ERK Signaling pp 235-241 | Cite as

Co-culture Activation of MAP Kinase in Drosophila S2 Cells

  • Josefa SteinhauerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)


Intercellular communication often involves phosphorylation of signal transduction proteins, including mitogen-activated protein kinases (MAPKs). Immunological detection of phosphorylated MAPK can be used to monitor signaling in vivo, identify novel pathway components, and assess ligand activity. In this chapter, I describe a cell co-culture method to assess activity of cell-bound extracellular ligands that result in phosphorylation of the ERK (extracellular signal-regulated kinase) MAPK in Drosophila. This protocol may be adaptable to other pathways and/or model systems.

Key words

MAP kinase ERK dp-ERK S2 cells Co-culture Western blot 



I thank Jean-Yves Roignant and Kevin Legent for help developing the ERK activation assay; Benny Shilo for the D2F cells; Jessica Treisman for support during the development of the protocol and critical comments on the manuscript. This work was supported by the March of Dimes Birth Defects Foundation (to Jessica Treisman) and the National Institutes of Health (grant number EY13777 to Jessica Treisman from the National Eye Institute).


  1. 1.
    Shilo BZ (2014) The regulation and functions of MAPK pathways in Drosophila. Methods 68:151–159CrossRefPubMedGoogle Scholar
  2. 2.
    Wortzel I, Seger R (2011) The ERK cascade: distinct functions within various subcellular organelles. Genes Cancer 2:195–209CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Carlson SM, Chouinard CR, Labadorf A et al (2011) Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3. Sci Signal 4(196):11CrossRefGoogle Scholar
  4. 4.
    Bandyopadhyay S, Chiang CY, Srivastava J et al (2010) A human MAP kinase interactome. Nat Methods 7:801–805CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kosako H, Yamaguchi N, Aranami C et al (2009) Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport. Nat Struct Mol Biol 16:1026–1035CrossRefPubMedGoogle Scholar
  6. 6.
    von Kriegsheim A, Baiocchi D, Birtwistle M et al (2009) Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 11:1458–1464CrossRefGoogle Scholar
  7. 7.
    Friedman A, Perrimon N (2007) Genetic screening for signal transduction in the era of network biology. Cell 128:225–231CrossRefPubMedGoogle Scholar
  8. 8.
    Futran AS, Link AJ, Seger R et al (2013) ERK as a model for systems biology of enzyme kinetics in cells. Curr Biol 23:R972–R979CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Friedman A, Perrimon N (2006) A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling. Nature 444:230–234CrossRefPubMedGoogle Scholar
  10. 10.
    Clemens JC, Worby CA, Simonson-Leff N et al (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A 97:6499–6503CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Brunner D, Oellers N, Szabad J et al (1994) A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways. Cell 76:875–888CrossRefPubMedGoogle Scholar
  12. 12.
    Biggs WH 3rd, Zipursky SL (1992) Primary structure, expression, and signal-dependent tyrosine phosphorylation of a Drosophila homolog of extracellular signal-regulated kinase. Proc Natl Acad Sci U S A 89:6295–6299CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gabay L, Seger R, Shilo BZ (1997) In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277:1103–1106CrossRefPubMedGoogle Scholar
  14. 14.
    Gabay L, Seger R, Shilo BZ (1997) MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124:3535–3541PubMedGoogle Scholar
  15. 15.
    Lim B, Samper N, Lu H et al (2013) Kinetics of gene derepression by ERK signaling. Proc Natl Acad Sci U S A 110:10330–10335CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lim B, Dsilva CJ, Levario TJ et al (2015) Dynamics of inductive ERK signaling in the Drosophila embryo. Curr Biol 25:1784–1790CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Miura GI, Roignant JY, Wassef M et al (2008) Myopic acts in the endocytic pathway to enhance signaling by the Drosophila EGF receptor. Development 135:1913–1922CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Friedman AA, Tucker G, Singh R et al (2011) Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling. Sci Signal 4(196):10Google Scholar
  19. 19.
    Ashton-Beaucage D, Udell CM, Gendron P et al (2014) A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila. PLoS Biol 12, e1001809CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Steinhauer J, Liu HH, Miller E et al (2013) Trafficking of the EGFR ligand Spitz regulates its signaling activity in polarized tissues. J Cell Sci 126:4469–4478CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Brachmann R, Lindquist PB, Nagashima M et al (1989) Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors. Cell 56:691–700CrossRefPubMedGoogle Scholar
  22. 22.
    Dong J, Opresko LK, Chrisler W et al (2005) The membrane-anchoring domain of epidermal growth factor receptor ligands dictates their ability to operate in juxtacrine mode. Mol Biol Cell 16:2984–2998CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Iwamoto R, Handa K, Mekada E (1999) Contact-dependent growth inhibition and apoptosis of epidermal growth factor (EGF) receptor-expressing cells by the membrane-anchored form of heparin-binding EGF-like growth factor. J Biol Chem 274:25906–25912CrossRefPubMedGoogle Scholar
  24. 24.
    Wong ST, Winchell LF, McCune BK et al (1989) The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell 56:495–506CrossRefPubMedGoogle Scholar
  25. 25.
    Schweitzer R, Shaharabany M, Seger R et al (1995) Secreted Spitz triggers the DER signaling pathway and is a limiting component in embryonic ventral ectoderm determination. Genes Dev 9:1518–1529CrossRefPubMedGoogle Scholar
  26. 26.
    Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27:353–365PubMedGoogle Scholar
  27. 27.
    Miura GI, Buglino J, Alvarado D et al (2006) Palmitoylation of the EGFR ligand Spitz by Rasp increases Spitz activity by restricting its diffusion. Dev Cell 10:167–176CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of BiologyYeshiva UniversityNew YorkUSA

Personalised recommendations