Ras proteins (H, N and K) localize at the inner leaflet of plasma membrane, where they activate diverse signal transduction pathways. K-Ras is farnesylated and contains a polybasic domain (KKKKKK) at the C-terminal domain. These elements are necessary for proper subcellular localization of this protein. 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, polyelectrolyte and Ca2+ concentration on membrane binding properties of K-Ras. Results from these experiments suggest that, electrostatic interactions contribute to plasma membrane association of this protein. 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 ionophore induced a significant and fast redistribution of both full-length and C-terminal domain of K-Ras fused to fluorescent proteins from plasma membrane to cytoplasm. This redistribution was inhibited when cells were incubated with Ca2+ chelators (BAPTA-AM, EGTA). Together, these results suggest that the dynamic nature of the interactions between K-Ras and membranes, and its modulation by intracellular Ca2+ might be relevant for subcellular localization and function of this protein.
