Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein.
p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. We show that 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 (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) were unaffected. Moreover, the simian virus 40 early promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppressed growth and E2F activity in cells lacking a functional pRb. Moreover, p21 protein (wild type but not mutant) specifically disrupted an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription.
Dimri
G P
GP
Department of Cancer Biology, Life Sciences Division, Berkeley National Laboratory, University of California 94720, USA.
Nakanishi
M
M
Desprez
P Y
PY
Smith
J R
JR
Campisi
J
J
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050704:E2F4 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic
C533431:Arid4a protein, mouse; C493956:CDKN1A protein, human; D002352:Carrier Proteins; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; C493754:E2f1 protein, mouse; C493763:E2f4 protein, mouse; D004791:Enzyme Inhibitors; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D018844:Cyclin-Dependent Kinases