Regulation of A-raf expression.
The Raf family proto-oncogenes encode cytoplasmic protein serine/threonine kinases which play a critical role in cell growth and development. A-raf shares several functional properties with Raf-1 including transforming activity, stimulation of the Raf/MAPK pathway and the ability of dominant negative versions to functionally block Ras signalling. A-raf transcripts are predominantly expressed in the mouse urogenital tissues. Interestingly, the human A-raf promoter region contains three potential glucocorticoid response elements GRE-1, GRE-2 and GRE-3, at positions -17, -34 and -168 respectively from the transcriptional start site. DNA sequence analysis of the mouse A-raf promoter region demonstrated that GRE-1 and -2 were conserved evolutionarily. To determine whether the human A-raf GREs represent functional motifs, an expression vector for the glucocorticoid receptor was cotransfected with A-raf promoter/reporter constructs into HeLa cells. A fivefold dexamethasone-dependent induction of A-raf promoter activity was observed using constructs containing all three GRE motifs whereas point mutations in the GREs either diminished or abolished dexamethasone induction. Electrophoretic mobility shift assays (EMSAs) using purified glucocorticoid receptor DNA binding domain (DBD) demonstrated that both GRE-2 and -3 motifs interact with DBD and oligonucleotide competition experiments established that these have different affinities for DBD. Using nuclear extracts from human and rodent cell lines in EMSAs, a specific protein-DNA complex was observed with GRE-1 which displayed binding properties unlike that of glucocorticoid receptor. These results demonstrate that the A-raf promoter is regulated in part by members of the glucocorticoid family of steroid hormone receptors and suggest a model for the regulation of A-raf expression in urogenital tissues.
Lee
J E
JE
Biological Carcinogenesis and Development Program, Program Resources, Inc.@Dyn Corp., Frederick, Maryland, USA
Beck
T W
TW
Wojnowski
L
L
Rapp
U R
UR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002457:Cell Extracts; D003907:Dexamethasone / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
D002457:Cell Extracts; D011518:Proto-Oncogene Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D003907:Dexamethasone; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf