Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading

Abstract

In this study we investigated the dynamics of R-Ras intracellular trafficking and its contributions to the unique roles of R-Ras in membrane ruffling and cell spreading. Wild type and constitutively active R-Ras localized to membranes of both Rab11- and transferrin-positive and -negative vesicles, which trafficked anterograde to the leading edge in migrating cells. H-Ras also co-localized with R-Ras in many of these vesicles in the vicinity of the Golgi, but R-Ras and H-Ras vesicles segregated proximal to the leading edge, in a manner dictated by the C-terminal membrane-targeting sequences. These segregated vesicle trafficking patterns corresponded to distinct modes of targeting to membrane ruffles at the leading edge. Geranylgeranylation was required for membrane anchorage of R-Ras, whereas palmitoylation was required for exit from the Golgi in post-Golgi vesicle membranes and trafficking to the plasma membrane. R-Ras vesicle membranes did not contain phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), whereas R-Ras co-localized with PtdIns(3,4,5)P3 in membrane ruffles. Finally, palmitoylation-deficient R-Ras blocked membrane ruffling, R-Ras/PI3-kinase interaction, enrichment of PtdIns(3,4,5)P3 at the plasma membrane, and R-Ras-dependent cell spreading. Thus, lipid modification of R-Ras dictates its vesicle trafficking, targeting to membrane ruffles, and its unique roles in localizing PtdIns(3,4,5)P3 to ruffles and promoting cell spreading.

An Increase in Tolerogenic Dendritic Cell and Natural Regulatory T Cell Numbers during Experimental Autoimmune Encephalomyelitis in Rras−/− Mice Results in Attenuated Disease

“…Ras proteins undergo prenylation, either farnesylation or geranylgeranylation, for anchoring to inner membranes and FTS functions by inhibiting the prenylation of Ras disrupting membrane localization thereby enhancing their degradation (82, 83). Although FTS blocks farnesylation and inhibits several members of the Ras family including H-Ras, N-Ras and K-Ras, it does not affect R-Ras activity, which unlike other Ras family proteins undergoes geranylgeranylation to anchor to membrane proteins (84). This differential anchoring mechanism provides a target for the development of specific R-Ras inhibitors.…”

The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration

“…One intriguing possibility relates to the role of RLIP76 as an effector of the small GTPase R-Ras[19]. We have found that R-Ras recruitment to the plasma membrane is required for its interaction with PI3K (also an R-Ras effector), localized generation of phosphatidylinositol (3,4,5) trisphospate (PIP 3 ), and formation of membrane ruffles and cell spreading[35]. ARNO and other Arf GEFs are recruited to the plasma membrane by interaction with PIP 3 via their pleckstrin homology (PH) domains, leading to localized Arf6 activation which may in turn be the downstream driver of cell spreading and motility[48,49,50,51,52].…”

“…NIH 3T3 cells were cultured and transfected, and processed for immunoprecipitations and western blotting as described[35]. …”

Inhibition of Galectin-1 Sensitizes HRAS-driven Tumor Growth to Rapamycin Treatment

“…Prolong Gold antifade mounting reagent was from Life Technologies (Carlsbad, CA, USA). GFP-RAS constructs were made as described elsewhere (12,16). GFP-HRAS-(G12V) (Addgene plasmid 18666) was a gift from K. Svoboda.…”