ACTIVITY AND NATURE OF P21(WAF1) COMPLEXES DURING THE CELL-CYCLE

Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibit or induce growth arrest. We have characterized a panel of monoclonal a ntibodies against human p21 in an effort to understand the dynamic reg ulatory interactions between this and other cellular proteins during t he cell cycle. The use of these reagents has allowed us to address sev eral important, yet unresolved, issues concerning the biological activ ity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found t hat the kinase activity of cyclin A/Cdk2 associated with p21 is signif icantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p2 1 complexes in vivo. In addition, we found that human fibroblasts rele ased from a quiescent state display abundant amounts of p21 devoid of associated proteins (''free'' p21), the levels of which decrease as ce lls approach S phase. Cyclin A levels increase as the amount of monome ric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes t hat are not bound to, or inactivated by, p21. Our data strengthen the notion that the G(1)-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.