IDENTIFICATION OF THE ACTIVE-REGION OF THE DNA-SYNTHESIS INHIBITORY GENE P21(SDI1 CIP1/WAF1)/

The cloning of the negative growth regulatory gene, p21(Sdi1), has led to the convergence of the fields of cellular senescence, cell cycle r egulation and tumor suppression. This gene was first cloned as an inhi bitor of DNA synthesis that was overexpressed in terminally non-dividi ng senescent human fibroblasts (SDI1) and later as a p53 transactivate d gene (WAF1) and a Cdk-interacting protein (CIP1, p21) that inhibited cyclin-dependent kinase activity. To identify the active region(s) of p21(Sdi1), cDNA constructs encoding various deleted forms of the prot ein were analyzed, Amino acids 22-71 were found to be the minimal regi on required for DNA synthesis inhibition. Amino acids 49-71 were invol ved in binding to Cdk2, and constructs deleted in this region expresse d proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amin o acids 60-76 of the p27(Kip1) protein, another Cdk inhibitor. Point m utations made in p21(Sdi1) in this region confirmed that amino acids c ommon to both proteins were involved in DNA synthesis inhibition. Addi tionally, a chimeric protein, in which amino acids 49-65 of p21(Sdi1) were substituted with amino acids 60-76 of p27(Kip1), had almost the s ame DNA synthesis inhibitory activity as the wild-type protein. The re sults indicate that the region of sequence similarity between p21(Sdi1 ) and p27(Kip1) encodes an inhibitory motif characteristic of this fam ily of Cdk inhibitors.