Regulation of the Pancreatic Duodenal Homeobox-1 Protein by DNA-dependent Protein Kinase

Abstract

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.First characterized as an endoderm-specific homeobox protein (1) and later as a transcription factor for somatostatin (2, 3) and insulin (4) genes, PDX-1 (pancreatic duodenal homeobox-1 protein) (also termed IPF-1, STF-1, and IDX-1) plays a critical role in pancreatic development. Although initially produced in both exocrine and endocrine compartments of the developing pancreas, PDX-1 expression shifts to beta cells in the fully formed pancreas (5), where it functions in glucose homeostasis. Targeted disruption of the pdx-1 gene leads to pancreatic agenesis in Pdx-1 Ϫ/Ϫ homozygotes (6). Pdx-1 ϩ/Ϫ mice develop normally and have wild type islet cell mass by morphometric analysis, but they display abnormal serum glucose levels after intraperitoneal glucose injection (7). Indeed, disruption of the pdx-1 gene in insulin-producing beta cells induces overt diabetes in these animals, with reduced expression of insulin and glucose transporter genes (8). Previous studies have demonstrated that the pdx-1 gene is modified in several type 2 diabetic families, leading to the establishment of MODY 4 (maturity onset diabetes of the young 4) (9, 10). Thus, PDX-1 is a key player in the development of the pancreas and in the maintenance of the function of pancreatic beta cells. Although the role of PDX-1 in beta cells is now well established, the mechanisms triggering its activation remain to be clarified. Several studies have suggested that its activation involves post-translational modifications of PDX-1. Taken together, the reported data suggest that serine/threonine phosphorylation could play a major role in th...

PARP-2 Interacts with TTF-1 and Regulates Expression of Surfactant Protein-B

“…Ku70 associates with Ku80 and DNA-protein kinase in a complex that interacts with various transcription factors containing homeodomains, including HoxC4, HoxD4, Dlx2, Oct-1, Oct-2, and PDX-1, suggesting that Ku70 and Ku80 may mediate transcription via the kinase activity of DNA-protein kinase (59,60). These findings suggest the concept that TTF-1 may interact with Ku70 and Ku80 to influence its activity.…”

A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis

“…nuclear-cytoplasmic shuttling) or function would allow for acute alterations to target gene transcription, where control on the order of seconds to minutes might be crucial. At least 3 different reversible post-translational modifications, including phosphorylation [86,108,[110][111][112][113][114], sumoylation [115], and glycosylation [116], have been proposed to explain nuclearcytoplasmic shuttling and/or other functions of Pdx1. Consistent with these modifications, different molecular weight species of Pdx1 have been described in immunoblots [115].…”

“…This modification could partially explain diminished β cell function during endoplasmic reticulum stress and oxidative stress (both of which occur in the diabetic state), where GSK3 activity is enhanced [111,119]. Similarly, diminished β cell function following DNA damage might be explained by phosphorylation of Thr11 of Pdx1 by DNA-dependent kinases; this modification has also been proposed to lead to its more rapid intracellular degradation [114].…”

Adaptation and failure of pancreatic β cells in murine models with different degrees of metabolic syndrome

“…First, we investigated whether markers of glucose transport into islets are altered in POKO islets compared with the islets of other genotypes studied. Glut2 is the major glucose transporter involved in glucose uptake in islets, and is under the regulatory control of the pancreatic and duodenal homeobox 1 transcription factor (PDX1) (Lebrun et al, 2005). Glut2 is considered to be a surrogate of the state of differentiation of  cells.…”