Sipthorp, J; Lebraud, H; Gilley, R; Kidger, AM; Okkenhaug, H; Saba-El-Lei, M; Meloche, S; ... Heightman, TD; + view all Sipthorp, J; Lebraud, H; Gilley, R; Kidger, AM; Okkenhaug, H; Saba-El-Lei, M; Meloche, S; Caunt, CJ; Cook, SJ; Heightman, TD; - view fewer (2017) Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe. Bioconjugate Chemistry , 28 (6) pp. 1677-1683. 10.1021/acs.bioconjchem.7b00152.

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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.

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