CDK5 knockdown increases TP53 and p27^Kip1 protein half-lives, increases nuclear localization of the two proteins, decreases p27^Kip1 p-T187 phosphorylation and induces p21^Cip1 in a TP53-dependent manner.

<p>(<b>A</b>) Transfection with CDK5 siRNA increased the half-life of p53 and p27^Kip1 protein. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h and then treated with 5 μg/mL of cycloheximide (CHX) for the indicated intervals. Cell lysates was subjected to Western analysis with the antibodies indicated. (<b>B</b>) CDK5 siRNA increased the nuclear localization of p53 and p27^Kip1. HEY cells were transfected with Control siRNA or CDK5 siRNA for 24 h and subjected to fractionation into cytoplasmic and nuclear components. Cytoplasmic (CE) and nuclear (NE) extracts were analyzed by immunoblotting. (<b>C</b>) CDK5 siRNA decreased the phosphorylation of p27Kip1 at T187. The cell Immunoblot analysis was performed with antibodies against phosphorylated and total p27Kip1. (<b>D</b>) CDK5 enhanced phosphorylation of TP53 and p27^Kip. A fluorescence based in vitro kinase assay was used to evaluate the interaction of CDK5 with TP53 and p27^Kip1. (<b>E</b>) CDK5 siRNA transcriptionally regulated the expression of p21^Cip1. HEY cells were transfected with CDK5 siRNA alone or in combination with TP53 siRNA and a luciferase reporter assay was used to measure p21^Cip1 promoter activity. (<b>F</b>) CDK5, p21^Cip1 and p27^Kip1 knockdown decreased the expression of CDK5, p21^Cip1 and p27^Kip1. HEY cells were treated with control siRNA or CDK5 or/and p21 plus p27 siRNA for 48 h and then with paclitaxel (6 nM) or diluent for 24 h. Immunoblot analysis was performed with antibodies indicated. (<b>G, H</b>) Knockdown of p21and p27 reduced CDK5 knockdown-induced apoptosis (G) and G1 arrest (H). HEY cells were treated as in (F) and cell cycle analyzed by flow cytometry after siRNA transfection. SubG1 cells were considered apoptotic cells. Data shown are mean values from three independent experiments.