Protein kinase A phosphorylates hepatocyte nuclear factor-6 and stimulates glucose-6-phosphatase catalytic subunit gene transcription.
Glucose-6-phosphatase is a multicomponent system that catalyzes the terminal step in gluconeogenesis. To examine the effect of the cAMP signal transduction pathway on expression of the gene encoding the mouse glucose-6-phosphatase catalytic subunit (G6Pase), the liver-derived HepG2 cell line was transiently co-transfected with a series of G6Pase-chloramphenicol acetyltransferase fusion genes and an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase A (PKA). PKA markedly stimulated G6Pase-chloramphenicol acetyltransferase fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple cis-acting elements were required for this response. One of these elements was mapped to the G6Pase promoter region between -114 and -99, and this sequence was shown to bind hepatocyte nuclear factor (HNF)-6. This HNF-6 binding site was able to confer a stimulatory effect of PKA on the expression of a heterologous fusion gene; a mutation that abolished HNF-6 binding also abolished the stimulatory effect of PKA. Further investigation revealed that PKA phosphorylated HNF-6 in vitro. Site-directed mutation of three consensus PKA phosphorylation sites in the HNF-6 carboxyl terminus markedly reduced this phosphorylation. These results suggest that the stimulatory effect of PKA on G6Pase fusion gene transcription in HepG2 cells may be mediated in part by the phosphorylation of HNF-6.
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
R S
RS
Streeper
L A
LA
Hornbuckle
C A
CA
Svitek
J K
JK
Goldman
J K
JK
Oeser
R M
RM
O'Brien
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D005952:Glucose-6-Phosphatase / Q000378:metabolism*; D051559:Hepatocyte Nuclear Factor 6; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D051559:Hepatocyte Nuclear Factor 6; D018398:Homeodomain Proteins; C496605:ONECUT1 protein, human; C496606:Onecut1 protein, mouse; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D005952:Glucose-6-Phosphatase