title: Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling.
abstract: Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, University Hospital, Jena, Germany.
authors.forename: Shu-Ping
authors.initials: SP
authors.lastname: Song
authors.forename: Anne
authors.initials: A
authors.lastname: Hennig
authors.forename: Katja
authors.initials: K
authors.lastname: Schubert
authors.forename: Robby
authors.initials: R
authors.lastname: Markwart
authors.forename: Philipp
authors.initials: P
authors.lastname: Schmidt
authors.forename: Ian A
authors.initials: IA
authors.lastname: Prior
authors.forename: Frank-Dietmar
authors.initials: FD
authors.lastname: Böhmer
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rubio
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000166:cytology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D054878:Lipoylation; D008565:Membrane Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D050505:Mutant Proteins / Q000819:agonists / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011994:Recombinant Proteins / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D019308:Palmitic Acid; D004815:Epidermal Growth Factor; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D018631:ras Proteins