Figure 10
HER2 ubiquitination is regulated by the PTPN18/β-Trcp complex in breast cancer cell lines and the HER2/PTPN18 ratio (expression ratio) is correlated with breast cancer stage. (A) Protein expression levels of PTPN18 and HER2 in seven breast cancer cell lines and HepG2. (B) Endogenous interaction between PTPN18 and β-Trcp was revealed by co-immunoprecipitation in MCF-7 cells. Left: PTPN18 was immunoprecipitated with protein A+G agarose beads and PTPN18 antibody with or without 100 ng/ml EGF stimulation for 30 min. PTPN18-associated β-Trcp was detected with a specific β-Trcp antibody. Right: reverse immunoprecipitation of PTPN18 with the β-Trcp antibody. (C) MCF-7 cells were transfected with HA-ubiquitin and PTPN18 siRNA. After 48 h, cells were stimulated with 100 ng/ml EGF for 4 h. Ubiquitinated HER2 was immunoprecipitated by anti-HA resin and detected with an anti-HER2 antibody. (D) Coordinated regulation of EGF-induced HER2 ubiquitination by β-Trcp and c-Cbl. MCF-7 cells were transfected with HA-ubiquitin and siRNAs directed against β-Trcp or c-Cbl. Ubiquitinated HER2 was examined with an anti-HER2 antibody as in C. (E) The relative ubiquitinated HER2 level in D was quantified and shown as a bar graph. *P < 0.05, **P < 0.01 compared with non-EGF-stimulated cells. #P < 0.05 compared with control siRNA. $P < 0.05 compared with Cbl siRNA. (F) Effects of siRNA-mediated knockdown of the β-Trcp expression on EGF-induced MCF-7 cell invasion. All statistics are from at least three independent experiments. *P< 0.05 compared with non-EGF-stimulated cells. #P < 0.05 compared with control. (G, H) The expression ratio of HER2 over PTPN18 (HER2/PTPN18) was significantly increased in tissues from breast cancer patients with stage III tumors compared with tissues from breast cancer patients with stage II tumors. (G), Relative HER2 expression divided by PTPN18 expression (See Supplementary information, Figure S10 for original western blots). (H) Statistics for G. (I) A proposed working model of PTPN18 regulated HER2 tyrosine phosphorylation and ubiquitination pathway. After activation, HER2 was phosphorylated on multiple sites at its C-terminus. PTPN18 specifically dephosphorylated HER2 pY1196 and pY1248 sites and inhibited HER2 signaling cascade for cell proliferation and invasion. Meanwhile, PTPN18 not only blocked the c-Cbl-mediated lysosome targeting of HER2 through dephosphorylation of HER2 on pY1112 site, but also recruited E3 ligase β-Trcp for K48-linked HER2 ubiquitination and proteasomal degradation. The interaction between PTPN18 and β-Trcp is phosphorylation regulated and requires AKT activation. The “HER2-AKT-PTPN18-HER2 ubiquitination” is a negative feedback loop for HER2 regulation.
