Publication — IRIC

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

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.

Publication date
June 21, 2017
Principal Investigators
Sipthorp J, Lebraud H, Gilley R, Kidger AM, Okkenhaug H, Saba-El-Leil M, Meloche S, Caunt CJ, Cook SJ, Heightman TD
PubMed reference
Bioconjug. Chem. 2017;28(6):1677-1683
PubMed ID
28449575
Affiliation
Signalling Laboratory, The Babraham Institute , Babraham Research Campus, Cambridge CB22 3AT, U.K.