Fig. 3: EGFR is a GEF for Rheb.
From: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1
a Afatinib decreases the level of GTP-bound Rheb in PC9 and HCC827 cells. PC9 and HCC827 cells treated with or without 25 nM erlotinib or 25 nM afatinib for 12 h, were subjected to immunoprecipitation using Rheb-GTP agarose and analyzed by western blotting. b The Rheb-D60V mutant preferentially interacts with endogenous WT EGFR. HEK-293T cells were transfected with empty vector, 3× FLAG-Rheb-WT, -D60V, or -Q64L as indicated, subjected to immunoprecipitation, and analyzed by western blotting. c EDTA increases the interaction between EGFR and Rheb at endogenous levels. HeLa cells were lysed in the absence or presence of EDTA, subjected to immunoprecipitation, and analyzed by western blotting. d EGFR preferentially interacts with nucleotide-free or GDP-bound Rheb in vitro. Purified EGFR-TKD was incubated with GST, nucleotide-free GST-Rheb, GST-Rheb with GDP, or GST-Rheb with GTP as indicated, and then precipitated with GST beads and subjected to SDS-PAGE analysis. Coomassie blue staining is shown. e Top, the reconstitution peaks in the size-exclusion chromatography analysis of the purified EGFR-TKD-WT and Rheb-D60V complex. The reconstitution peak for the complex is shown by a dotted line and observed at 10.22 mL. Bottom, SDS-PAGE analysis of each reconstitution sample. The peak for the complex of EGFR-TKD and Rheb is indicated. f EGFR induces Rheb nucleotide exchange from the GDP-bound to the GTP-bound state. A guanine nucleotide exchange assay was performed in vitro using purified EGFR-TKD and Rheb. The relative fluorescence reflects the guanine nucleotide exchange activity. The initial fluorescence intensity was set to 1. Human RhoGEF Dbs (hDbs) and RhoA were used as positive controls. Curves are representative of three independent experiments. Data are presented as means ± SEM. g EGFR induces Rheb nucleotide exchange in a dose-dependent manner. An in vitro guanine nucleotide exchange assay was performed using purified Rheb and a concentration gradient of EGFR-TKD-WT as indicated. h LAMP2-V5-HER2-ICD, LAMP2-V5-IGF1R-ICD, or LAMP2-V5-c-MET-ICD failed to activate mTORC1. HEK-293T cells stably expressing LAMP2-V5-EGFR-TKD-WT, LAMP2-V5-HER2-ICD, LAMP2-V5-IGF1R-ICD, or LAMP2-V5-c-MET-ICD were serum-starved for 24 h and analyzed by western blotting.
