Advertisement

Assaying Activation and Subcellular Localization of ERK in Cells and Tissues

  • Carme CaellesEmail author
  • Carles Bayod
  • Melisa Morcillo
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)

Abstract

The extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are the focus of many studies due to their involvement in numerous physiological and pathological processes, such as cell proliferation and differentiation, and oncogenic transformation, respectively. ERK1/2 belong to the mitogen-activated protein kinase (MAPKs) family, which are serine/threonine kinases that participate in signal transduction and are activated by dual phosphorylation in the Thr-X-Tyr motif located in their activation loop. In addition, ERK activation induces its dimerization and translocation into the nucleus. On the basis of this knowledge, different assays and tools have been developed to determine ERK activity or monitor its activation. In this chapter, we describe methods to assay ERK activity based on the ability of ERK immunocomplexes to phosphorylate a substrate, as well as on immunoblot analysis using antibodies that recognize ERK1/2 phosphorylated in the Thr-X-Tyr motif. In addition, we describe an immunocytochemistry procedure to reveal stimuli-induced nuclear translocation of ERK1/2.

Key words

ERK1 ERK2 ERK activity Phospho-ERK ERK nuclear translocation Immunocomplex assay Immunoblot analysis Immunocytochemistry analysis 

Notes

Acknowledgments

This work was supported by the Plan Nacional I + D grants SAF2013-40832-P and SAF2014-57856-P from the Spanish Ministry of Economy and Competitivity. C.B. was supported by an ADR predoctoral fellowship from University of Barcelona, Spain.

References

  1. 1.
    Avruch J (2007) MAP kinase pathways: the first twenty years. Biochim Biophys Acta 1773:1150–1160CrossRefPubMedGoogle Scholar
  2. 2.
    Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of MAPKs. Oncogene 26:3100–3112CrossRefPubMedGoogle Scholar
  3. 3.
    González MV, González-Sancho JM, Caelles C et al (1999) Hormone-activated nuclear receptors inhibit the stimulation of the JNK and ERK signalling pathways in endothelial cells. FEBS Lett 459:272–276CrossRefPubMedGoogle Scholar
  4. 4.
    Caelles C, Bruna A, Morales M et al (2002) Glucocorticoid receptor antagonism of AP-1 activity by inhibition of MAPK family. In: Cato ACB, Schäcke H, Asadullah K (eds) Recent advances in glucocorticoid receptor action. Springer, BerlinGoogle Scholar
  5. 5.
    Anderson NG, Maller JL, Tonks NK et al (1990) Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651–653CrossRefPubMedGoogle Scholar
  6. 6.
    Crews CM, Alessandrini A, Erikson RL (1992) The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science 258:478–480CrossRefPubMedGoogle Scholar
  7. 7.
    Khokhltchev AV, Canagarajah B, Wilsbacher J et al (1998) Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93:605–615CrossRefGoogle Scholar
  8. 8.
    Chen RH, Sarnecki C, Blenis J (1992) Nuclear translocation and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol 12:915–927CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Jacobs D, Glossip D, Xing H et al (1999) Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev 13:163–175CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Casar B, Sanz-Moreno V, Yazicioglu MN et al (2007) Mxi2 promotes stimulus-independent ERK nuclear translocation. EMBO J 26:635–646CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sun H, Charles CH, Lau LF, Tonks NK (1993) MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75:487–493CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and PhysiologySchool of Pharmacy, University of BarcelonaBarcelonaSpain

Personalised recommendations