Abstract
Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.
Highlights
Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells
Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant
Design and Construction of Mutant Ras Proteins—In order to evaluate the individual and combined contributions of either the methionine-containing CAAX (M-CAAX) motif characteristic of N-Ras and K-Ras4B or the polybasic domain (K6) unique to K-Ras4B, we generated a comprehensive set of chimeric Ras mutants (Fig. 1)
Summary
Generation of Ras Mutants—Ras constructs were generated by PCR using primers (University of North Carolina Lineberger Comprehensive Cancer Center Nucleic Acids Core Facility) coding for the desired mutant COOH-terminal sequence (Fig. 1). After glycerol shock, cells were returned to complete medium for 48 h in the presence of a 3 or 10 M concentration of the FTI L-744,832 (Biomol; a kind gift from Jay Gibbs and Allen Oliff (Merck)) or FTI-277 (Calbiochem; a kind gift from Saıd Sebti (University of South Florida) and Andy Hamilton (Yale)) or of vehicle (Me2SO or Me2SO plus 10 M dithiothreitol, respectively) These two FTIs are structurally distinct CAAX peptidomimetics that are competitive with the Ras protein. All assays were performed at least three times, and data are displayed Ϯ S.D. Soft Agar Assays—The ability of cells to grow in the absence of attachment to substrate is one of the hallmarks of a transformed phenotype and can be induced by oncogenically mutated H-, N-, or K-Ras. To determine the sensitivity of anchorage-independent growth to FTI treatment, NIH 3T3 fibroblasts stably transfected with constructs encoding H-, N-, or K-Ras4B mutants were removed from dishes by trypsinization and triturated to ensure a single-cell suspension.
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