title: The efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane conformer-specific nanoclustering.
abstract: Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling. Using quantitative conventional and super-resolution fluorescence methods, as well as mathematical modeling, we investigated nanoclustering of H-ras helix α4 and hypervariable region mutants that have different bona fide conformations on the membrane. By following the emergence of conformer-specific nanoclusters in the plasma membrane of mammalian cells, we found that conformers impart distinct nanoclustering responses depending on the cytoplasmic levels of the nanocluster scaffold galectin-1. Computational modeling revealed that complexes containing H-ras conformers and galectin-1 affect both the number and lifetime of nanoclusters and thus determine the specific Raf effector recruitment. Our results show that mutations in Ras can affect its nanoclustering response and thus allosterically effector recruitment and downstream signaling. We postulate that cancer- and developmental disease-linked mutations that are associated with the Ras membrane conformation may exhibit so far unrecognized Ras nanoclustering and therefore signaling alterations.
authors.affiliation: From the Turku Centre for Biotechnology, Åbo Akademi University, Tykistökatu 6B, 20520 Turku, Finland.
authors.forename: Camilo
authors.initials: C
authors.lastname: Guzmán
authors.forename: Maja
authors.initials: M
authors.lastname: Šolman
authors.forename: Alessio
authors.initials: A
authors.lastname: Ligabue
authors.forename: Olga
authors.initials: O
authors.lastname: Blaževitš
authors.forename: Débora M
authors.initials: DM
authors.lastname: Andrade
authors.forename: Luc
authors.initials: L
authors.lastname: Reymond
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
authors.forename: Daniel
authors.initials: D
authors.lastname: Abankwa
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D006224:Cricetinae; D037483:Galectin 1 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D009369:Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D037483:Galectin 1; D048490:raf Kinases; D015689:Oncogene Protein p21(ras)
keywords: Cancer; Fluorescence Correlation Spectroscopy; Fluorescence Recovery after Photobleaching (FRAP); Fluorescence Resonance Energy Transfer (FRET); Fluorescence-lifetime Microscopy Imaging (FLIM); Membrane; Nanocluster; Ras; Signaling; Stimulated Emission Depletion (STED)