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. 2021 Jan 7;220(2):e201902073. doi: 10.1083/jcb.201902073

Figure 1.

Figure 1.

pY374-PKCδ regulates EGFR recycling and degradation but not endocytosis. (a) Time course of EGFR endocytosis, induced by addition of EGF (20 ng/ml), in BT-549 cells transiently transfected with either control (siCtrl) or PTPN14 (siPTPN14) siRNA (mean fluorescence intensity ± SD; n = 3 replicates) with linear regression line of the time course. Two independent experiments are shown. Inset: Rates of endocytosis (change in mean fluorescence intensity over time ± range; n = 2) calculated by linear regression analysis of each time course. (b and c) Phosphorylation on Y374 on PKCδ is required to promote EGFR recycling in PTPN14-KO cells. BT-549 cells overexpressing either WT (wt) or Y374F-PKCδ (Fig. S2 b) were prelabeled with the mouse EGFR-ECD antibody in the presence of 20 ng/ml EGF (4°C), and endocytosis was initiated by incubation at 37°C. After stripping any EGFR-ECD antibody and EGF remaining on the cell surface with an acid wash at 4°C after the pulse of endocytosis, EGFR recycling was initiated by incubation at 37°C and allowed to continue for 30 min. Representative immunofluorescence micrographs show either cell surface EGFR in nonpermeabilized cells or intracellular EGFR in permeabilized cells as indicated (b) and quantified data from n = 2 independent experiments (c; mean ± SD; **, P < 0.01 using multiple t tests; Prism). (d–f) Loss of PTPN14 represses, whereas concomitant PKCδ knockdown derepresses, EGFR degradation in BT-549 breast cancer cells. Representative Western blot showing EGFR levels (top panel) and vinculin as a loading control (lower panel) in pCas9Vec control or PTPN14-KO cells, pretreated with cycloheximide for 30 min before stimulation with 100 ng/ml EGF for the indicated times (d). Representative Western blot showing PKCδ knockdown in PTPN14-KO cells (e) and quantitation of EGFR levels, relative to total protein loaded, at each time point after EGF stimulation (mean ± SD from n = 5 independent experiments for pCas9Vec/siCtrl and PTPN14-KO/siCtrl and n = 4 independent experiments using four different PKCδ siRNAs for PTPN14-KO/siPKCδ; f). The t1/2 ± SD of EGFR was obtained using a nonlinear one-phase decay fit of the time course; ***, P < 0.001; **, P < 0.01, Student’s t test.