A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling (original) (raw)

Nature volume 444, pages 230–234 (2006)Cite this article

Abstract

Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs1 or downstream pathway components such as Ras and Raf2. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components—in multiple cell types with different RTK ligands and oncogenic stimuli—validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.

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References

  1. Krause, D. S. & Van Etten, R. A. Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. 353, 172–187 (2005)
    Article CAS PubMed Google Scholar
  2. Hilger, R. A., Scheulen, M. E. & Strumberg, D. The Ras-Raf-MEK-ERK pathway in the treatment of cancer. Onkologie 25, 511–518 (2002)
    CAS PubMed Google Scholar
  3. DasGupta, R., Kaykas, A., Moon, R. T. & Perrimon, N. Functional genomic analysis of the Wnt-wingless signaling pathway. Science 308, 826–833 (2005)
    Article ADS CAS PubMed Google Scholar
  4. Gabay, L., Seger, R. & Shilo, B. Z. MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124, 3535–3541 (1997)
    CAS PubMed Google Scholar
  5. Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503 (2000)
    Article ADS CAS PubMed PubMed Central Google Scholar
  6. Kiger, A. et al. A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2 27 doi: 10.1186/1475-4924-2 (1 October, 2003)
  7. York, R. D. et al. Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392, 622–626 (1998)
    Article ADS CAS PubMed Google Scholar
  8. Luschnig, S., Krauss, J., Bohmann, K., Desjeux, I. & Nusslein-Volhard, C. The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases. Mol. Cell 5, 231–241 (2000)
    Article CAS PubMed Google Scholar
  9. Arevalo, J. C., Pereira, D. B., Yano, H., Teng, K. K. & Chao, M. V. Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation. J. Biol. Chem. 281, 1001–1007 (2006)
    Article CAS PubMed Google Scholar
  10. Johnson, S. M. et al. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647 (2005)
    Article CAS PubMed Google Scholar
  11. Roignant, J-Y., Hamel, S., Janody, F. & Treisman, J. E. The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway. Genes Dev. 20, 795–806 (2006)
    Article CAS PubMed PubMed Central Google Scholar
  12. Westbrook, T. F. et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848 (2005)
    Article CAS PubMed Google Scholar
  13. Chang, F. et al. Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review). Int. J. Oncol. 22, 469–480 (2003)
    CAS PubMed Google Scholar
  14. Asha, H. et al. Analysis of Ras-induced overproliferation in Drosophila hemocytes. Genetics 163, 203–215 (2003)
    CAS PubMed PubMed Central Google Scholar
  15. Nebreda, A. R., Gannon, J. V. & Hunt, T. Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes. EMBO J. 14, 5597–5607 (1995)
    Article CAS PubMed PubMed Central Google Scholar
  16. Chen, X. et al. Cyclin D-Cdk4 and cyclin E-Cdk2 regulate the Jak/STAT signal transduction pathway in Drosophila. . Dev. Cell 4, 179–190 (2003)
    Article CAS PubMed Google Scholar
  17. Yang, S. H., Sharrocks, A. D. & Whitmarsh, A. J. Transcriptional regulation by the MAP kinase signaling cascades. Gene 320, 3–21 (2003)
    Article CAS PubMed Google Scholar
  18. Lipsick, J. S. synMuv verité—Myb comes into focus. Genes Dev. 18, 2837–2844 (2004)
    Article CAS PubMed Google Scholar
  19. Jordan, K. C. et al. Genome wide analysis of transcript levels after perturbation of the EGFR pathway in the Drosophila ovary. Dev. Dyn. 232, 709–724 (2005)
    Article CAS PubMed Google Scholar
  20. Irie, H. Y. et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial–mesenchymal transition. J. Cell Biol. 171, 1023–1034 (2005)
    Article CAS PubMed PubMed Central Google Scholar
  21. Zimmermann, S. & Moelling, K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286, 1741–1744 (1999)
    Article CAS PubMed Google Scholar
  22. Puig, O. & Tjian, R. Transcriptional feedback control of insulin receptor by dFOXO/FOXO1. Genes Dev. 19, 2435–2446 (2005)
    Article CAS PubMed PubMed Central Google Scholar
  23. Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev. 16, 427–438 (2002)
    Article CAS PubMed PubMed Central Google Scholar
  24. Dan, I., Watanabe, N. M. & Kusumi, A. The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol. 11, 220–230 (2001)
    Article CAS PubMed Google Scholar
  25. Halfar, K., Rommel, C., Stocker, H. & Hafen, E. Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity. Development 128, 1687–1696 (2001)
    CAS PubMed Google Scholar
  26. Ory, S., Zhou, M., Conrads, T. P., Veenstra, T. D. & Morrison, D. K. Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr. Biol. 13, 1356–1364 (2003)
    Article CAS PubMed Google Scholar
  27. Sung, V. et al. The Ste20 kinase MST4 plays a role in prostate cancer progression. Cancer Res. 63, 3356–3363 (2003)
    CAS PubMed Google Scholar
  28. Baril, C. & Therrien, M. Alphabet, a Ser/Thr phosphatase of the protein phosphatase 2C family, negatively regulates RAS/MAPK signaling in. Drosophila. Dev. Biol. 294, 232–245 (2006)
    Article CAS PubMed Google Scholar
  29. Cheng, A., Kaldis, P. & Solomon, M. J. Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2Cα and β2 isoforms. J. Biol. Chem. 275, 34744–34749 (2000)
    Article CAS PubMed Google Scholar
  30. Takekawa, M., Maeda, T. & Saito, H. Protein phosphatase 2Calpha inhibits the human stress-responsive p38 and JNK MAPK pathways. EMBO J. 17, 4744–4752 (1998)
    Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank A. Philippakis and P. Hong for helpful discussions of statistical approaches, M. Melnick for dpERK antibody assistance, L. Kockel for pioneering the phospho-specific antibody high-throughput screen approach in our laboratory and help with in vivo analysis, C. Micchelli and R. Binari for assistance with genetic manipulations and interpretation, and other current and former members of the Perrimon lab for reagents and discussions. We thank L. Kockel, C. Micchelli, M. Kulkarni, B. Neel, R. Dasgupta, R. Binari and B. Mathey-Prevot for critical manuscript review. A.F. is a recipient of the Medical Scientist Training Program (MSTP) grant. N.P. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, 77 Avenue Louis Pasteur, Massachusetts, 02115, USA
    Adam Friedman & Norbert Perrimon

Authors

  1. Adam Friedman
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  2. Norbert Perrimon
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Corresponding author

Correspondence toNorbert Perrimon.

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Competing interests

Full datasets and dsRNA sequence information are available at the DRSC website (http://www.flyrnai.org). Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains the Supplementary Methods, Supplementary Figures and Legends 1–6, Supplementary Notes, and six of nine Supplementary Tables of data discussed in the text. (PDF 3675 kb)

Supplementary Table 1

This file contains the complete list of hits from the primary RNAi screen, sorted by gene name, and including amplicon identifiers and Z-score at baseline and under insulin stimulus. This list can also be obtained at www.flyrnai.org. (XLS 190 kb)

Supplementary Table 2

This file contains the complete secondary screen validated gene list, sorted by gene name, and including amplicon identifiers (both original primary screen-scoring amplicon and secondary amplicon, if available), and the percent of negative control (luciferase) normalized dpERK signal under each condition tested. (XLS 86 kb)

Supplementary Table 9

This file contains the complete epistasis data for validated genes screened in RasV12 and Gap1 experiments (see Supplementary Methods for details). (XLS 53 kb)

Supplementary Data

This file contains Gene keywords associated with this study. (DOC 19 kb)

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Friedman, A., Perrimon, N. A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling.Nature 444, 230–234 (2006). https://doi.org/10.1038/nature05280

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