Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the translocation of RBD Raf1 -GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems.
|Number of pages||10|
|Journal||Molecular and Cellular Biochemistry|
|State||Published - Feb 2014|
Bibliographical noteFunding Information:
Acknowledgments We thank Gus Wright for help in editing the manuscript. This work was supported by a NIH Grant R01 GM66152 to HPS and R01 GM080370 to CJ.
- Ras function inhibitors
ASJC Scopus subject areas
- Molecular Biology
- Clinical Biochemistry
- Cell Biology