Akt1 sequentially phosphorylates p27 at S10 and a neighboring serine. A, Akt1 does not phosphorylate hp27S10A. Upper panel: Recombinant Akt1 was incubated in a kinase reaction with the indicated forms of p27 as described. Samples were separated by SDS/PAGE and visualized by autoradiography. Bottom panel: His-tagged cyclin-E-CDK2 was purified from transfected HEK293 cells (see Methods), then incubated with indicated forms of p27 in the presence of [32P]-γ-ATP. Samples were separated by SDS-PAGE and visualized by autoradiography. Commassie staining shows equal amounts of hwtp27 and p27S10A were present in the reactions. B, Akt1 phosphorylates hp27S10T. Kinase reaction was performed with recombinant Akt1 and indicated substrates. Upper panel: autoradiograph showing phosphorylated p27. Bottom panel: western blot showing similar levels of p27 in the reaction. C, Phospho-amino acid analysis comparing hwtp27 and hp27S10T. Kinase reaction described in B was repeated and radiolabeled p27 was subjected to PAA as in Figure 3C. Top panel: scheme representing migration of phospho-amino acid standards. Second panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin. Third panel: phospho-amino acid analysis of radiolabeled hwtp27. Bottom panel: phospho-amino acid analysis of hp27S10T. Radiolabeled peptides and amino acids were detected by phosphoimager.