FIG. 6.

Akt1, and not Akt2, phosphorylates p21 and releases cdk2 from p21. A. Purified active Akt1 and -2 isoforms were incubated in vitro with p21 immunoprecipitated from synchronized mouse 3T3 cells in a kinase reaction buffer as described in the experimental procedures. After 15 min of incubation, reactions were stopped and samples were analyzed by PAGE. After transfer to nitrocellulose and autoradiography, samples were probed for p21 by immunoblotting. The upper panel is an autoradiogram of the phosphorylated region around p21, and the lower panel shows the corresponding immunoblot for p21 revealed by Western blotting (WB) using p21 antibody. B. The same amounts of kinase used in panel A were incubated with purified GST-tagged crosstide or a mutant form (R replaced by K). Samples were analyzed by PAGE, and shown is an autoradiogram of the incorporation of ³²P. C. p21 immunoprecipitation from cytoplasmic and nuclear cell fractions followed by Akt's in vitro kinase assay. The upper panel shows immunoprecipitated (IP) p21 detected by Western blotting for p21, and the lower panel represents p21 phosphorylation as detected by autoradiography for ³²P. D. Western blots for cdk2 (upper panel) or p21 in total cell nuclear and cytoplasmic extracts (lanes 1 to 2) or in p21 immunoprecipitates from cytoplasmic or nuclear extracts incubated with Akt1 (lanes 3 to 4), Akt2 (lanes 5 to 6), kinase buffer (lanes 7 to 8), or with ADP as a control (not shown) for 30 min before continued precipitation of p21. The resulting pellets were probed for cdk2 and p21 as a control for immunoprecipitation.