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.