FIGURE 3.

CDK2 blocks the p21-cdc20 interaction and prevents p21 ubiquitylation mediated by APC/C^Cdc20. A, interaction of p21 with components of various E3 ligase. Whole cell extracts of D1NLS/CDK4-infected cardiomyocytes were immunoprecipitated (IP) with anti-p21 antibody or control IgG, and the immunocomplexes were assayed by Western blotting (WB). B, Western blotting of immunoprecipitates using anti-p21 antibody. Cardiomyocytes were infected with adenoviruses for D1NLS/CDK4 and wild type CDK2. Whole cell extracts were immunoprecipitated with anti-p21 antibody and immunoblotted with indicated antibodies. C, in vitro binding analysis of p21 with Cdc20. Whole cell extracts (WCE) (40 μg of protein) from D1NLS/CDK4 cells were incubated with or without GST-CDK2/FLAG-cyclin E (CDK2/CycE) at 4 °C for 2 h and immunoprecipitated using anti-p21 antibody. Bound Cdc20 and GST-CDK2 proteins were assayed by Western blot. Right panel represents quantitative data of Cdc20 expression in anti-p21 immunoprecipitates. D, immunopurification of APC/C complex. Whole cell extracts were immunoprecipitated with anti-Cdc27 antibody and immunoblotted with indicated antibodies. E, in vitro ubiquitylation analysis using immunopurified APC/C complex (D) or whole cell extracts (WCE) in the absence or presence of GST-CDK2/FLAG-cyclin E (CDK2/CycE). In vitro translated p21 protein was used as a substrate. The reaction products were immunoprecipitated with anti-p21 antibody, followed by immunoblotting with anti-GST antibody. Asterisk indicates light chain of IgG. F, inability of p21CK− to bind to CDK2. GST-CDK2/cyclin E complex was incubated with p21WT or p21CK− at 4 °C for 30 min. The protein lysates were immunoprecipitated with anti-p21 antibody (left panel) or incubated with glutathione-Sepharose beads (middle panel), and the binding of p21 and GST-CDK2 was assayed by Western blot. G, in vitro ubiquitylation analysis using in vitro translated p21 or p21CK− as a substrate and immunopurified APC/C complex represented in D.