Membrane Interactions of a Constitutively Active GFP-Ki-Ras 4B and Their Role in Signaling

doi:10.1074/jbc.274.3.1606

Membrane Interactions of a Constitutively Active GFP-Ki-Ras 4B and Their Role in Signaling

EVIDENCE FROM LATERAL MOBILITY STUDIES*

From the Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

Abstract

Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonistS-trans,trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, 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 preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between 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 followed 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 associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites.

Footnotes

↵* This work was supported in part by Grant 97-00141 from the United States-Israel Binational Science Foundation, Jerusalem, Israel (to Y. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed. Tel.: 972-3-640-9699; Fax: 972-3-640-7643; E-mail: kloog{at}post.tau.ac.il.
↵2 G. Elad, A. Paz, R. Haklai, D. Marciano, and Y. Kloog, unpublished observations.
↵3 Y. Egozi, M. Gana-Weisz, and Y. Kloog, unpublished results.
↵4 H. Niv and Y. Kloog, unpublished observations.
Abbreviations:

FTS

S-trans,trans-farnesylthiosalicylic acid

AFC

N-acetyl-S-trans,trans-farnesyl-l-cysteine

BSA

bovine serum albumin

D

lateral diffusion coefficient

DiIC₁₆

1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocynanine perchlorate

DMEM

Dulbecco’s modified Eagle’s medium

FCS

fetal calf serum

FPR

fluorescence photobleaching recovery

GFP

green fluorescent protein

GFP-Ki-Ras(12V)

GFP-tagged constitutively active Ki-Ras 4B(V12)

GPI

glycosylphosphatidylinositol

GTS

S-geranylthiosalicylic acid

HBSS

Hanks’ balanced salt solution

PBS

phosphate-buffered saline

R18

octadecyl rhodamine B chloride

R_F

mobile fraction.
- Received August 7, 1998.
- Revision received September 28, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.