The C-terminal Polylysine Region and Methylation of K-Ras Are Critical for the Interaction between K-Ras and Microtubules

doi:10.1074/jbc.M006687200

The C-terminal Polylysine Region and Methylation of K-Ras Are Critical for the Interaction between K-Ras and Microtubules*

From the Departments of ^‡Biochemistry and ^§Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686 and the ^‖Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and the Department of Medicine, University of California, San Francisco, California 94141-9100

Abstract

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants GM46372 (to P. J. C.) and AG15451 (to S. G. Y.), American Cancer Society Grant BE-117 (to P. J. C.), and a University of California Tobacco-related Disease Research Program grant (to S. G. Y.).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.
↵¶ Supported by a fellowship from the Leukemia Society of America.
↵** To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710-3686. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail: casey006@mc.duke.edu.
Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M006687200
Abbreviations:

FTase

farnesyltransferase

FPP

farnesyl diphosphate

DTT

dithiothreitol

GFP

green fluorescent protein

Rce1

a CAAX-specific prenyl-protein endoprotease

ICMT

isoprenylcysteine methyltransferase

ER

endoplasmic reticulum

AdoMet

S-adenosylmethionine

PCR

polymerase chain reaction

Pipes

1,4-piperazinediethanesulfonic acid
- Received July 26, 2000.
- Revision received September 12, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.