Figure 5.
Phosphorylation of NPAT by cyclin E–CDK2. (A) Lysates from U2OS cells (1 mg of total protein) or from U2OS cells (0.25 mg of total protein) transfected with plasmids expressing indicated proteins were precipitated with anti-cyclin E (HE 67) or a control antibody (pAb 419) as indicated. The immunoprecipitates were incubated with [γ-32p]ATP in the absence (−) or presence (+) of purified CDK inhibitor p21 protein (20 ng) as indicated. The phosphorylation of NPAT was determined by reimmunoprecipitation of the phosphorylated protein with anti-NPAT antibody (DH3). The amounts of expression plasmids used in the transfection experiments were 15 μg of NPAT, 4 μg of cyclin E, and 6 μg of CDK2 and CDK2dn, respectively. (B) U2OS cells were transfected with indicated expression plasmids, and the cellular phosphoproteins were labeled with [32P]orthophosphate. Lysates were immunoprecipitated with anti-NPAT antibody (DH3) or a control antibody as indicated. The immunoprecipitates were denatured and reimmunoprecipitated with anti-HA antibody (12CA5). Fifteen micrograms of HA-tagged NPAT, 4 μg of cyclin E, 6 μg of CDK2/CDK2dn, 4 μg of cyclin D1, and 6 μg of CDK4 expression plasmids were used in the transfection experiments.