Fig. 2: EGFR-TKD directly binds Rheb.
From: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1
a Co-localization of endogenous mutant EGFR with Rheb and LAMP1 (top) or mTOR (bottom). Immunofluorescence analysis of endogenous Rheb (green), EGFR (pink) and LAMP1 (top) or mTOR (bottom) (red) in EGFR-mutated cells using super-resolution SIM. Scale bar, 1 μm (enlarged view, 0.1 μm). b Afatinib inhibits the interaction between EGFR and Rheb in cells. PC9 cells treated with or without 25 nM erlotinib or afatinib for 12 h were subjected to immunoprecipitation and analyzed by western blotting. c The interaction between EGFR and Rheb is independent of EGFR kinase activity. HEK-293T cells stably expressing empty vector, HA-tagged EGFR WT or KD were subjected to immunoprecipitation and analyzed by western blotting. d Afatinib disrupts the EGFR–Rheb interaction in vitro. Purified EGFR-TKD was incubated with GST or GST-tagged Rheb1–169 (Rheb, unless specified) with or without 100 μM erlotinib or 100 μM afatinib, precipitated with GST beads, and subjected to SDS-PAGE analysis. Coomassie blue staining is shown.
