title: Raft protein clustering alters N-Ras membrane interactions and activation pattern.
abstract: 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.
authors.affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Sharon
authors.initials: S
authors.lastname: Eisenberg
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Beckett
authors.forename: Ian A
authors.initials: IA
authors.lastname: Prior
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Dekker
authors.forename: Christian
authors.initials: C
authors.lastname: Hedberg
authors.forename: Herbert
authors.initials: H
authors.lastname: Waldmann
authors.forename: Marcelo
authors.initials: M
authors.lastname: Ehrlich
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D011905:Genes, ras; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D005353:Fibronectins; D017261:Glycosylphosphatidylinositols; D039081:Integrin alpha5beta1; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D018631:ras Proteins