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. 2021 May 25;220(7):e202102005. doi: 10.1083/jcb.202102005

Figure 3.

Figure 3.

Serine 1006 and the R1 serine/threonine cluster are necessary for p38-induced, LL1010/1011-mediated EGFR internalization. (A) Schematic representation of EGFR region encompassed by residues 999–1020 including the dileucine motif and surrounding clusters of serines and threonines. Residues 1015–1018 are designated as the R1 cluster per Tanaka et al. (2018). Key mutations and mutant names are indicated. (B) PAE cells stably expressing EGFR-GFP (WT or indicated mutants) were treated with 100 nM anisomycin for 15 min and immunolabeled with the EEA1 antibody. 3D images were acquired through the 488-nm (green, EGFR-GFP) and 640-nm (red, EEA1) channels. Maximum intensity projections of three consecutive confocal sections are shown. Scale bars, 10 µm. (C) Quantification of the fraction of EGFR-GFP colocalized with EEA1 endosomes in images exemplified in B. Scatter dot plot represents mean values with SDs (n = 8–15). P values were determined by multiple-comparison one-way ANOVA. P values against “WT plus anisomycin” are shown in black. P values against LL1010/11A mutant are shown in red. (D and E) PAE cells stably expressing EGFR-GFP (WT or mutants as indicated) were treated with anisomycin for 15 min and lysed, and EGFR-GFP was immunoprecipitated. In D, total cell lysates (TCL) and immunoprecipitates (IP) were probed by Western blotting (WB) with antibodies to α-adaptin subunit of AP2 (α-Ad), pS1015, phosphorylated p38 (p-p38), total phosphoserine (pSer), and GFP. The identity of α-adaptin band was confirmed by immunoblotting analysis of cells depleted of AP2 by μ2 siRNA (Fig. S2 F). In E, the amount of AP2 coimmunoprecipitated with EGFR-GFP was normalized by the amount of immunoprecipitated EGFR-GFP, and the values of the AP2/EGFR-GFP ratio were further normalized to this value obtained in immunoprecipitates from anisomycin-treated WT cells in each independent experiment. Bar graph represents mean values with SEM (n = 2–4 independent experiments). P values against WT treated with anisomycin were determined by the unpaired Student’s t test.