Analysis of wild-type and mutant p21^WAF-1 gene activities

The p21^WAF-1 gene is positively regulated by the wild-type p53 protein, p21^WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G₁ cyclin-dependent kinases (cdks). Mutational analysis with the c21^WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21^WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21^WAF-1 family members, p27^kip-1 and p57^kip-2. The same p21^WAF-121,24 mutant also fails to bind to cyclin D₁-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21^WAF-1-cdk-cyclin trimeric complex interactions. The p21^WAF-1 wild-type protein will suppress tumor cell growth in culture while p21^WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21^WAF-1 protein in cells. These results provide evidence that p21^WAF-1 acts through cyclin D₁-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21^WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.