Fig. 1. Akt phosphorylates CDK2 both in vitro and in vivo. (A) A non-radioactive in vitro kinase assay was performed with GST-CDK2 alone, using a recombinant active Akt or inactive Akt, and the phosphorylation of CDK2 was detected by immunoblotting with an anti-phospho substrate Akt antibody (upper panel). The levels of GST-CDK2 were detected by immunoblotting with CDK2 antibodies. Constitutively active Akt (Akt-CA) or catalytically inactive Akt (Akt-K179M) immunoprecipitated from 293T cells were used in a similar kinase assay using GST-CDK2 as a substrate, and the phosphorylation of CDK2 was detected as above (lower panel). (B) CDK2 wild-type (WT) proteins or mutated ones at T39, T160 or both were used in a kinase assay with a recombinant active Akt (Rec. Akt) or immunoprecipitated active Akt, and the CDK2 phosphorylation was detected by immunoblotting as in A. The expression of GST-CDK2 wild-type and mutant proteins was monitored by immunoblotting with CDK2 antibody. (C) The CDK2 phosphorylation in vivo was monitored by transfecting 293T cells with either pSR-CDK2 wild-type vector or the mutant proteins all tagged with HA epitope, immunoprecipitated using anti-HA antibodies after 48 hours and blotted with anti-phospho-Akt substrate antibody. The expression of CDK2 wild-type and mutant proteins was detected by anti-HA antibodies. (D) Murine 3T3 fibroblasts were transiently transfected with a wild-type CDK2 or CDK2-T39A mutant and serum-starved for 30 hours, then incubated with 20 ng/ml EGF, in combination with EGF and wortmannin (5 nM) or 10% fetal calf serum. The phosphorylation of CDK2 was detected as in A. Total CDK2 expression, phosphorylated Akt and total Akt were detected by the respective antibodies. (E) 3T3 fibroblasts were serum-starved for 30 hours, then treated exactly as in D. The phosphorylation of endogenous CDK2 was detected by immunoprecipitating with CDK2 antibodies and then immunoblotting with phospho-Akt substrate-specific antibody. Total CDK2 expression was detected by anti-CDK2 antibody. (F) Murine 3T3 fibroblasts were transfected with HA-tagged CDK2 wild-type plasmid and then serum-starved for 30 hours. Then they were incubated with a combination of EGF (20 ng/ml) and specific Akt inhibitor (210 nM, Calbiochem) or EGF alone or in combination with EGF and rapamycin. The phosphorylation of CDK2 was detected by immunoprecipitating with CDK2 antibodies and then immunoblotting with phospho-AKT substrate-specific antibody. Total CDK2 expression was detected by CDK2 antibodies. (G) 3T3 fibroblasts were transfected with HA-tagged CDK2 wild-type plasmid and then serum-starved for 30 hours. The phosphorylation of CDK2 was detected by immunoprecipitating with CDK2 antibodies and then immunoblotting with phospho-Akt substrate-specific antibody after stimulating the cells with EGF at different indicated time points. For detection of phosphorylated GSK3, cell lysates at indicated time points were immunoblotted with phospho-GSK3 antibody (cell signaling). Total GSK3 and CDK2 were detected using respective antibodies.