Biochemical Characterization of Complexes with p21, a CDK Inhibitor

The growth suppressor protein p2lWAFl p21 is a cyclin-dependent kinase inhibitor CKI that promotes growth arrest by tight-binding to several cyclinCDK complexes and the DNA replicationrepair protein PCNA. Loss of p21 from such complexes is known to occur in cancer cells, including several mammary tumors we have surveyed. We have also recently uncovered and investigated interactions between p21 and a transcription factor, E2F, known to promote cell cycle progression. First, we detected a specific interaction between these proteins in vivo and in vitro. We observe that only a single member of the E2F family forms complexes with p21 in vivo, although there are at least six known members of this family. We have detected the E2F-p21 interaction in vitro by transfection and by capturing native complexes on promoter fragments bearing E2F consensus binding sites. Using this approach, we have mapped the region of p21 needed for association with E2F. In addition, we have mapped the region in E2F required for association. Interestingly, this region corresponds to one conserved between the entire family, suggesting that there are subtle differences in this domain that confer binding to p21. Having established this, we are now testing the effects of p21 on E2F-mediated transcription.