Further Characterization of the Glucocorticoid Response Unit in the Phosphoenolpyruvate Carboxykinase Gene. The Role of the Glucocorticoid Receptor-Binding Sites

The minimal DNA sequence required for glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells was defined. This novel glucocorticoid response unit (GRU) spans about 110 base pairs (bp) and includes two receptor-binding elements plus two accessory factor-binding elements. Purified glucocorticoid receptor bound to two regions (GR1 and GR2) between -395 and -349 bp relative to the transcription start site. Factors in crude rat liver nuclear extract bound to DNA in the regions -455 to -431 and -420 to -403 bp, which are designated accessory factor 1 (AF1) and accessory factor 2 (AF2) elements, respectively. Gel retardation analysis revealed that at least two proteins bound to AF1 and that they were distinct from the protein(s) that bound to AF2. Various combinations of GR1, GR2, AF1, and AF2 were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a glucocorticoid receptor expression plasmid (pSVGR1) into H4IIE cells to identify the functional GRU. Neither the glucocorticoid receptor binding region nor the accessory factor binding region alone was sufficient to confer glucocorticoid responsiveness. The two components of the glucocorticoid receptor binding region functioned independently, and each accounted for half of the maximal response, provided the accessory factor elements were present. Similarly, deletion of either AF1 or AF2 diminished glucocorticoid induction of the PEPCK gene to approximately half of the maximum. We propose that the complex PEPCK gene GRU provides the stringent regulation required of this critical enzyme in liver.

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The Phosphoenolpyruvate Carboxykinase Gene Glucocorticoid Response Unit: Identification of the Functional Domains of Accessory Factors HNF3 (Hepatic Nuclear Factor-3 ) and HNF4 and the Necessity of Proper Alignment of Their Cognate Binding Sites

Molecular Endocrinology ◽

10.1210/me.13.4.604 ◽

1999 ◽

Vol 13 (4) ◽

pp. 604-618 ◽

Cited By ~ 31

Author(s):

J.-C. Wang

Keyword(s):

Binding Sites ◽

Phosphoenolpyruvate Carboxykinase ◽

Functional Domains ◽

Nuclear Factor ◽

Response Unit ◽

Glucocorticoid Response ◽

Accessory Factors ◽

Phosphoenolpyruvate Carboxykinase Gene

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The Phosphoenolpyruvate Carboxykinase Gene Glucocorticoid Response Unit: Identification of the Functional Domains of Accessory Factors HNF3β (Hepatic Nuclear Factor-3β) and HNF4 and the Necessity of Proper Alignment of Their Cognate Binding Sites

Molecular Endocrinology ◽

10.1210/mend.13.4.0269 ◽

1999 ◽

Vol 13 (4) ◽

pp. 604-618

Author(s):

Jen-Chywan Wang ◽

Per-Erik Strömstedt ◽

Takashi Sugiyama ◽

Daryl K. Granner

Keyword(s):

Binding Sites ◽

Phosphoenolpyruvate Carboxykinase ◽

Functional Domains ◽

Nuclear Factor ◽

Response Unit ◽

Glucocorticoid Response ◽

Accessory Factors ◽

Phosphoenolpyruvate Carboxykinase Gene

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Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate carboxykinase gene.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4712 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4712-4719 ◽

Cited By ~ 192

Author(s):

E Imai ◽

P E Stromstedt ◽

P G Quinn ◽

J Carlstedt-Duke ◽

J A Gustafsson ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Receptor Binding ◽

Phosphoenolpyruvate Carboxykinase ◽

Binding Region ◽

Factor Binding ◽

Response Unit ◽

Accessory Factor ◽

Glucocorticoid Response ◽

Glucocorticoid Induction ◽

Receptor Binding Region

The minimal DNA sequence required for glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells was defined. This novel glucocorticoid response unit (GRU) spans about 110 base pairs (bp) and includes two receptor-binding elements plus two accessory factor-binding elements. Purified glucocorticoid receptor bound to two regions (GR1 and GR2) between -395 and -349 bp relative to the transcription start site. Factors in crude rat liver nuclear extract bound to DNA in the regions -455 to -431 and -420 to -403 bp, which are designated accessory factor 1 (AF1) and accessory factor 2 (AF2) elements, respectively. Gel retardation analysis revealed that at least two proteins bound to AF1 and that they were distinct from the protein(s) that bound to AF2. Various combinations of GR1, GR2, AF1, and AF2 were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a glucocorticoid receptor expression plasmid (pSVGR1) into H4IIE cells to identify the functional GRU. Neither the glucocorticoid receptor binding region nor the accessory factor binding region alone was sufficient to confer glucocorticoid responsiveness. The two components of the glucocorticoid receptor binding region functioned independently, and each accounted for half of the maximal response, provided the accessory factor elements were present. Similarly, deletion of either AF1 or AF2 diminished glucocorticoid induction of the PEPCK gene to approximately half of the maximum. We propose that the complex PEPCK gene GRU provides the stringent regulation required of this critical enzyme in liver.

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First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Journal of Bacteriology ◽

10.1128/jb.186.14.4620-4627.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4620-4627 ◽

Cited By ~ 29

Author(s):

Wakao Fukuda ◽

Toshiaki Fukui ◽

Haruyuki Atomi ◽

Tadayuki Imanaka

Keyword(s):

Binding Sites ◽

Phosphoenolpyruvate Carboxykinase ◽

Cation Binding ◽

Activity Levels ◽

Binding Region ◽

Hyperthermophilic Archaeon ◽

Dependent Activity ◽

Additional Role

ABSTRACT Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (Pck Tk ). Pck Tk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in Pck Tk . However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant Pck Tk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of Pck Tk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of Pck Tk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.

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