Figure 3

Akt1 phosphorylates p27 at multiple serines within the N-terminus. A, Phospho-peptide maps comparing hwtp27 to hp27T157A. The kinase assay was performed as described with indicated forms of p27. After SDS/PAGE separation (upper panel) radiolabeled p27 was subjected to two-dimensional phospho-peptide analysis (lower panel) (See Methods for details). Direction of electrophoresis and chromatography are indicated by arrows. B, Schematic representation of phospho-peptide pattern. Ellipses 1–3 correspond to phospho-peptides in 3A, while number 4 represents [³²P]. C, Akt1 phosphorylates p27 at serines. Radiolabeled human wtp27 was hydrolyzed to constituent amino acids and then separated by electrophoresis on a TLC plate as described in Methods. Top panel: scheme representing migration of the phospho-amino acid standards (phospho-S and phospho-T). Middle panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin (see Methods). Bottom panel: phospho-amino acid analysis of [³²P]hwtp27 visualized by phosphoimager. D, Akt1 phosphorylates p27(1–86). Kinase assay was performed as described with His-purified p27(1–86) and p27(87–198). Samples were separated by SDS/PAGE and visualized by autoradiography. Commassie staining shows the levels of deletion mutants were similar. E, Phospho-peptide maps comparing hwtp27 to hp27(1–86). Radiolabeled hwtp27 and p27(1–86) were analyzed as in A.