Molecular and Cellular Biochemistry

, Volume 387, Issue 1–2, pp 177–186 | Cite as

Identification of a farnesol analog as a Ras function inhibitor using both an in vivo Ras activation sensor and a phenotypic screening approach

  • Kamalakkannan Srinivasan
  • Thangaiah Subramanian
  • H. Peter Spielmann
  • Chris Janetopoulos
Article

Abstract

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.

Keywords

Ras function inhibitors RBD Polarity Development 

Notes

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.

Supplementary material

Supplementary movie-1 Random motility of m-CN-AGOH-treated cells (MPEG 5190 kb)

Supplementary movie-2 Random motility of DMSO-treated cells Random motility of DMSO-treated cells (MPEG 1490 kb)

Supplementary movie-3 Random motility of m-CN-AGOH-treated and -washed cells (MPEG 1546 kb)

11010_2013_1883_MOESM4_ESM.ppt (1 mb)
Supplementary Figure 1 Cells expressing RBD Raf1 -GFP were treated with m-CN-AGOH and control cells expressing LimE-RFP were treated with DMSO. Cells were then both stimulated uniformly with 100 μM folic acid. m-CN-AGOH (Compound-8, Table 1) inhibited RBD Raf1 -GFP translocation to membrane, whereas there was no inhibition in the control cells expressing LimE-RFP. C and T indicate control and treated cells, respectively. Arrow indicates the recruitment of LimE-RFP to the plasma membrane in response to folic acid stimulation (bar, 5 μm). Supplementary Figure 2 Cells expressing RBD Raf1 -GFP and LimE-RFP were treated with putative inhibitors (Compounds-1-3, 5-9, 11, Table 1). Control cells expressing RBD Raf1 -GFP alone were treated with DMSO. These compounds did not inhibit translocation of RBD Raf1 -GFP or LimE-RFP to the plasma membrane. C and T indicate control and test, respectively. Arrow indicates plasma membrane RBD Raf1 -GFP and LimE-RFP responses to folic acid (bar, 5 μm). (PPT 1049 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kamalakkannan Srinivasan
    • 1
  • Thangaiah Subramanian
    • 3
  • H. Peter Spielmann
    • 3
    • 4
    • 5
    • 6
  • Chris Janetopoulos
    • 1
    • 2
  1. 1.Department of Biological SciencesVanderbilt UniversityNashvilleUSA
  2. 2.Cell and Developmental BiologyVanderbilt UniversityNashvilleUSA
  3. 3.Department of Molecular and Cellular BiochemistryUniversity of KentuckyLexingtonUSA
  4. 4.Markey Cancer CenterUniversity of KentuckyLexingtonUSA
  5. 5.Kentucky Center for Structural BiologyUniversity of KentuckyLexingtonUSA
  6. 6.Department of ChemistryUniversity of KentuckyLexingtonUSA

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