Ras proteins were described localizing at the inner leaflet of plasma membrane, where they activate diverse signal transduction pathways. There are three isoforms of Ras proteins, H-, N- and K-Ras, which undergo different lipid modifications, at the COOH-terminus. H- and N-Ras are farnesylated and palmitoylated, whereas K-Ras is farnesylated, contains a polybasic domain (KKKKKK) and is not palmitoylated. These post-translational modifications confer them the capacity to associate with plasma membrane. To evaluate the significance of membrane properties on plasma membrane association of K-Ras, constructs were engineered to express the full length and C-terminal domain of K-Ras fused to fluorescent proteins. By biochemical approaches, we evaluated in vitro the effect of ionic strength, H+, Ca2+ and polyelectrolyte concentration on membrane binding properties of K-Ras. Results from these experiments strongly suggest that plasma membrane association of K-Ras relies on reversible electrostatic interactions. We next extended the analysis of membrane association of K-Ras to evaluate the subcellular distribution of this protein in cells submitted to different conditions to perturb membrane properties. By using confocal microscopy, we did not observe significant changes in the intracellular distribution K-Ras neither in K+-depolarized PC12 cells nor in CHO-K1 cells incubated with neuraminidase to remove negatively charged sialic acid groups from the outer leaflet of plasma membrane. The externalization of phosphatidylserine, an anionic phospholipid, at early stages of staurosporine induced-apoptosis in CHO-K1 cells induced a slight redistribution of K-Ras from plasma membrane to cytoplasm. This redistribution was more evident at late stages of apoptosis, when fragmented nuclei were observed. To investigate the role of calcium on the subcellular distribution of K-Ras, we analyzed in live cells the effect of the calcium ionophore A23187 on the plasma membrane localization of K-Ras. The increase of intracellular calcium concentration induced a significant and fast redistribution of K-Ras from plasma membrane to cytoplasm. Together, these results suggest that the dynamic nature of the interactions between K-Ras and membranes, and its modulation by intracellular Ca2+ and changes in lipid asymmetry of target membranes, might be relevant for subcellular localization and function of this protein.
