Regulation of the pancreatic duodenal homeobox-1 protein by DNA-dependent protein kinase.
The transcription factor PDX-1 plays a crucial role during pancreatic development and in the function of insulin-producing beta cells. Disruption of the pdx-1 gene in these cells induces overt diabetes in mice, and this gene is modified in several type 2 diabetic families. It is thus crucial to determine the molecular mechanisms involved in the regulation of PDX-1 expression and/or activation. We identified new proteins associated with PDX-1 by mass spectrometry. These proteins, Ku70 and Ku80, are regulatory subunits of DNA-dependent protein kinase (DNA-PK). We determined that the interaction between PDX-1 and Ku70 or Ku80 is dependent on the homeodomain of PDX-1. Most interestingly, we demonstrated in vitro that the DNA-PK phosphorylates PDX-1 on threonine 11. Although this residue is located in the transactivation domain, this phosphorylation does not seem to be implicated in the transcriptional activation of PDX-1. However, in response to radiation, which activates DNA-PK, a second form of the PDX-1 protein appears rapidly. This form is phosphorylated on threonine and seems to drive PDX-1 degradation by the proteosome. In correlation with this degradation, we observed a subsequent reduction in the activation of the insulin promoter and a decrease in PDX-1-mediated gene expression, i.e. glut2 and glucokinase. Our study demonstrates that radiation, through the activation of DNA-PK, may regulate PDX-1 protein expression.
Lebrun
Patricia
P
INSERM U145, Faculty of Medicine, IFR50 Nice, France. plebrun@unice.fr
Montminy
Marc R
MR
Van Obberghen
Emmanuel
E
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005720:Gamma Rays; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000378:metabolism; D051273:Glucose Transporter Type 2 / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism / Q000502:physiology*; D047468:Immunoprecipitation; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism; D000072200:Ku Autoantigen; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013912:Threonine / Q000737:chemistry; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014466:Ultraviolet Rays
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D051273:Glucose Transporter Type 2; D018398:Homeodomain Proteins; D007328:Insulin; D012333:RNA, Messenger; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D013912:Threonine; D005982:Glutathione Transferase; D005941:Glucokinase; D051747:DNA-Activated Protein Kinase; D046988:Proteasome Endopeptidase Complex; C000606510:Xrcc6 protein, mouse; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen