Genetic and functional characterization of putative Ras/Raf interaction inhibitors in C. elegans and mammalian cells
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González Pérez, Vanessa, et al. Genetic and Functional Characterization of Putative Ras/raf Interaction Inhibitors In C. Elegans and Mammalian Cells. BioMed Central Ltd, 2010. https://doi.org/10.17615/afe3-8166APA
González Pérez, V., Reiner, D., Alan, J., Mitchell, C., Edwards, L., Khazak, V., Der, C., & Cox, A. (2010). Genetic and functional characterization of putative Ras/Raf interaction inhibitors in C. elegans and mammalian cells. BioMed Central Ltd. https://doi.org/10.17615/afe3-8166Chicago
González Pérez, Vanessa, David Reiner, Jamie K Alan, Cicely Mitchell, Lloyd Edwards, Vladimir Khazak, Channing Der et al. 2010. Genetic and Functional Characterization of Putative Ras/raf Interaction Inhibitors In C. Elegans and Mammalian Cells. BioMed Central Ltd. https://doi.org/10.17615/afe3-8166- Creator
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González-Pérez, Vanessa
- Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
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Reiner, David
- Affiliation: N.C. Cancer Hospital, UNC Lineberger Comprehensive Cancer Center, School of Medicine, Department of Pharmacology
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Alan, Jamie K
- Affiliation: School of Medicine, Department of Pharmacology
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Mitchell, Cicely
- Affiliation: Gillings School of Global Public Health, Department of Biostatistics
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Edwards, Lloyd
- Affiliation: Gillings School of Global Public Health, Department of Biostatistics
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Khazak, Vladimir
- Other Affiliation: NexusPharma, Inc., 253-13 Summit Sq. Ctr., Langhorne, PA, 19047
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Der, Channing
- Affiliation: N.C. Cancer Hospital, UNC Lineberger Comprehensive Cancer Center, School of Medicine, Curriculum in Genetics and Molecular Biology
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Cox, Adrienne
- Affiliation: N.C. Cancer Hospital, UNC Lineberger Comprehensive Cancer Center, School of Medicine, Curriculum in Genetics and Molecular Biology
- Abstract
- Abstract Background Activation of the mammalian Ras-Raf-MEK-ERK MAPK signaling cascade promotes cellular proliferation, and activating Ras mutations are implicated in cancer onset and maintenance. This pathway, a therapeutic target of interest, is highly conserved and required for vulval development in C. elegans. Gain-of-function mutations in the Ras ortholog lead to constitutive pathway signaling and a multivulva (Muv) phenotype. MCP compounds were identified in a yeast two-hybrid screen for their ability to disrupt Ras-Raf interactions. However, this had not been confirmed in another system, and conflicting results were reported regarding selective MCP-mediated blockade of Ras- and Raf-mediated biological activities in mammalian cells. Here we used the easily-scored Muv phenotype as an in vivo readout to characterize the selectivity of MCP110 and its analogs, and performed biochemical studies in mammalian cells to determine whether MCP treatment results in impaired interaction between Ras and its effector Raf. Results Our genetic analyses showed significant dose-dependent MCP-mediated reduction of Muv in C. elegans strains with activating mutations in orthologs of Ras (LET-60) or Raf (LIN-45), but not MAP kinases or an Ets-like transcription factor. Thus, these inhibitors selectively impair pathway function downstream of Ras and upstream of or at the level of Raf, consistent with disruption of the Ras/Raf interaction. Our biochemical analyses of MCP110-mediated disruption of Ras-Raf interactions in mammalian cells showed that MCP110 dose-dependently reduced Raf-RBD pulldown of Ras, displaced a fluorescently-tagged Raf-RBD probe from plasma membrane locations of active Ras to the cytosol and other compartments, and decreased active, phosphorylated ERK1/2. Conclusions We have effectively utilized C. elegans as an in vivo genetic system to evaluate the activity and selectivity of inhibitors intended to target the Ras-Raf-MAPK pathway. We demonstrated the ability of MCP110 to disrupt, at the level of Ras/Raf, the Muv phenotype induced by chronic activation of this pathway in C. elegans. In mammalian cells, we not only demonstrated MCP-mediated blockade of the physical interaction between Ras and Raf, but also narrowed the site of interaction on Raf to the RBD, and showed consequent functional impairment of the Ras-Raf-MEK-ERK pathway in both in vivo and cell-based systems.
- Date of publication
- February 23, 2010
- DOI
- Identifier
- Resource type
- Article
- Rights statement
- In Copyright
- Rights holder
- Vanessa González-Pérez et al.; licensee BioMed Central Ltd.
- License
- Journal title
- Journal of Molecular Signaling
- Journal volume
- 5
- Journal issue
- 1
- Page start
- 2
- Language
- English
- Is the article or chapter peer-reviewed?
- Yes
- ISSN
- 1750-2187
- Bibliographic citation
- Journal of Molecular Signaling. 2010 Feb 23;5(1):2
- Publisher
- BioMed Central Ltd
- Access right
- Open Access
- Date uploaded
- August 23, 2012
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