The transcription factor PDX-1 is post-translationally modified by O-linked N-acetylglucosamine and this modification is correlated with its DNA binding activity and insulin secretion in min6 beta-cells.
The pancreatic/duodenal homeobox-1 protein (PDX-1, also called STF-1, IPF-1) is a transcription factor that plays an important role in pancreatic function and development. Here, we have overexpressed and purified PDX-1 from baculovirus/sf-9 cells, transiently transfected Cos-7 cells and native Min6 cells and demonstrated that the protein is posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). The approaches we used include binding of the protein to the lectin WGA, labeling with galactosyltransferase and UDP-[(3)H]gal and probing with the O-GlcNAc-specific antibody, RL-2. PNGase F treatment and structural analysis indicate that the carbohydrate is beta-linked O-GlcNAc. Mapping of [(3)H]gal-labeled tryptic peptides indicates that PDX-1 has two major sites for O-GlcNAcylation. In Min6 cells, elevated glucose concentration leads to an increase in protein O-GlcNAcylation and this hyperglycosylation correlates with an increase in DNA binding activity of PDX-1 and insulin secretion. On the other hand, the GFAT inhibitor azaserine reduces intracellular O-GlcNAc levels and profoundly attenuates glucose-stimulated insulin secretion. These data suggest that O-GlcNAcylation may be involved in the regulation of PDX-1 DNA binding activity and in glucose-stimulated insulin secretion in beta-cells.
Gao
Yuan
Y
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.
Miyazaki
Jun-ichi
J
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry; D005786:Gene Expression Regulation; D005947:Glucose / Q000494:pharmacology; D018398:Homeodomain Proteins; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009841:Oligonucleotides / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D015534:Trans-Activators / Q000096:biosynthesis* / Q000737:chemistry
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005947:Glucose; D000117:Acetylglucosamine