v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element.
We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.
Xie
W
W
Department of Biological Chemistry, UCLA School of Medicine 90024.
Fletcher
B S
BS
Andersen
R D
RD
Herschman
H R
HR
D016475:3T3 Cells; D051698:Activating Transcription Factor 2; D000818:Animals; D001483:Base Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D051546:Cyclooxygenase 2; D004252:DNA Mutational Analysis; D015971:Gene Expression Regulation, Enzymologic; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D014157:Transcription Factors; D014158:Transcription, Genetic
D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D008156:Luciferases; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015688:Oncogene Protein pp60(v-src); D015689:Oncogene Protein p21(ras)