title: Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements.
abstract: The cis elements involved in the cAMP regulation of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) were studied by introducing a series of block mutations (10-15 base pairs of random sequence) into eight of the protein binding domains in a region of the promoter between -490 and +73. Each mutant promoter was ligated to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected into HepG2 cells. Transcription of PEPCK-CAT was stimulated 4-fold by the addition of 8-bromo-cAMP (8-Br-cAMP), whereas overexpression of the catalytic subunit of protein kinase A in these cells increased transcription from the PEPCK promoter 30-fold. Several elements within the PEPCK promoter acted synergistically to mediate this effect. These include CRE-1 (-92 to -82) and a complex unit from -220 to -280 composed of multiple binding sites termed P3(I) (-250 to -234), P3(II) (-260 to -250), and P4 (-286 to -270). Mutation of both CRE-1 and P3(I) resulted in the complete elimination of transcriptional induction by either 8-Br-cAMP or the catalytic subunit of protein kinase A. To examine the proteins involved in this response, we replaced CRE-1, which binds both C/EBP and cAMP-responsive element binding protein (CREB), with an optimal C/EBP binding sequence which significantly decreased the binding of CREB, but maintained the affinity for C/EBP. Transcription from this modified promoter was induced by 8-Br-cAMP and the catalytic subunit of protein kinase A (PKA) to a similar extent as noted with the native PEPCK promoter. However, the results of experiments involving cotransfection of PEPCK-CAT with expression vectors for PKA and either C/EBP or CREB suggest that CREB is capable of mediating a greater responsiveness to PKA than C/EBP. Our results indicate that multiple cis elements are involved in the cAMP induction of PEPCK gene transcription and that C/EBP and CREB are potentially involved in this response.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
authors.forename: J S
authors.initials: JS
authors.lastname: Liu
authors.forename: E A
authors.initials: EA
authors.lastname: Park
authors.forename: A L
authors.initials: AL
authors.lastname: Gurney
authors.forename: W J
authors.initials: WJ
authors.lastname: Roesler
authors.forename: R W
authors.initials: RW
authors.lastname: Hanson
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D010729:Phosphoenolpyruvate Carboxykinase (GTP)