Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways
Fig 7
p17 stabilizes PTEN by promoting Rak-PTEN association and by stimulating phosphorylation of PTEN to protect PTEN from NEDD4-1 targeting.
(A) In reciprocal co-immunoprecipation experiments, the amounts of Rak and PTEN association were examined in ARV-infected or p17-transfected cells. Western blot assay of PTEN, and NEDD4-1 contained in PTEN or Rak immunoprecipates was carried out. The p17-transfected, mock-transfected, mock-infected, and ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection for Western blot assays. Data shown represent the mean± SD calculated from three independent experiments. The amounts of Rak-PTEN and NEDD4-1-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (B) In reciprocal co-immunoprecipation experiments, the binding of β-arrestin to PTEN was examined in either ARV-infected or p17-transfected Vero cells. Western blot of β-arrestin, PTEN, and NEDD4-1 contained in PTEN or β-arrestin immunoprecipates was carried out. Data shown represent the mean± SD calculated from three independent experiments. The amounts of β-arrestin-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (C) In coimmunoprecipation experiments, the binding of β-arrestin to PTEN was examined in p17-transfected Vero cells. Western blot of PTEN and β-arrestin contained in PTEN immunoprecipates was carried out in presence and absence of Rock-1 shRNA. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at vector only. The level of indicated proteins at vector only was considered 1-fold. (D) The interaction between PTEN and β-arrestin as well as between NEDD4-1 and PTEN were examined in p17-transfected and mock-transfected Vero cells. Proteins immunoprecipated with an anti-PTEN antibody from Vero cell lysates treated with Rak shRNA, Y-27632, and TBB, respectively were resolved by SDS-PAGE and immunbloted with the indicated antibodies. Data shown represent the mean± SD calculated from three independent experiments. (E) Cells were co-transfected with pcDNA3.1-p17 plasmid with either Rak or Rock-1 shRNA for 24 hours. The cells were harvested and washed twice in PBS buffer and scraped in 200 μl of lysis buffer. About 500 ug of cellular proteins was incubated with 4 ug of anti-PTEN antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot assay, and then proteins were detected with anti-ubiquitin antibody. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. (F) A model illustrates the PTEN regulation by p17.