Fig. 3: PDZK1 promotes EGFR degradation by enhancing c-Cbl-mediated ubiquitination and inhibits EGFR phosphorylation by hindering EGFR dimerisation in TNBC.

A PDZK1 overexpression promoted EGFR degradation. Vector and PDZK1 expression plasmid were transfected into MDA-MB-231 cells stably overexpressing EGFR-WT or EGFR-MT for 24 h, then cells were treated with CHX at 0 h, 24 h or 36 h, EGFR protein level was detected by WB. Data were presented as mean ± SD of three independent experiments and analysed by repeated measures ANOVA. *P < 0.05, **P < 0.01. B PDZK1 overexpression promoted EGF-induced EGFR ubiquitination. MDA-MB-231 cells co-transfected with PDZK1 and EGFR-WT/EGFR-MT were stimulated with EGF, anti-EGFR antibody coupled with beads was used to precipitate lysates and ubiquitinated EGFR was detected by WB. C PDZK1 interacted with c-Cbl. PDZK1-WT, PDZK1-MT_S1, PDZK1-MT_S1,4 or PDZK1-MT_S2,3 were transfected into MDA-MB-231 cells stably overexpressing c-Cbl, and the interaction between PDZK1 and c-Cbl was detected by Co-IP. D PDZK1 promoted the binding of c-Cbl with EGFR. Myc-c-Cbl plasmid and PDZK1-Flag plasmid were transfected into MDA-MB-231 cells stably overexpressing EGFR-WT or EGFR-MT and were stimulated with EGF. EGFR was precipitated and E3 ubiquitin ligase c-Cbl was detected by WB. E Vector or PDZK1-HA expression plasmid were transfected into MDA-MB-231 cells overexpressing Myc and Flag tagged EGFR, and the level of EGFR dimerisation was detected by Co-IP. Data are the representative of three independent experiments (A–E).