Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe

James Sipthorp, Honorine Lebraud, Rebecca Gilley, Andrew M. Kidger, Hanneke Okkenhaug, Marc Saba-El-Leil, Sylvain Meloche, Christopher J. Caunt, Simon J. Cook, Tom D. Heightman

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4 Citations (Scopus)

Abstract

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.

Original languageEnglish
Pages (from-to)1677-1683
Number of pages7
JournalBioconjugate Chemistry
Volume28
Issue number6
DOIs
Publication statusPublished - 21 Jun 2017

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ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Sipthorp, J., Lebraud, H., Gilley, R., Kidger, A. M., Okkenhaug, H., Saba-El-Leil, M., ... Heightman, T. D. (2017). Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe. Bioconjugate Chemistry, 28(6), 1677-1683. https://doi.org/10.1021/acs.bioconjchem.7b00152