Ras induces p21Cip1/Waf1 cyclin kinase inhibitor transcriptionally through Sp1-binding sites.
p21Cip1/Waf1 cyclin-dependent kinase inhibitor (p21) is inducible by Raf and mitogen-activated protein kinase kinase (MAPKK), but the level of regulation is unknown. We show here by conditional and transient Ras-expression models that Ras induces p21. Induction of p21 in conditionally Ras-expressing cells is posttranscriptional utilizing mitogen-activated protein kinase (MAPK) pathway. Transient, high-level Ras-expression induces transcriptional activation of p21 mediated by a GC-rich region in p21 promoter -83-54 bp relative to the transcription initiation site containing binding sites for Sp1-family transcription factors. Mutation of either Sp1-binding site 2 or 4 in this region decreases the magnitude of induction of promoter activity by Ras, but only the simultaneous mutation of both sites abolishes fully the induction. Electrophoretic mobility shift assays using an oligonucleotide corresponding to Sp1-binding site 2 indicate that both Sp1 and Sp3 transcription factors bind to this region. The results demonstrate that the central cytosolic growth regulator Ras is a potent transcriptional and posttranscriptional inducer of the nuclear growth inhibitor p21.
Kivinen
L
L
Haartman Institute, Department of Virology, University of Helsinki, Finland.
Tsubari
M
M
Haapajärvi
T
T
Datto
M B
MB
Wang
X F
XF
Laiho
M
M
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D019556:COS Cells; D002522:Chlorocebus aethiops; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005419:Flavonoids; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D016283:Proto-Oncogene Proteins p21(ras); C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one