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.
Mesh Terms:
Amino Acid Sequence, Animals, Antigens, Nuclear, Cell Line, Chromatin Immunoprecipitation, DNA Damage, DNA-Activated Protein Kinase, DNA-Binding Proteins, Gamma Rays, Gene Expression Regulation, Enzymologic, Glucokinase, Glucose Transporter Type 2, Glutathione Transferase, Homeodomain Proteins, Immunoprecipitation, Insulin, Insulin-Secreting Cells, Mass Spectrometry, Mice, Molecular Sequence Data, Phosphorylation, Polymerase Chain Reaction, Promoter Regions, Genetic, Proteasome Endopeptidase Complex, Protein Binding, Protein Structure, Tertiary, RNA, Messenger, Rats, Threonine, Trans-Activators, Transcription, Genetic, Transcriptional Activation, Transfection, Ultraviolet Rays
Amino Acid Sequence, Animals, Antigens, Nuclear, Cell Line, Chromatin Immunoprecipitation, DNA Damage, DNA-Activated Protein Kinase, DNA-Binding Proteins, Gamma Rays, Gene Expression Regulation, Enzymologic, Glucokinase, Glucose Transporter Type 2, Glutathione Transferase, Homeodomain Proteins, Immunoprecipitation, Insulin, Insulin-Secreting Cells, Mass Spectrometry, Mice, Molecular Sequence Data, Phosphorylation, Polymerase Chain Reaction, Promoter Regions, Genetic, Proteasome Endopeptidase Complex, Protein Binding, Protein Structure, Tertiary, RNA, Messenger, Rats, Threonine, Trans-Activators, Transcription, Genetic, Transcriptional Activation, Transfection, Ultraviolet Rays
J. Biol. Chem.
Date: Nov. 18, 2005
PubMed ID: 16166097
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