TY - JOUR
T1 - Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe
AU - Sipthorp, James
AU - Lebraud, Honorine
AU - Gilley, Rebecca
AU - Kidger, Andrew M.
AU - Okkenhaug, Hanneke
AU - Saba-El-Leil, Marc
AU - Meloche, Sylvain
AU - Caunt, Christopher J.
AU - Cook, Simon J.
AU - Heightman, Tom D.
PY - 2017/6/21
Y1 - 2017/6/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85021164682&partnerID=8YFLogxK
UR - http://dx.doi.org/10.1021/acs.bioconjchem.7b00152
U2 - 10.1021/acs.bioconjchem.7b00152
DO - 10.1021/acs.bioconjchem.7b00152
M3 - Article
AN - SCOPUS:85021164682
SN - 1043-1802
VL - 28
SP - 1677
EP - 1683
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 6
ER -
