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 pro moters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region be tween -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60(v-src) induction in 3T3 cells. This region co ntains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGT CA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competitio n experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG seq uence can bind proteins at both the ATF/CRE, and E-box sequences. Domi nant-negative CREB and Myc proteins that bind DNA, but do not transact ivate, block v-src induction of a luciferase reporter driven by the fi rst 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis dis tinguishes which TIS10/PGS2 cis-acting element mediates pp60(v-src) in duction, E-box mutation has no effect on the fold induction in respons e to pp60(v-src). In contrast, ATF/CRE mutation attenuates the pp60(v- src) response. Antibody supershift and methylation interference experi ments demonstrate that CREB and at least one other ATF transcription f actor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expres sion of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60(v-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 ele ment.