Figure 9

Stress-activated Akt phosphorylates p27 at serine 10 in cells. A, Overexpressed Akt1 phosphorylates S10 in HEK293 cells. hwtp27 was transiently overexpressed in HEK293 alone or in the presence of co-transfected Akt1. S10 phosphorylation was determined by western blot analysis after 36 hrs. B, CuSO₄-dependent activation of Akt leads to p27S10 phosphorylation. Cells were transiently transfected as in A and treated with vehicle or CuSO₄ for 2 hours prior to harvest. Samples were analyzed by western blot. Akt inhibitor was added 30 minutes prior to CuSO₄. C, Endogenous Akt1 activated by oxidative stress phosphorylates p27S10. HEK293 cells were transfected with p27 and treated with CuSO₄ or FBS for 2 hours prior to harvest. Inhibitors were added 30 minutes before treatment. Samples were analyzed by western blot. D, H₂O₂-activated endogenous Akt phosphorylates p27S10. Experiment was performed as in C treating the cells with H₂O₂. E, Growth factor withdrawal leads to an Akt-dependent phosphorylation of p27S10. Hela cells were grown in 10% FBS (prolif.) or serum starved (G0) in the presence or absence of Akt inhibitor. Cells were harvested and phosphorylation of S10 analyzed by western blot. F, Mitogen-activated Akt does not target p27S10. HDF cells were synchronized in G0 by serum deprivation and re-fed with 20% FBS. Cells were lysed at the indicated time points and analyzed by western blot.