title: The site of cAMP action in the insulin induction of gene expression of acetyl-CoA carboxylase is AP-2.
abstract: Insulin induction of acetyl-CoA carboxylase (ACC) and differentiation of 30A5 preadipocytes into adipocytes requires a brief exposure of the cells to cAMP. Using the techniques of DNase I footprinting, DNA band shift, and analysis of the point and deletion mutations, a region from -113 to -95 has been identified as the site through which cAMP sensitizes the cell for the response to insulin. One sequence-specific DNA-protein complex, b3, is formed in the DNA-mobility shift assay when nuclear extract from 30A5 cells is mixed with the oligonucleotide representing this region. Purified human AP-2 also generates the complex corresponding to b3 with the same ACC PII probe or with the AP-2 consensus sequence probe from SV40 promoter. Substitution of A for G in the sequence GGGGCTGGG abolishes the formation of b3 sequence-specific complex. Stably transfected 30A5 cells with the same mutations in the plasmid no longer respond to insulin in spite of their exposure to cAMP. These results establish that the 21 base pair region in ACC promoter II and the binding of AP-2 protein to this sequence are required for cAMP action. cAMP-dependent protein kinase phosphorylates AP-2 both in vitro and in vivo and the phosphorylation of AP-2 does not affect its binding activity.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
authors.forename: K
authors.initials: K
authors.lastname: Park
authors.forename: K H
authors.initials: KH
authors.lastname: Kim
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000096:biosynthesis* / Q000235:genetics; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002999:Clone Cells; D000242:Cyclic AMP / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D007328:Insulin; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D000103:Acetyl-CoA Carboxylase