Full length Akt1 phosphorylates p27 at serine 10 in vitro and in cells. A, Expression and activation of Akt1 in HEK293 cells. Increasing amounts of HA-tagged wtAkt1 were transfected in HEK293 cells as described in the Methods. After 36 hours active and total levels of Akt1 were determined by western blot analysis. B, Full length wild type Akt1 phosphorylates p27. Overexpressed HA-Akt1 was purified by immunoprecipitation (IP-Akt1) and incubated alone or with indicated forms of His-p27 in the presence of [32p]-γ-ATP as described in Methods. Autoradiograph shows full length Akt1 phosphorylates human and mouse p27 as well as the hp27T157A mutant. C, Wild type Akt1 targets S10. The kinase reaction was performed by incubating IP-Akt1 with non-radiolabeled ATP and indicated substrates. Western blot shows full length Akt phosphorylates S10. D, Specific Akt inhibitor blocks S10 phosphorylation. A kinase reaction was performed as in C incubating full length His-purified Akt1 with p27 in the presence or absence of Akt inhibitor. S10 phosphorylation was determined by phospho-specific antibody. E, Full length Akt1 fails to phosphorylate hp27S10A. Full length His-Akt1 was incubated in a kinase reaction with the indicated forms of p27 in the presence of radiolabeled ATP. Samples were separated by SDS/PAGE and visualized by autoradiography. Akt inhibitor was added where indicated.