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J. Biol. Chem., Vol. 283, Issue 40, 27279-27288, October 3, 2008

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Differential Interference of Chlorpromazine with the Membrane Interactions of Oncogenic K-Ras and Its Effects on Cell Growth^*

Sharon Eisenberg¹, Klaudia Giehl^¶, Yoav I. Henis², and Marcelo Ehrlich^||3

From the Departments of Neurobiology and ^||Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel and the Institute of Pharmacology and Toxicology and the ^¶Department of Internal Medicine I, University of Ulm Medical Center, 89070 Ulm, Germany

Membrane anchorage of Ras proteins is important for their signaling and oncogenic potential. K-Ras4B (K-Ras), the Ras isoform most often mutated in human cancers, is the only Ras isoform where a polybasic motif contributes essential electrostatic interactions with the negatively charged cytoplasmic leaflet. Here we studied the effects of the cationic amphiphilic drug chlorpromazine (CPZ) on the membrane association of oncogenic K-Ras(G12V), cell proliferation, and apoptosis. Combining live cell microscopy, FRAP beam size analysis, and cell fractionation studies, we show that CPZ reduces the association of GFP-K-Ras(G12V) with the plasma membrane and increases its exchange between plasma membrane and cytoplasmic pools. These effects appear to depend on electrostatic interactions because the membrane association of another related protein that has a membrane-interacting polybasic cluster (Rac1(G12V)) was also affected, whereas that of H-Ras was not. The weakened association with the plasma membrane led to a higher fraction of GFP-K-Ras(G12V) in the cytoplasm and in internal membranes, accompanied by either cell cycle arrest (PANC-1 cells) or apoptosis (Rat-1 fibroblasts), the latter being in correlation with the targeting of K-Ras(G12V) to mitochondria. In accord with these results, CPZ compromised the transformed phenotype of PANC-1 cells, as indicated by inhibition of cell migration and growth in soft agar.

Received for publication, June 16, 2008 , and in revised form, August 5, 2008.

^* This work was supported in part by an award from the Jacqueline Seroussi Memorial Foundation for Cancer Research (to Y. I. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by a Nehemia Levtzion Ph.D. Fellowship awarded by The Council for Higher Education, Israel.

² Incumbent of the Zalman Weinberg Chair in Cell Biology. To whom correspondence may be addressed. Tel.: 972-3-640-9053; Fax: 972-3-640-7643; E-mail: henis{at}post.tau.ac.il.

³ To whom correspondence may be addressed. Tel.: 972-3-640-9406; Fax: 972-3-642-2046; E-mail: marceloe{at}tauex.tau.ac.il.

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