Fig. 9: The Arl4A–VPS36 interaction attenuates EGFR degradation.
From: Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36
a HeLa cells were transfected with siControl or siVPS36 as well as the indicated plasmids. After 48 h of transfection, the cells were starved and stimulated with EGF (50 ng/ml) for the indicated times. b Quantification of EGFR levels was performed using ImageJ software. The results represent the mean ± SD of three independent experiments and the p-values were assessed by one-way ANOVA with Tukey’s test. c The lysates of the cells used in Fig. 9a were analyzed by Western blotting with antibodies against VPS36 and α-tubulin. d C33-A cells were transfected with either the control or VPS36 siRNA and combined with EGFR-Myc and the indicated plasmid. After 48 h, the cells were starved and stimulated with EGF (50 ng/ml) for the indicated times. Cell lysates were immunoprecipitated with Myc-Trap and analyzed by Western blotting with antibodies against ubiquitin. e Quantification of ubiquitin-EGFR was performed using ImageJ software. The graph resulted from densitometric scanning of the 60-min ubiquitin-EGFR signal relative to the 15-min ubiquitin-EGFR signal. The relative amount of ubiquitinated EGFR at 60 min was compared by one-way ANOVA. The results represent the mean ± SD of five independent experiments and the p-values were assessed by one-way ANOVA with Tukey’s test. f The lysates of the cells used in Fig. 9d were analyzed by western blotting with antibodies against VPS36 and α-tubulin. Source data are provided as a Source Data file. g Model showing how Arl4A attenuates EGFR degradation. Endosomal Arl4, through the interaction of ESCRT-II component VPS36, mediates EGFR ubiquitination levels, sorting to MVBs, and lysosomal degradation. Arl4A-depletion enhances the endocytosed EGFR transport to late endosomes/lysosomes and accelerates EGF-stimulated EGFR degradation. Specifically, the Arl4A-VPS36 interaction facilitates the association of VPS36 with MVBs and sustains the association of VPS36 and the ESCRT-III component CHMP2A, resulting in a reduction of CHMP2A-deubiquitinating enzyme USP8 interaction, which could further affect EGFR ubiquitination levels.
