INHIBITION OF E2F ACTIVITY BY THE CYCLIN-DEPENDENT PROTEIN-KINASE INHIBITOR P21 IN CELLS EXPRESSING OR LACKING A FUNCTIONAL RETINOBLASTOMA PROTEIN

p21(Sdi1/WAF1/Cip1) inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the ph osphorylation of growth-regulatory proteins, including the retinoblast oma tumor suppressor protein (pRb). The ultimate effector(s) of p21 gr owth inhibition, however, is largely a matter of conjecture. We show t hat p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppressed the activity of E2F-responsive promoters (dihydrof olate reductase and cdc2), but E2F-unresponsive promoters (c-fos and s imian virus 40 early) were unaffected. Moreover, the simian virus 40 e arly promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 suppressed a wild-type, but not mutant, E2F-1 promoter via its autoregulatory E2F binding site; p21 d eletion mutants showed good agreement in their abilities to inhibit E2 F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. D espite the central role for pRb in regulating E2F, p21 suppressed grow th and E2F activity in cells lacking a functional pRb. Moreover, p21 p rotein (wild type but not mutant) specifically disrupted an E2F-cyclin -dependent protein kinase 2-p107 DNA binding complex in nuclear extrac ts of proliferating cells, whether or not they expressed normal pRb. T hus, E2F is a critical target and ultimate effector of p21 action, and pRb Is not essential for the inhibition of growth or E2F-dependent tr anscription.