THE C-TERMINAL REGION OF P21(SDI1 WAF1/CIP1) IS INVOLVED IN PROLIFERATING CELL NUCLEAR ANTIGEN-BINDING BUT DOES NOT APPEAR TO BE REQUIRED FOR GROWTH-INHIBITION/

The cyclin-dependent kinase (Cdk) inhibitor p21(SDI1/WAF1/CIP1) has be en found to be involved in cell senescence, cell cycle arrest, and dif ferentiation. p21(SD11) inhibits the activity of several Cdks, in cont rast to other inhibitors such as p15(INK4B) and p16(INK4A), which act on specific cyclin-Cdk complexes, Of interest were reports that p21(SD 11) also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but n ot DNA repair in vitro, To better understand the function of this inte raction in vivo, we first determined the region of p21(SD11) that was needed for PCNA binding. Analysis of deletion mutants of p21(SD11), wh ich covered the majority of the protein, revealed that deletion of eit her amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutage nesis in this region led to the identification of the PCNA binding mot if RQXXMTXFYXXXR and demonstrated that mutation of either amino acid M et-147 or Phe-150 resulted in almost complete ablation of PCNA binding . Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and /or PCNA, we found that loss of binding to PCNA did not affect inhibit ory activity, whereas lack of Cdk2 binding greatly reduced the same. T his result suggests that the primary mechanism for inhibition of DNA s ynthesis by p21(SD11) occurs via inhibition of Cdk activity.