CHARACTERIZATION OF P21(CIP1 WAF1) PEPTIDE DOMAINS REQUIRED FOR CYCLIN E/CDK2 AND PCNA INTERACTION/

The cyclin-dependent kinase inhibitor p21(Cip1/Waf1) is responsible fo r the p53-dependent growth arrest of cells in G1 phase following DNA d amage, In the present study we investigated regions of p21 involved in inhibition of the G(1)/S phase cyclin-dependent kinase, cyclin E/Cdk2 , as well as regions of p21 important for binding to this kinase and r ecombinant PCNA, To perform these studies we synthesized a series of o verlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombina nt p21 and PCNA proteins, One amino-terminal p21 peptide spanning amin o acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase, Antagonism of p21 binding was, however, lost in a similar pep tide lacking amino acids 15-20, or in a peptide in which cysteine-18 w as substituted for a serine, These results suggest that this peptide r egion is important for p21 interaction with cyclin E/Cdk2, A second pe ptide (amino acids 58-77) also antagonized p21-activity, but this pept ide did not affect the ability of p21 to interact with cyclin E/Cdk2, A region in p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2, We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a re quirement for amino acids 26 through 45 for this interaction, A p21 pe ptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interac tion, Conformational analysis revealed that peptides spanning amino ac ids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein, Taken together, our resu lts provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction, Our results also suggest that a potentia l p21 dimerization domain may lie in the amino-terminus of p21, Contin ued exploration of these domains could prove useful in assessing p21-m imetic strategies for cancer treatment.