Fig. 5.

Inhibition of CDK2 kinase activity and H4 gene transcription by p57^KIP2. (A) In Cos7 lysates, p27^KIP1 is more potent than p57^KIP2 or p21^WAF1/CIP1 in blocking CDK2 activity. CDK2 kinase activity was measured using γ-³²P-ATP and histone H1 as a substrate in CDK2 immunoprecipitates (respectively, 2, 5 and 10 μl of beads) obtained with lysates of mock-transfected cells or cells expressing p57^KIP2, p27^KIP1 or p21^WAF1/CIP1. (B) Selective inhibition of HiNF-P/p220^NPAT signaling at the H4 promoter by p57^KIP2. Co-transfection experiments with a wild type H4 promoter-luciferase reporter gene in the absence (first bar) or presence of vectors for HiNF-P (200 ng/well) or p220^NPAT (150 ng/well) (remaining bars) in cells expressing exogenous CKIs (25 or 50 ng vector) as indicated. H4 promoter-luciferase activities were measured within 24 h after transfection and plotted as a function of p57^KIP2 vector concentration (right panel). Western blot analysis (left panel) was used to examine CKI expression in whole cell lysates (20 μg protein). Note that Cos7 cells do not express endogenous p57^KIP2. (C) The HiNF-P binding site is sufficient for p57^KIP2 dependent inhibition of the H4 gene promoter. Cos7 cells were co-transfected with Luciferase reporters controlled by wild type or mutant HiNF-P elements fused to a minimal TATA box promoter. Relative promoter activity was assessed in the presence (+) or absence (−) of vectors expressing HiNF-P, p220^NPAT or p57^KIP2 (25 ng/ml) as above. (D) Summary of data presented in Figs. 1 to 3 with + signs indicating the relative effectiveness of CKIs to affect the activities in the left column.