Kinetic analysis of p27 phosphorylation. A, Time course of p27 phosphorylation. Kinase reaction was performed as described with hwtp27 or p27T157A and stopped at indicated times. Phosphorylation is shown by autoradiograph and total p27 by western blot. The amount of [32P] incorporated was measured by densitometry, expressed as OD, and plotted vs. reaction time. [32P] incorporation was normalized against the highest value arbitrarily set at 1. B, Phosphorylation rate as a function of p27 concentration. Indicated dilutions of hwtp27 and p27T157A were utilized in the kinase reaction. Autoradiographs show [32P] incorporation. Graph shows radiolabeled p27 quantitated as in A vs. protein dilution. C, Rate of S10 phosphorylation. Indicated forms of His-tagged p27 were incubated with Akt1 and non-labeled ATP. Samples were separated by SDS-PAGE and visualized by western blotting with the indicated antibodies. D, p27(87–198) inhibits Akt1 phosphorylation of p27. Kinase reaction was performed with hwtp27, Akt1 and [32P]-γ-ATP in the presence of indicated amounts of p27(87–198). Upper panel: autoradiograph. Bottom panel: western blot showing levels of full length and C-terminus of p27. E, p27(87–198) inhibits Akt1 phosphorylation of p27S10. Kinase reaction was performed as described in D with non-labeled ATP in the presence of p27(87–198) or GSK. S10 phosphorylation was determined by western blot analysis.