Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptor.
We have previously defined the promoter of human urokinase-type plasminogen activator receptor (uPAR) gene in a 188-bp fragment between bases -141 and +47 relative to the translation start site. Here, we report that a novel nuclear factor-kappaB (NF-kappaB)-like sequence (5'-GGGAGGAGTC-3') at -45 is located in the uPAR promoter and one of the two DNase I-protected regions, region I between bases -51 and -30. This NF-kappaB-like motif differs at positions 7-9 from the decameric consensus sequences of NF-kappaB (5'-GGGRNNYYCC-3' where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) and at positions 1 and 7-9 from the kappaB-like motifs (5'-HGGARNYYCC-3' where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF-kappaB-like site at position -45. Mutation of the NF-kappaB-like motif decreased the binding of transcription factor NF-kappaB and reduced the uPAR promoter activity in comparison with the wild-type sequences. Co-transfection with a dominant negative I-kappaB kinase-2 expression vector reduced uPAR promoter activity by 65-75%. These results demonstrate that a previously uncharacterized NF-kappaB motif is required for uPAR promoter activity.
Wang
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Y
Division of Molecular Medicine, John Curtin School of Medical Research, The Australian National University, Canberra, Australia. y.wang@unsw.edu.au
Dang
J
J
Wang
H
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Allgayer
H
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Murrell
G A
GA
Boyd
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D
D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003110:Colonic Neoplasms / Q000473:pathology; D016384:Consensus Sequence; D018983:DNA Footprinting; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D051550:I-kappa B Kinase; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics*; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D020121:5' Untranslated Regions; D016328:NF-kappa B; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human