Fig. 4: EGFR-Glu804 is a potential glutamic finger indispensable for GEF activity.
From: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1
a AlphaFold2-Multimer prediction of EGFR-TKD complexed with Rheb (amino acids 1–184). Glu804 of EGFR is shown as a stick-and-ball model and is in close proximity to the nucleotide-binding pocket of Rheb (left), which is similar to the cytohesin-2–Arf1 complex structure (PDB: 1R8Q). GDP Guanosine diphosphate, G3P Guanosine-3’-monophosphate-5’-diphosphate. b EGFR-E804K does not bind Rheb in cells. HEK-293T cells stably expressing SFB-Rheb were transfected with HA-tagged EGFR-WT, -KD, or -E804K, then subjected to immunoprecipitation using anti-HA antibody, and analyzed by western blotting. c EGFR-E804K does not directly bind Rheb in vitro. Purified EGFR-TKD-WT or -E804K was incubated with GST or GST-tagged Rheb as indicated, precipitated with GST beads, and subjected to SDS-PAGE. Coomassie blue staining is shown. d EGFR-E804K failed to induce Rheb nucleotide exchange. An in vitro guanine nucleotide exchange assay was performed using purified EGFR-TKD-WT or -E804K and Rheb as indicated and analyzed as described in Fig. 3f. e LAMP2-V5-EGFR-TKD-WT or -KD, but not LAMP2-V5-EGFR-TKD-E804K, triggers the activation of mTORC1. HEK-293T cells stably expressing the indicated plasmids were serum-starved for 24 h and analyzed by western blotting. f Afatinib, but not erlotinib, impairs the EGFR-mediated Rheb nucleotide exchange. An in vitro guanine nucleotide exchange assay was performed using purified Rheb and EGFR-TKD-WT with the addition of erlotinib or afatinib as indicated and analyzed as described in Fig. 3f. g Afatinib inhibits mTORC1 activation in TSC2-deficient MEFs. WT and TSC2-deficient MEFs were serum-starved, treated with MK-2206 (10 μM), erlotinib (10 μM), or afatinib (10 μM) for 24 h, stimulated with 100 ng/mL EGF for 30 min and analyzed by western blotting. h Afatinib decreases the level of GTP-bound Rheb. TSC2-deficient MEFs treated with or without 10 μM erlotinib or 10 μM afatinib as indicated for 24 h were subjected to immunoprecipitation using Rheb-GTP agarose and analyzed by western blotting. i MK-2206 does not inhibit mTORC1 activation in PC9 and HCC827 cells. PC9 and HCC827 cells were treated with MK-2206 (1 μM), erlotinib (25 nM), or afatinib (25 nM) for 12 h as indicated, and analyzed by western blotting.
