Identification and function of phosphorylation in the glucose-regulated transcription factor ChREBP

Abstract

In the liver, induction of genes encoding enzymes involved in de novo lipogenesis occurs in response to increased glucose metabolism. ChREBP (carbohydrate-response-element-binding protein) is a basic helix-loop-helix/leucine zipper transcription factor that regulates expression of these genes. To evaluate the potential role of ChREBP phosphorylation in its regulation, we used MS to identify modified residues. In the present paper, we report the detection of multiple phosphorylation sites of ChREBP expressed in hepatocytes, several of which are only observed under high-glucose conditions. Mutation of each of these serine/threonine residues of ChREBP did not alter its ability to respond to glucose. However, mutation of five N-terminal phosphoacceptor sites resulted in a major decrease in activity under high-glucose conditions. These phosphorylated residues are located within a region of ChREBP (amino acids 1-197) that is critical for glucose regulation. Mutation of Ser(56) within this region to an aspartate residue resulted in increased nuclear accumulation and activity under high-glucose conditions. Together, these data suggest that ChREBP activity is regulated by complex multisite phosphorylation patterns involving its N-terminal regulatory region.

ChREBP Mediates Glucose Repression of Peroxisome Proliferator-activated Receptor α Expression in Pancreatic β-Cells

“…Furthermore, AMPK has been reported to impair the transcriptional activity of ChREBP in primary mouse hepatocytes and to inhibit the DNA binding activity of mouse ChREBP by phosphorylating ChREBP on Ser 566 in vitro (39). Moreover, the same site was found to be phosphorylated in primary rat hepatocytes in an independent study (59). Based on these findings, we propose a model for glucose-regulated gene expression in pancreatic ␤-cells as shown in Fig.…”

Salt-inducible kinase 2 links transcriptional coactivator p300 phosphorylation to the prevention of ChREBP-dependent hepatic steatosis in mice

“…In the case of ChREBP, Lys672, which has a high impact on ChREBP transcriptional activity in response to p300, is located within the basic region of the DNA-biding domain ( Figure 4A) and is adjacent to two arginine (R) residues (R673 and R674) previously described to be important to support the glucose response by ChREBP (28). Indeed, mutation of R673 and R674, respectively, to alanine and glutamine totally abolished the binding of ChREBP to its target gene promoter and inhibited its transcriptional activity.…”

Dietary carbohydrate and control of hepatic gene expression: mechanistic links from ATP and phosphate ester homeostasis to the carbohydrate-response element-binding protein

“…For example mutation of residues that are substrates for phosphorylation by cyclic AMP dependent protein kinase and AMP-dependent protein kinase (S196, S626, T666; S566) and for dephosphorylation by glucose (8) , did not generate a glucose-insensitive ChREBP indicating involvement of additional mechanisms (77) . Covalent modification of ChREBP by O-GlcNAc which is dependent on UDP-N-acetylglucosamine generated by the hexosamine pathway caused ChREBP protein stabilisation and transcriptional activation (77,78) . However, increased flux through the hexosamine pathway at low glucose does not activate ChREBP target genes (43) .…”

“…ChREBP is transcribed as either a fulllength ChREBP-α (864 residues), which is cytoplasmic at low glucose and activated by high glucose which causes translocation to the nucleus, recruitment to target genes and transcriptional activation, or as a shorter ChREBP-β isoform (lacking the first 117-residues at the N-terminus), that is constitutively active at low glucose (75) . Several aspects of the post-transcriptional regulation of ChREBP-α have been explored, including covalent modification by phosphorylation (8,(76)(77)(78) , O-GlcNAc-modification (79,80) and acetylation (81) ; mutational studies for mapping of the N-terminal glucose sensory domain and 14-3-3 binding sites (82)(83)(84) , and identification of putative metabolites that mediate the response to high glucose. The proposed metabolites include: G6P (85,86) , xylulose 5-P (8) , the regulatory metabolite F2,6P 2 ( 43,87) , ketone bodies (88) and the product of the hexosamine pathway UDP-N-acetylglucosamine (79,80) which is the substrate for O-GlcNAc-modification of proteins.…”