H. Niv et al., Membrane interactions of a constitutively active GFP-Ki-Ras 4B and their role in signaling - Evidence from lateral mobility studies, J BIOL CHEM, 274(3), 1999, pp. 1606-1613
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.