Abstract
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.
Highlights
Glucose-6-phosphatase is a multicomponent system located in the endoplasmic reticulum that catalyzes the terminal step in gluconeogenesis and hepatic glycogenolysis [1, 2]
An Element Located between Ϫ129 and Ϫ85 in the glucose-6-phosphatase catalytic subunit (G6Pase) Promoter Contributes to the Full Stimulatory Effect of the cAMP Signal Transduction Pathway on G6Pase-CAT Fusion Gene Expression—To examine the molecular mechanisms by which the cAMP signal transduction pathway stimulates mouse G6Pase gene expression, the liver-derived HepG2 cell line was transiently co-transfected with a series of G6PaseCAT fusion genes and an expression vector encoding protein kinase A (PKA)
This strategy led to the identification of a cAMP response element (CRE) in the mouse G6Pase promoter located between Ϫ162 and Ϫ155 [27]
Summary
G6Pase, glucose-6-phosphatase catalytic subunit; HNF, hepatocyte nuclear factor; PKA, catalytic subunit of contained within the lumen of the endoplasmic reticulum [1, 2]. A modest overexpression of G6Pase in rats, again using recombinant adenovirus, resulted in approximately a 1.6 –3-fold increase in hepatic G6Pase enzymatic activity that was associated with glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral triglyceride stores, changes similar to those found in early stage type 2 diabetic patients [17]. These observations [16, 17] suggest that G6Pase is a major control point in the protein kinase A; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; IPTG, isopropyl--D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; CRE, cAMP response element; CREB, cAMP-response element-binding protein; CRU, cAMP response unit. This paper shows that one of these elements is a binding site for hepatocyte nuclear factor (HNF)-6 and demonstrates that HNF-6 is a substrate for the catalytic subunit of protein kinase A (PKA)
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