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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Cancer Discov. 2012 Mar 31;2(5):458–471. doi: 10.1158/2159-8290.CD-11-0284

FIGURE 4. Type II EGFR kinase inhibitors more effectively displace ATP from the EGFR kinase domain of EGFR ectodomain mutants than type I inhibitors.

FIGURE 4

A. Schematic of ATP competition-binding assessment. Lysates of cells expressing the ATP-binding protein under study are simultaneously treated with a targeted agent (e.g. EGFR TKI) or vehicle, and a biotinylating ATP probe. All ATP-binding proteins will be biotinylated unless the ATP-binding pocket is occupied (e.g. with EGFR TKI). Lysates are subjected to avidin pulldown. The ability of the test compound to compete with ATP for binding to the target protein is assessed by immunoblot of the pulldown using antibodies against the target protein (e.g. EGFR). B. and C. Results of ATP competition assay in lysates from (B) EGFR EC mutant GBM cells and (C) EGFR KD mutant lung cancer cells. (B) Lapatinib more effectively competes with ATP for binding to the EGFR-TK in SKMG3 (EGFR A289D) cell lysates than erlotinib. (C.) Erlotinib more effectively competes with ATP for binding to the EGFR-TK in H3255 cell lysates (EGFR L858R KD mutation) than lapatinib. Cell lysates were carried through the assay described in A. The ATP probe was competed with the indicated doses of erlotinib or lapatinib. Following the avidin pulldown, samples were analyzed by immunoblot with antibodies for EGFR. Immunoblots were also probed with antibodies for Src as a control. D. A model of ligand-induced changes in EGFR conformation. In the absence of ligand (serum-free), the conformational equilibrium of EGFR-EC mutants is shifted towards to “inactive” conformation which is preferentially bound by type II inhibitors. In ligand-occupied receptor (EGF), the conformational equilibrium shifts towards the “active” conformation, which is the preferred substrate of type I inhibitors. E. EGF “desensitizes” the A289D EGFR EC mutant from lapatinib and “sensitizes” it to erlotinib. SKMG3 GBM cells were serum-starved, stimulated with EGF or vehicle, and subsequently treated with the indicated doses of erlotinib and lapatinib (while still under EGF treatment). Cell were lysed 30 minutes of drug treatment and analyzed by immunoblot with the indicated antibodies.