Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix͞leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser 196 and Thr 666 were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser 196 in the cytoplasm and also stimulated the DNAbinding activity by dephosphorylation of Thr 666 in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP. T he liver is the principal organ responsible for conversion of excess dietary carbohydrate to triglycerides. A high carbohydrate diet leads to activation of several regulatory enzymes of glycolysis and lipogenesis including L-type pyruvate kinase (L-PK), acetyl CoA carboxylase, and fatty acid synthase (1). Excess carbohydrate also results in post-translational activation of several key enzymes involved in carbohydrate metabolism and lipogenesis (1). Until recently it was thought that hormones such as insulin and glucagon regulate the transcription of genes. It has only been appreciated recently that nutrients themselves play an important role in the regulation.Glucose-stimulated L-PK gene expression in liver is mediated through the glucose or carbohydrate response element (ChRE) that is located in the region Ϫ183 to Ϫ96 base pairs upstream from the cap site of the L-PK gene (2). The binding site for the ChRE contains an E-box sequence, CACGGG, separated by 5 bases that corresponds to the consensus binding site (CACGTG) for upstream stimulatory factors and their related family members. Several transcription factors binding the ChRE have been reported previously (3-5), but none of these factors vary with diet, and the mechanisms of regulation are still unclear.We recently purified, identified, and cloned a transcription factor that binds specifically to the ChRE of the L-PK gene (6). We termed this new transcription factor ChRE-binding protein (ChREBP; ref. 6). ChREBP is expressed specifically in liver and is responsive to excess carbohydrate, i.e., ChREBP is activated by high glucose diet and inhibited by high fat diet. Overexpression of ChREBP in primary cultured hepatocytes results in increased transcription activity of the L-PK gene in response to high glucose.ChREBP is a member of the basic helix-loop-helix͞leucine zipper (bHLH͞ZIP) f...

Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB␦C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and longterm regulation of glucose metabolism and fat synthesis.

Carbohydrate-responsive element-binding protein (ChREBP) is a new transcription factor that binds to the carbohydrate-responsive element of the L-type pyruvate kinase gene (L-PK). The aim of this study was to investigate the mechanism by which feeding high fat diets results in decreased activity of ChREBP in the liver (Yamashita, H., Takenoshita, M., Sakurai, M., Bruick, R. K., Henzel, W. J., Shillinglaw, W., Arnot, D., and Uyeda, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9116 -9121). We cloned the rat liver ChREBP gene for use throughout this study. Acetate, octanoate, and palmitate inhibited the glucose-induced activation of L-PK transcription in ChREBP-overexpressed hepatocytes. In these hepatocytes, the cytosolic AMP concentration increased 30-fold and AMP-activated protein kinase activity was activated 2-fold. Similarly to the fatty acids, 5-amino-4-imidazolecarboxamide ribotide, a specific activator of AMP-activated protein kinase (AMPK) also inhibited the L-PK transcription activity in ChREBPoverexpressed hepatocytes. Using as a substrate a truncated ChREBP consisting of the C-terminal region, we demonstrated that phosphorylation by AMPK resulted in inactivation of the DNA binding activity. AMPK specifically phosphorylated Ser 568 of ChREBP. A S568A mutant of the ChREBP gene showed tight DNA binding and lost its fatty acid sensitivity, whereas a S568D mutant showed weak DNA binding and inhibited L-PK transcription activity even in the absence of fatty acid. These results strongly suggested that the fatty acid inhibition of glucose-induced L-PK transcription resulted from AMPK phosphorylation of ChREBP at Ser 568 , which inactivated the DNA binding activity. AMPK was activated by the increased AMP that was generated by the fatty acid activation.Glucose metabolism in liver is inhibited by administration of fatty acids, the so-called "glucose sparing" effect (1-3). Fatty acids inhibit genes of key enzymes of glycolysis and lipogenesis such as L-pyruvate kinase (L-PK), 1 acetyl-CoA carboxylase, and fatty acid synthetase. L-PK, regulating the flux of metabolites through the pyruvate-phosphoenolpyruvate cycle (4), is known to play an important role in hepatic glucose and lipid metabolism. The activity of L-PK is subject to acute control by covalent modification and allosteric effectors (5). On the other hand, long term control of L-PK is achieved by regulating L-PK gene transcription (5). Fatty acids inhibit transcription of L-PK and other enzymes in glycolysis and lipogenesis pathways, whereas excess glucose induces expression of these genes (6). However, the mechanism of fatty acid inhibition of transcription is not understood. We recently identified a new transcription factor, which binds specifically to the carbohydrate-responsive element of the L-PK gene, and we have termed this new transcription factor, "carbohydrate-responsive element-binding protein" (ChREBP) (7). ChREBP is expressed specifically in liver and is responsive to diet. ChREBP is activated by a high carbohydrate diet and inhibited by a h...

Prolyl aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.

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