Lipid-Anchored Ras Proteins Sense/Modulate Plasma Membrane Curvature in an Isoform- Specific Manner

Abstract

Ras proteins, including isoforms H-, N- and K-Ras, are lipid-anchored small GTPases and play key roles in cell growth, division, proliferation, and migration. Constitutively active Ras mutants are oncogenic and found in 20% of all human tumors. Ras signaling is highly compartmentalized to the plasma membrane (PM), making their trafficking critical to their biological function. Ras trafficking is still poorly understood. Ras C-termini are lipid-modified, which potentially allows direct sensing and modulation of PM curvature, a key ultrastructural event in membrane trafficking. We measured Ras lateral oligomerization, an essential step in Ras signaling, on giant plasma membrane vesicles (GPMVs) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FLIM-FRET). Nanometer-scaled membrane curvature changes in the form of thermal undulations were manipulated via altering osmotic pressure. Hypotonic conditions disrupted local clustering of H-Ras anchor but enhanced K-Ras oligomerization, suggesting that H-Ras favors curved membrane and conversely for K-Ras. Super resolution electron microscopy-spatial mapping was used to quantify the lateral oligomerization of Ras on intact cell PM. Expression of positive curvature-inducing amphiphysin 2 BAR domain (BARamph2) enhanced H-Ras lateral oligomerization, but disrupted K-Ras oligomerization. On the other hand, expressing negative curvature-inducing IRS53p BAR domain (BARIRS53p) almost completely abolished H-Ras oligomerization, while having little effect on K-Ras. EM-bivariate co-localization analysis shows that H- or K-Ras co-localized extensively with both BAR domains. This was correlated with elevated mitogenic signaling (preferentially regulated by K-Ras) but diminished PI3K signaling (preferentially regulated by H-Ras) when exposed to hypotonic medium. Atomic force microscopy shows that expressing H-Ras anchor induced more PM protrusions and decreased the elastic Young's modulus of apical PM of live kidney cells, suggesting that H-Ras anchor induces PM curvature. Our data suggest that Ras senses and modulates membrane curvature in an isoform-specific manner.