Fig. 6.

p57^KIP2 and p220^NPAT form specific complexes. (A) Wild type p220^NPAT forms a complex with p57^KIP2 via residues involved in CDK2 phosphorylation. Immuno-complexes were obtained from Cos7 cells transfected with expression vectors for p57^KIP2 (25 ng/well) and wild type or mutant p220^NPAT proteins (200 ng/well). Whole cell protein (~100 μg) was precipitated with anti-p220^NPAT antibody (1 μg) antibody, and analyzed by western blotting using an anti-rabbit-p57^KIP2 antibody (1:3,000 dilution; secondary goat anti-rabbit IgG antibody= 1:5,000 dilution). Wild type p220^NPAT and two p220^NPAT mutants (LisH, deletion of aa 3-38; LoxP1: alanine substitutions between aa 121-145, respectively) interact with p57^KIP2, but the p220^NPAT -ΔCDK2 mutant with alanine substitution in five C-terminal CDK2 phosphorylation sites (S/T) does not. (B) The N-terminal cyclin binding of wild type p57^KIP2 supports interactions with p220^NPAT. Wild type p57^KIP2 and a p57^KIP2 mutant with amino acid substitutions in the cyclin binding domain (see C) were expressed in Cos7 cells, and immunoprecipitates were obtained as described above (see A). Complexes with p220^NPAT are only formed with wild type p57^KIP2 but not with p57^KIP2-CCT as indicated. The immunoprecipitation results presented here were correlated with those obtained for HiNF-P and p220^NPAT previously (lower panel). (C) Inhibition of H4 gene transcription requires the cyclin binding domain of p57^KIP2. Co-activation assays for p220^NPAT/HiNF-P were performed with cells co-transfected with vectors expressing wild type or mutant p57^KIP2 (i.e., T, CC and CCT; 25 ng/well) and luciferase activity for each mutant is plotted. (D) As in Panel C, but using reciprocal in the C-termini of p57^KIP2 and p27^KIP1 are swapped.