Membrane interactions of a constitutively active GFP-Ki-Ras 4B and their role in signaling - Evidence from lateral mobility studies

Membrane anchorage of Ras proteins in the inner leaflet of the plasma membr ane is an important factor in their signaling and oncogenic potential. Desp ite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these inter actions at the surface of intact cells, To investigate Ras-membrane interac tions in live cells, we employed studies on the lateral mobility of a const itutively active Ras isoform to characterize its membrane dynamics, and exa mined the effects of the Ras-displacing antagonist S-trans,trans-farnesylth iosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marc iano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306-1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP- Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was prefere ntially localized to the plasma membrane and displayed transforming activit y. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange betwee n membrane bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was fol lowed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associa ted with the plasma membrane. Control experiments with other S-prenyl analo gs showed that these effects are specific for FTS. These results have impli cations for the interactions of Ki-Ras with specific membrane anchorage dom ains or sites.