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

Research output: Contribution to journalArticle

  • 3 Citations

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.

LanguageEnglish
Pages1677-1683
Number of pages7
JournalBioconjugate Chemistry
Volume28
Issue number6
DOIs
StatusPublished - 21 Jun 2017

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Visualization
Proteins
Oncology
Cycloaddition
MAP Kinase Signaling System
Mitogen-Activated Protein Kinase Kinases
Phosphatases
Cycloaddition Reaction
Anchors
Fluorescent Dyes
Phosphoric Monoester Hydrolases
Antibodies
Fusion reactions
Dyes
Electrons
Staining and Labeling
Imaging techniques
Molecules
Neoplasms
Experiments

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. DOI: 10.1021/acs.bioconjchem.7b00152

Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe. / Sipthorp, James; Lebraud, Honorine; Gilley, Rebecca; Kidger, Andrew M.; Okkenhaug, Hanneke; Saba-El-Leil, Marc; Meloche, Sylvain; Caunt, Christopher J.; Cook, Simon J.; Heightman, Tom D.

In: Bioconjugate Chemistry, Vol. 28, No. 6, 21.06.2017, p. 1677-1683.

Research output: Contribution to journalArticle

Sipthorp, J, Lebraud, H, Gilley, R, Kidger, AM, Okkenhaug, H, Saba-El-Leil, M, Meloche, S, Caunt, CJ, Cook, SJ & Heightman, TD 2017, 'Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe' Bioconjugate Chemistry, vol. 28, no. 6, pp. 1677-1683. DOI: 10.1021/acs.bioconjchem.7b00152
Sipthorp J, Lebraud H, Gilley R, Kidger AM, Okkenhaug H, Saba-El-Leil M et al. Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe. Bioconjugate Chemistry. 2017 Jun 21;28(6):1677-1683. Available from, DOI: 10.1021/acs.bioconjchem.7b00152
Sipthorp, James ; Lebraud, Honorine ; Gilley, Rebecca ; Kidger, Andrew M. ; Okkenhaug, Hanneke ; Saba-El-Leil, Marc ; Meloche, Sylvain ; Caunt, Christopher J. ; Cook, Simon J. ; Heightman, Tom D./ Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe. In: Bioconjugate Chemistry. 2017 ; Vol. 28, No. 6. pp. 1677-1683
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