
authors.initials: F
authors.lastname: McCormick
authors.affiliation: Departments of Physics and Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305 nan@ohsu.edu grayjo@ohsu.edu mccormic@cc.ucsf.edu schu@stanford.edu.
authors.forename: Steven
authors.initials: S
authors.lastname: Chu
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D006160:Guanosine Triphosphate; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins
keywords: MAPK signaling; Ras dimer; cancer; single molecule imaging; superresolution microscopy