Figure 7.

Presenilins regulate EGFR expression, signaling and transformation through Fbw7. (a) Control and PS–/– fibroblasts stably transfected with empty (–) or Fbw7 small interfering RNAs (siRNAs) (+) pSuper vectors were analyzed by immunoblotting to detect endogenous Fbw7, EGFR, c-jun, Notch-4 and PS1 (NTF). Fbw7 siRNA reduced the levels of Fbw7 targets as well as EGFR. (b) PS–/– fibroblasts stably expressing control or Fbw7 siRNAs were transiently transfected with NICD-Myc cDNA by using LipofectAMINE 2000 and treated with CHX for the indicated times. NICD stability was analyzed by western blotting with 9E10 antibody. (c) PS–/– fibroblasts stably expressing pSuper or pSuper-Fbw7 siRNA were cultured in the absence (c) or presence of EGF for 0–30 min. Inactivation of Fbw7 reduced EGFR and pERK1/2 levels. The images are representative of three independent experiments. (d) PS1/PS2 and PS–/– fibroblasts stably expressing pSuper or pSuper-Fbw7 siRNA (n = 3) were cultured in soft-agar and colonies were quantified at day 15. Mean values±s.e.m. are shown. *P<0.05, compared with PS–/– pSuper cells; **P<0.001, compared with PS1/PS2 pSuper cells. (e) Model depicting the role of PS on mediating the dual regulation of EGFR and NICD by the ubiquitin ligase Fbw7α. According to this model, PS mediate NICD generation and degradation through γ-secretase and Fbw7, respectively. By contrast, PS negatively modulate EGFR ubiquitination and stability by affecting Fbw7 directly or indirectly through an unknown effector (X).