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Originally published In Press as doi:10.1074/jbc.M504842200 on September 15, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38203-38210, November 18, 2005
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Regulation of the Pancreatic Duodenal Homeobox-1 Protein by DNA-dependent Protein Kinase*

Patricia Lebrun{ddagger}§1, Marc R. Montminy§, and Emmanuel Van Obberghen{ddagger}2

From the {ddagger}INSERM U145, Faculty of Medicine, IFR50 Nice, France and §The Salk Institute for Biological Studies, La Jolla, California 92037

Received for publication, May 3, 2005 , and in revised form, September 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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–/– homozygotes (6). Pdx-1+/– 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 the nuclear translocation and function of PDX-1 (11–13). However, it is not clear how those phosphorylations regulate PDX-1 in vivo, which kinases are implicated in the phosphorylation process, and which stimuli promote PDX-1 phosphorylation.

Work from Haché and co-workers (14) has shown that homeobox proteins can interact with the DNA-dependent protein kinase (DNA-PK)3 and can be phosphorylated by its catalytic activity in vitro. A member of the phosphatidylinositol 3-kinase-related family, DNA-PK is a Ser/Thr kinase composed of two Ku regulatory subunits (Ku70/Ku80) and a large catalytic subunit (DNA-PKcs) (15). This kinase is implicated in nonhomologous end-joining (NHEJ), which is the major repair process in cells (16). In response to DNA damage, DNA-PK is recruited to DNA ends, activated, and joined to the DNA repair complex. DNA-PK-deficient mice are immunodeficient, radiosensitive, and more susceptible to tumor development (17–19). DNA-damaging stimuli result in profound changes in gene expression causing the inhibition of proliferation and differentiation of cells (that allow DNA repair) thus preventing transmission of chromosomal aberrations. Several investigators suggest that aside from its role in DNA repair, DNA-PK may also be implicated in cell survival (20–22) and may regulate the activity and/or the stability of transcription factors. Among these transcription factors, interferon regulatory factor-3 (IRF-3) is phosphorylated on threonine by DNA-PK in response to viral infection (23). This leads to the retention of IRF-3 in the nucleus, thereby protecting it from proteosomal degradation. More recently, it has been demonstrated that DNA-PK may down-regulate histone H2B expression in response to DNA damage by regulating the stability and activity of the homeobox transcription factor Oct-1 (octamer transcription factor-1) (24).

To define new molecular mechanisms that could be implicated in PDX-1 regulation, we have performed an analysis of proteins associated with PDX-1 by mass spectrometry. We demonstrate here that PDX-1 interacts with the two regulatory subunits of the DNA-PK, Ku70 and Ku80. These results corroborate those published previously showing an interaction of the homeodomain proteins Oct-1 and Hox4 with the Ku proteins (14). Our present data also show that the DNA-PK is able to phosphorylate PDX-1 on threonine 11. Threonine phosphorylation on PDX-1 has also been detected in response to UV light and {gamma}-radiation, which are known to activate DNA-PK. Finally, we show in this report that radiation is able to decrease the protein level of PDX-1 and the expression of its major target genes such as glut2 and glucokinase. The interaction of PDX-1 with Ku subunits and its phosphorylation on threonine 11 by the DNA-PK appear to be implicated in its degradation by the proteosome. Thus, the DNA-PK-dependent phosphorylation of PDX-1 may regulate its function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Antibodies—MIN6 and RIN-5AH cells were grown in Dulbecco's modified Eagle's medium, 24 mM glucose with 10% (v/v) fetal bovine serum. MIN6 cells were used between passage 25 and 30. Lipofectamine 2000 (Invitrogen) was used for transfection. For immunoprecipitation experiments, PDX-1 antibody (generated in the Montminy's laboratory) was covalently cross-linked to protein A beads by using a standard protocol (25). Immunoprecipitation of FLAG-tagged proteins was performed with the FLAG-agarose beads from Promega (Madison, WI). Rabbit polyclonal anti-Ku70 and rabbit polyclonal anti-Ku80 were purchased from Serotec (Raleigh, NC) (AHP316 and AHP317, respectively). For the coimmunoprecipitation of Ku80 with the endogenous PDX-1, goat polyclonal anti-Ku86 (M-20) from Santa Cruz Biotechnology (Santa Cruz, CA) was used. Detection of CREB was performed by using an antibody generated in the Montminy's laboratory, and antibody revealing PBX1 (P-20) was purchased from Santa Cruz Biotechnology. The phospho-(Ser/Thr)ATM/ATR substrate and phosphothreonine (42H4) antibodies are from Cell Signaling (Beverly, MA). The p53, phospho-specific (Ser-15) antibody 3 was purchased from Oncogene Research Products (San Diego, CA).

Treatments—For UV treatment, cells were exposed to 50 J/m2 of a 254 nm light. Cells were alternatively exposed to 20 gray of {gamma}-radiation using a 60Co source (Gammabeam 150-C). Treatment of cells with caffeine (0.1, 1, and 10 mM) or MG132 (50 µM) commenced 30–60 min before radiation. Forskolin (10 µM) was given 30 min before harvesting the cells.

Lysis Buffers, Immunoprecipitations, and GST-Pull Down—For coimmunoprecipitation experiments, lysates were prepared using a 300 mM NaCl, 50 mM Hepes, 100 mM NaF, 10 mM EDTA, 10 mM Na4P2O7, 2 mM NaVO4, 1% (v/v) Nonidet P-40 buffer, pH 7.5, supplemented with protease inhibitors (pepstatin and leupeptin). Lysates were homogenized by sonication. During the 4 °C incubation for immunoprecipitation, the NaCl concentration was reduced to 150 mM. For GST-pull down experiments, cell lysates were incubated with 2 µg of GST protein beads for 2 h. We tested the interaction of Ku subunits with GST/full-length PDX-1-(1–283), GST/N-terminal domain of PDX-1-(1–143), GST/homeodomain of PDX-1-(143–212), or GST/C-terminal domain of PDX-1-(212–283).

In Vitro Phosphorylation—The partially purified DNA-PK was purchased from Promega (catalogue number V5811). The phosphorylation assay was performed according to the manufacturer's instructions. Briefly, 2 µg of GST proteins were incubated in the presence or absence of purified DNA-PK with unlabeled or [{gamma}-32P]ATP at 30 °C for 20 min. The experiment was also performed in the presence or absence of DNA fragments (sonicated calf DNA) and 1 µM wortmannin. After the reaction, GST protein beads were incubated or not with 20 units of calf intestinal alkaline phosphatase (CIP; New England Biolabs, Beverly, MA) at 37 °C for 1 h. The samples were separated by SDS-PAGE, and the phosphorylated proteins were revealed by autoradiography or Western blotting using a phosphoserine/threonine antibody.

Luciferase Assays—The transcriptional activities of PDX-1 wild type and mutant were measured using pM1Gal4-PDX-1 constructs. MIN6 cells were transfected with pM1Gal4-PDX-1WT or T11A and the reporter vector pFR-Luc. 40 h after transfection, the cells were treated with UV radiation (50 J/m2) and harvested 2 h after radiation. The activity of the different constructs was estimated by using a luciferase assay normalized by the expression of exogenous {beta}-galactosidase. The activation of the insulin gene promoter was measured using a 250-bp fragment of the rat insulin-1 promoter subcloned upstream of the luciferase cDNA in the pCAT3 basic vector. The Trp-53-Luc construct was kindly provided by Dr. Puri (26).

Chromatin Immunoprecipitation—The cross-linking was performed by 1% (v/v) formaldehyde for 30 min at room temperature. The cells were then scraped off the plate, spun down, and lysed in 5 mM Hepes, pH 8.0, 85 mM KCl, 0.5% (v/v) Nonidet P-40 complemented with protease inhibitors. The nuclei were spun down and resuspended in 50 mM Tris-Cl, pH 8.1, 10 mM EDTA, 1% (v/v) SDS, with protease inhibitors. To generate chromatin fragments of about 600 bp, the nuclei lysate was sonicated for a total of 30 s. The chromatin was then precleared by incubation with protein A-Sepharose beads for 15 min, and immunoprecipitations were prepared by using a nonimmune serum or an anti-PDX-1 antibody. After reversion of the cross-linking at 65 °C, DNA was extracted from the immunoprecipitates by phenol/chloroform extraction and precipitated with ethanol. PCR using Insulin promoter primers was subsequently performed.

Quantitative PCR—Total RNA was extracted from cells exposed or not to UV radiation (50 J/m2) by using the RNeasy kit from Qiagen. The extraction was performed 2 h or 5 h and 30 min after radiation treatment. cDNA generated by Superscript II enzyme (Invitrogen) was analyzed by Q-PCR using a SYBR Green PCR kit and an ABIPRISM 7700 sequence detector (PerkinElmer Life Sciences). All data were normalized to ribosomal 36B4 expression. The following primers were used for PCRs: PDX-1, 5'-AAAACCGTCGCATGAAGTGG-3' forward and 5'-CCCGCTACTACGTTTCTTATCTTCC-3' reverse; Glut2, 5'-CCAGCTCCCTGGGATGAAGAGGAG-3' forward and 5'-GATGAGGGCGTGTGCCGGTCC-3' reverse; glucokinase, 5'-GGTGAATGACACGGTGGCC-3' forward and 5'-CTCCAGCAGGAACTCGTCCAGC-3' reverse; YWHAH, 5'-GCCTCTTAGCCAAACAAGCCTTC-3' forward and 5'-TTCTTCATCCTGCTGGTCGCTC-3' reverse; MAFK, 5'-CCTAATTTATTGCTGTACATGTTGCC-3' forward and 5'-TTTATTGCAAAGATACAAAAGCAGTCAC-3' reverse; and 36B4, 5'-CCACGAAAATCTCCAGAGGCAC-3' forward and 5'-ATGATCAGCCCGAAGGAGAAGG-3' reverse.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of PDX-1 with Ku70 and Ku80—To reveal new cofactors of the transcription factor PDX-1, we have overexpressed a FLAG-tagged PDX-1 in 293T cells. FLAG-PDX-1 was immunoprecipitated using a FLAG antibody, and the immunoprecipitates were analyzed by SDS-PAGE. The coimmunoprecipitated proteins were visualized by Coomassie Blue staining and analyzed by mass spectrometry. Two proteins with molecular masses of 70 and 80 kDa appeared to be Ku70 and Ku80, respectively (data not shown). Because the expression of PDX-1 is mainly restricted to pancreatic beta cells, it was important to verify that this interaction occurred in beta cells. To do so, MIN6 cells were transfected with the FLAG-PDX-1 construct, and an immunoprecipitation was performed. As expected, we found that both Ku70 and Ku80 coimmunoprecipitated with FLAG-PDX-1 (Fig. 1A, lane 1), and this interaction is outcompeted by the presence of an excess of the FLAG peptide (lane 2). To test the specificity of the interaction of Ku with PDX-1, we overexpressed two other FLAG-tagged transcription factors, PBX-1 and CREB, in MIN6 cells. PBX-1 contains a homeodomain and CREB a leucine zipper protein without a homeodomain. By using a FLAG antibody, we did not detect coimmunoprecipitation of Ku80 with FLAG-PBX-1 and FLAGCREB (Fig. 1B). This result confirms that the Ku subunits associate with PDX-1 and that the coimmunoprecipitation observed is not because of an artifactual interaction of Ku with the FLAG tag. To demonstrate that Ku is also able to interact with the endogenous PDX-1, we performed an immunoprecipitation of PDX-1 expressed in MIN6 using an antibody to PDX-1. As shown in Fig. 1C, Ku80 coimmunoprecipitates with endogenous PDX-1 (lane 2). As expected, no coimmunoprecipitation is observed using a nonimmune serum (Fig. 1C, lane 1). Because it has been demonstrated previously that Ku subunits can interact with the homeodomain of Oct-1 and HoxC4 (14), two other homeobox transcription factors, we investigated whether the homeodomain of PDX-1 plays a role in the association of Ku70 and Ku80. To do so, we performed a GST-pull down using the following constructs of GST-PDX-1: GST/full-length PDX-1-(1–283) (GST/FL), GST/N-terminal domain of PDX-1-(1–143) (GST/N), GST/homeodomain of PDX-1-(144–243) (GST/HD), and GST/C-terminal domain of PDX-1-(244–283) (GST/C) (Fig. 1D). We found that GST/FL and GST/HD can pull down Ku70 and Ku80, but GST/N and GST/C do not, suggesting that the homeodomain of PDX-1 mediates its interaction with the Ku subunits. As a control we showed an interaction of GST-PDX-1 full length with the homeodomain protein PBX-1, which is known to associate with the conserved pentapeptide (YPWMK) located upstream of the PDX-1 homeodomain (27, 28).



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  		FIGURE 1.
PDX-1 associates with Ku70 and Ku80. A, MIN6 cells were transfected with a FLAG-tagged construct of PDX-1. FLAG-PDX-1 was immunoprecipitated (IP) with an anti-FLAG antibody linked to agarose beads in the presence (+) or not (–) of 25 µg of the FLAG peptide. Immunoprecipitated proteins are separated by SDS-PAGE. Immunoblots with anti-FLAG, anti-Ku70, and anti-Ku80 antibodies are shown. Immunoprecipitates are compared with FLAG-PDX-1, Ku70, and Ku80 expressed in MIN6 (whole cell lysate, lane 3). B, FLAG-PDX1, FLAG-PBX1, or FLAG-CREB were overexpressed in MIN6 cells. An immunoprecipitation with an anti-FLAG antibody and an immunoblot with an anti-Ku80 antibody reveals that Ku80 associates specifically with PDX-1 in islet cells. C, whole cell extracts from MIN6 are incubated with a nonimmune (NI) serum or PDX-1 antibodies. Immunoblotting using anti-Ku80 antibodies showed coimmunoprecipitation of Ku80 with endogenous PDX-1. D, nuclear extracts from MIN6 cells are incubated with different GST/PDX-1 constructs: GST/full-length PDX-1 (GST/FL), GST/N-terminal domain of PDX-1 (GST/N), GST/homeodomain of PDX-1 (GST/HD), or GST/C-terminal domain of PDX-1 (GST/C). Retained proteins are separated by SDS-PAGE, and immunoblots with anti-Ku70, anti-Ku80, and PBX1 antibodies have been performed. Each panel shows a representative Western blot of three separate experiments.

 
In Vitro Phosphorylation of PDX-1 by DNA-PK—Ku70 and Ku80 are the regulatory subunits of DNA-PK, and it has been demonstrated previously that this kinase is able to phosphorylate other homeodomain proteins like Oct-1 (14, 24). DNA-PK phosphorylates many substrates on serine/threonine residues followed by glutamine ((S/T)Q) (29, 30). The analysis of the PDX-1 sequence shows a consensus site for DNA-PK phosphorylation in the N-terminal region. This site contains threonine 11 and is conserved in most species, with the exception of Zebrafish in which another consensus site exists at serine 29 (Fig. 2A). To determine whether DNA-PK can phosphorylate PDX-1, we performed an in vitro kinase assay using GST constructs of PDX-1 with purified DNA-PK containing the catalytic subunit and Ku proteins. We performed this experiment in the presence of unlabeled or radioactive ATP. As shown in Fig. 2B, left panel (lane 2), DNA-PK is able to phosphorylate PDX-1 wild type in vitro. As expected this phosphorylation occurs on serine and/or threonine residues as demonstrated by Western blotting using an antibody to phosphoserine/threonine. However, no phosphorylation was detected on PDX-1 mutated on threonine 11 (Fig. 2B, lane 4). To verify that this phosphorylation was generated by the DNA-PK activity, we performed the same experiment in the presence of 1 µM of wortmannin, which inhibits DNA-PK. As shown in Fig. 2B, right panel, the phosphorylation of PDX-1 by DNA-PK is abolished in presence of wortmannin (lane 3). This phosphorylation does not occur in the absence of DNA fragments, which is consistent with the fact that DNA-PK is activated by its interaction with DNA ends (Fig. 2B, right panel, lane 4) (31, 32). We also show that the phosphorylated form of GST/PDX-1WT is no longer detected after incubation with the alkaline phosphatase CIP (Fig. 2B, right panel, lane 5). To summarize, we demonstrated here that DNA-PK is able to phosphorylate PDX-1 in vitro and that this phosphorylation occurs on threonine 11.

PDX-1 Is Phosphorylated on Threonine in Response to DNA Damage—To determine which stimuli induce threonine phosphorylation of PDX-1 in intact cells, we investigated whether the stimuli able to activate DNA-PK (such as DNA-damaging radiation) have an effect on PDX-1 phosphorylation. First, we treated MIN6 cells with {gamma}-radiation (20 gray) or UV radiation (50 J) and immunoprecipitated endogenous PDX-1. We found that PDX-1 is indeed modified in response to both {gamma}- and UV radiation. This modification appears as a new form of PDX-1 that migrates more slowly on SDS-PAGE (Fig. 3A). As a serine/threonine-phosphorylated protein usually runs slower on SDS-PAGE than the nonphosphorylated form, we investigated whether the modification of PDX-1 observed after DNA damage corresponds to its phosphorylation on threonine. To do so, MIN6 cells were treated with UV light, and endogenous PDX-1 was immunoprecipitated. By using an antibody to phosphothreonine, we showed that UV radiation can induce threonine phosphorylation of PDX-1. As expected, treatment of immunoprecipitated PDX-1 with the alkaline phosphatase CIP abolished the threonine phosphorylation of PDX-1 and consistently inhibited the formation of the upper form of PDX-1 (Fig. 3B). As a control, we confirmed that UV radiation induced p53 phosphorylation (33). These experiments demonstrate that treatment with DNA-damaging agents results in PDX-1 phosphorylation on threonine in islet cells.



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  		FIGURE 2.
PDX-1 is phosphorylated by DNA-PK on threonine 11. A, alignment of PDX-1 N-terminal amino acid sequences of different species. Consensus sites for DNA-PK phosphorylation are indicated in italic. B, left panel, a DNA-PK assay was performed in the presence of GST/PDX-1 wild type or mutated on the threonine 11 (GST/T11A) by using a purified DNA-PK. The reaction was carried out in presence of [{gamma}-32P]ATP (middle panel) or in presence of nonradioactive ATP (upper panel) for 20 min at 30 °C. Subsequent to the nonradioactive reaction, the GST proteins were separated on a polyacrylamide gel and transferred onto a nitrocellulose membrane. An immunoblot using antibodies to phosphoserine/phosphothreonine residues shows that PDX-1 wild type, but not the mutant, is phosphorylated in the presence of DNA-PK. Right panel, the same cold reaction was performed in presence or absence of DNA fragments or 1 µM wortmannin. GST/PDX-1 shown in the lane 5 was incubated with CIP phosphatase for 1 h at 37 °C after the kinase reaction. Coomassie staining shows the levels of GST proteins. Each panel shows a representative Western blot of four individual experiments.

 



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  		FIGURE 3.
PDX-1 is phosphorylated on threonine in response to DNA damage. A, DNA damage has been induced in MIN6 cells by 20 gray of {gamma}-radiation or 50 J/m2 of UV radiation. Cells were harvested 4 h after radiation. An immunoprecipitation (IP) using a nonimmune serum (NI) or an anti-PDX-1 antibody was performed. After SDS-PAGE and transfer onto a nitrocellulose membrane, an anti-PDX-1 immunoblotting was performed. B, MIN6 cells were treated with UV radiation (50 J/m2), and an immunoprecipitation with PDX-1 was performed 5 h after radiation. The immunoprecipitate was incubated (+) or not (–) with the phosphatase CIP for 1 h at 37 °C and was separated on a polyacrylamide gel. Immunoblots using anti-PDX-1 and anti-phosphothreonine antibodies were performed. Phosphorylation of p53 in response to UV light was observed in immunoblots of whole cell lysates. C, MIN6 cells were treated with increasing concentrations of caffeine (0.1–10 mM) prior to UV radiation (50 J/m2). Cells were harvested 3 h after radiation, and proteins from whole cell lysates were separated by SDS-PAGE. The two forms of PDX-1 and the phosphorylated form of p53 were revealed by Western blot. Each panel shows a representative Western blot of several individual experiments.

 
To investigate further the role of DNA damage kinases in threonine phosphorylation of PDX-1 induced by DNA damage, we first treated MIN6 cells with caffeine. At low doses (up to 0.5 mM) caffeine is known to be a specific inhibitor of ATM and ATR kinases. However, starting at 5 mM, this drug is no longer specific for ATM and ATR and can also inhibit other phosphatidylinositol 3-kinase-like kinases such as DNA-PK (34). Preceding UV radiation, MIN6 cells were treated with increasing concentrations of caffeine (0.1 to 10 mM). As shown in Fig.3C, only a high dose of caffeine (10 mM) is able to inhibit the PDX-1 shift (lane 5). As a control of efficiency for UV light and caffeine treatments, a Western blot using an anti-phospho-p53 was performed. As expected, p53 was phosphorylated after UV radiation, and this phosphorylation was strongly inhibited after treatment with high doses of caffeine. This experiment suggested that neither ATM nor ATR are implicated in PDX-1 phosphorylation and that DNA-PK inhibition might correlate with the reduction of the upper form of PDX-1. However, we cannot exclude that another pathway inhibited by 10 mM caffeine was also implicated in the PDX-1 threonine phosphorylation induced by DNA damage.

The Phosphorylation of PDX-1 on Threonine 11 Does Not Affect Its Transcriptional Activity or Its DNA Binding—To investigate the putative in vivo role of PDX-1 phosphorylation, we studied whether DNA-damaging treatment like UV radiation has an effect on PDX-1 DNA binding and/or on its transcriptional activity. To do so, we first treated RIN-5AH cells with UV radiation and extracted chromatin as described under the "Materials and Methods." After immunoprecipitation and by using a nonimmune serum or a PDX-1-specific antibody, PCR amplifying the PDX-1-linked-insulin promoter region was performed. As shown in Fig. 4A, no change in PDX-1 DNA binding was observed after UV treatment. The same result was obtained with the glucokinase promoter (data not shown). These results suggest that PDX-1 threonine phosphorylation does not modulate the binding of PDX-1 to the promoters of its target genes. To search further for a role of threonine 11 on PDX-1 transcriptional activity, we generated a Gal4 construct containing PDX-1 mutated on threonine 11. The transcriptional activity of this construct has been tested in MIN6 cells, in parallel with the wild type form of PDX-1, subsequent to treatment or not with UV radiation. As indicated by Fig. 4B, only a modest decrease in PDX-1 wild type and mutant activities was seen in response to UV radiation. This is consistent with the results described previously concerning the binding of PDX-1 to its target promoters. Moreover, we observed that the threonine 11 mutant presents the same transcriptional activity as wild type PDX-1. These results suggest that the phosphorylation of PDX-1 on threonine 11 induced after DNA-damaging treatment is not implicated in the regulation of PDX-1 transcriptional activity.



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  		FIGURE 4.
UV radiation does not affect PDX-1 binding to the insulin promoter nor its transcriptional activity. A, RIN-5AH cells were treated with 50 J/m2 of UV radiation. 3 h later cells were fixed, and chromatin was extracted. We then performed immunoprecipitations (IP) using a nonimmune (NI) serum or antibodies specific for PDX-1. PCR using specific primers for the insulin promoter revealed the binding of PDX-1 to this promoter. As expected, no amplification of the insulin promoter was obtained in the nonimmune precipitates. A representative picture of several separate experiments is shown. B, MIN6 cells were transfected with Gal4-PDX-1WT or Gal4-PDX-1T11A and treated with 50 J/m2 of UV radiation. WT, wild type. 2 h after treatment, a luciferase assay was performed on cell lysates revealing the transcriptional activity of each construct. A histogram representative of three individual experiments is shown.

 
UV Radiation Induces a Decrease in PDX-1 Protein Expression—To investigate the biological effect of radiation on PDX-1, we studied PDX-1 protein expression at different times after UV radiation. MIN6 cells were irradiated with UV light 50 J/m2 and cells were harvested 1, 5, and 10 h after irradiation. Expression of PDX-1 was then revealed by Western blot. As shown in Fig. 5A, PDX-1 protein expression starts to decrease within 5 h after UV treatment and is dramatically reduced after 10 h (upper panel). As expected, the phosphorylation of p53 was detected as early as 1 h, confirming the effect of UV treatment (Fig. 5A, lower panel). In contrast to our observations concerning PDX-1, no decrease in CREB protein expression was obtained in response to UV radiation (Fig. 5A, middle panel), suggesting that the effect observed for PDX-1 was not because of a general shut down of the translational machinery.

Next, we wanted to determine whether the phosphorylated form of PDX-1 obtained after treatment with DNA-damaging agents was implicated in the decreased protein expression. To do so, MIN6 cells were treated or not with 50 µM MG132, a proteosome inhibitor, and exposed or not to 50 J/m2 of UV radiation. We demonstrated that the amount of the upper form of PDX-1 was increased by MG132 treatment (Fig. 4B) suggesting that the form of PDX-1, corresponding to the threonine phosphorylated form, could be at least partially degraded by the proteosome. These experiments demonstrate that the PDX-1 protein is degraded in response to UV radiation and that the threonine phosphorylation of PDX-1 may be implicated in this degradation via the proteosomal machinery.

Down-regulation of Islet-specific Genes in Response to Radiation—PDX-1 is one of the major transcription factors implicated in the metabolism of pancreatic beta cells. In particular, PDX-1 is important for the activation of the insulin promoter in response to glucose (4, 35–37). We further investigated the effect of radiation downstream of PDX-1. We first determined the activation of the insulin promoter in response to UV or {gamma}-radiation using a luciferase construct of the proximal insulin promoter. As a control, we used a chromatin-integrated p53-responsive reporter, which is known to be activated after DNA damage (26). As shown in Fig. 6A, the activation of the insulin promoter is decreased after {gamma}- and UV radiation. This result is consistent with the decrease in PDX-1 protein expression demonstrated previously. As expected, the Trp-53-Luc is activated in response to radiation (Fig. 6B).

We further investigated whether radiation had an effect only on PDX-1 and insulin expression or whether this could also affect additional islet-specific genes such as the PDX-1 target genes, glut2, and glucokinase. By using quantitative PCR, we analyzed the expression level of PDX-1, Glut2, and glucokinase mRNAs at two time points after 50 J/m2 UV radiation. As controls we quantified the mRNA expression of two genes not related to islet, the CREB target YWHAH, and MAF kinase (Fig. 7A). We observed a substantial decrease in the expression of the three islet-related genes (pdx-1, glut2, and glucokinase) with in 2 h of UV treatment. This decrease reached 80/90% after 5.5 h. The expression of non-islet-related genes ywhah and mafk was also decreased, but to a lesser extent because this decrease did not reach more than 55% after 5.5 h. To verify that the decrease in islet-specific gene expression in response to radiation is because of the reduced expression of PDX-1, we have performed the same experiment in MIN6 cells overexpressing or not exogenous PDX-1-T11A. As shown in Fig. 7B, the expression of Glut2 is restored in presence of PDX-1-T11A. However, no significant effect of PDX-1-T11A has been observed on glucokinase expression (data not shown). This suggests that other factors implicated in glucokinase gene expression may be down-regulated in response to UV radiation. Finally, to verify that the observed effects were not because of a general shut-down of the biological response, we analyzed the ability of forskolin to induce CREB phosphorylation after UV radiation. The cells were exposed to two different doses of UV light (12.5 J and 50 J/m2) and harvested 5 or 10 h after irradiation. 30 min before harvesting, cells were treated with 10 µM forskolin. As shown in Fig. 7C, the phosphorylation of CREB induced by forskolin is unchanged after UV radiation, indicating that the cells are able to respond normally even 10 h after a high dose of UV treatment. These results demonstrated that after UV radiation, the cells preferentially inhibit the expression of genes that play a crucial role in metabolism.



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  		FIGURE 5.
UV radiation decreases PDX-1 protein level, and the slow migrating form of PDX-1 is degraded by the proteosome. A, MIN6 cells were treated by UV light (50 J/m2), and cells were harvested 1, 5, and 10 h after treatment. The protein expression levels of PDX-1 and CREB and the phosphorylation of p53 were evaluated by immunoblot. B, MIN6 cells were pretreated for 1.5 h with the proteosome inhibitor MG132 (50 µM). Cells were then treated with UV light (50 J/m2) and harvested 3.5 h later. Proteins from whole cell lysates were separated by SDS-PAGE, and expression of PDX-1 was revealed by immunoblot. For each panel, a Western blot representative of several separate experiments is shown.

 



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  		FIGURE 6.
Radiation decreases promoter activation of the insulin gene. MIN6 cells were transfected with InsPr-Luc or Trp53-Luc constructs and treated with {gamma}- or UV radiation. 10 h later, cell lysate was extracted, and luciferase assays revealed the activation of InsPr-Luc (A) and Trp53-Luc (B) in response (black bars) or not (white bars) to radiation. A histogram representative of three individual experiments is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our present study defines Ku70 and Ku80 as proteins interacting with PDX-1 and reveals a new phosphorylation site on PDX-1, which is a substrate for the DNA-PKcs. The Ku70 and Ku80 proteins are DNA-binding regulatory subunits of DNA-PK, which is composed of a 470-kDa catalytic subunit (DNA-PKcs) and Ku proteins (15). Ku70 and Ku80 proteins act as regulatory components of DNA-PK and initiate the repair process of double-stranded breaks in DNA by activating DNA-PKcs after binding to the DNA double-stranded ends (31, 32). Although the role of DNA-PK in NHEJ is now well established, strong evidence exists for a role of DNA-PK independent of DNA repair. It has been demonstrated, for example, that Ku70 may have a cytoprotective function. Indeed, Matsuyama and co-workers (22) demonstrated that Ku70 can interact with the pro-oncogene Bax and consequently inhibit the apoptotic translocation of Bax to the mitochondria. Ku70 may be a newly identified Bax suppressor and may protect cells against apoptosis (22). Most interestingly, another study demonstrates a similar role for Ku70 and Ku80 in the pancreatic cells AR42J (21). In these cells, oxidative stress, responsible for cell death, induces a decrease in Ku70 and Ku80 in the nucleus. Overexpression of Ku proteins prevents oxidative stress-induced apoptosis. Moreover, it has been demonstrated that nitric oxide treatment leads to an increased expression of DNA-PK, which protects cells against oxidative stress and DNA-damaging agents (38). Taken together, these results demonstrate that the role of DNA-PK is not restricted to DNA repair. Indeed, the DNA-PK appears to exert other cellular functions, including a role in blocking apoptosis.

Our present results demonstrate that both Ku proteins, Ku70 and Ku80, are able to interact with PDX-1 in pancreatic beta cells. Moreover, we found that this interaction implicates the PDX-1 homeodomain, which is also involved in PDX-1 binding to its DNA-responsive elements. This result is in agreement with those published by Haché and co-workers who showed an interaction of homeodomain proteins like HoxD4, Oct-1, and Oct-2 with Ku70 and Ku80 (14). Similarly to what we have found, these interactions appear to implicate the homeodomain. Indeed, Haché and co-workers reported that the DNA-PK is able to phosphorylate Oct-1 in vitro, but they did not reveal the phosphorylation site. Here we demonstrate that DNA-PK phosphorylates PDX-1 in vitro on threonine 11, which is the unique conserved consensus site for the DNA-PK catalytic subunit ((S/T)Q). It has been demonstrated previously that DNA-PK phosphorylates proteins on serine and threonine, and among these target proteins many are transcription factors (14, 39–42). However, except for IRF-3 (23), these phosphorylations have been shown in vitro and very little is known about their function. Most interestingly, we found that threonine phosphorylation of PDX-1 occurs also in intact cells in response to UV or {gamma}-radiation. Those stimuli are known to induce DNA damage and by doing so activate DNA-PK. We therefore visualized the effect of DNA-damaging stimuli on PDX-1 by the appearance of a slow migrating form of PDX-1, which corresponds to a species phosphorylated on threonine. In addition, our experiments demonstrate that threonine phosphorylation of PDX-1 is inhibited by caffeine, an inhibitor of DNA-PK at the dose used. However, we cannot conclude that DNA-PK is the only kinase implicated in this phosphorylation. Indeed, in response to DNA damage other serine/threonine kinases such as ATM and ATR are activated. These kinases phosphorylate their substrate on the same consensus site as DNA-PK and may also be inhibited by caffeine. However, at the doses of caffeine described previously to inhibit ATM and ATR, the slow migrating form of PDX-1 is still observed (Fig. 3C, lanes 3 and 4), suggesting that these kinases are not implicated in DNA damage-induced PDX-1 phosphorylation. Although further investigations are needed to clearly define the specific role of DNA-PK on PDX-1 phosphorylation in response to DNA damage, and whether additional kinases are involved in this phosphorylation, our results strongly suggest that PDX-1 may be an endogenous substrate of DNA-PK in pancreatic beta cells. Because threonine 11 is part of the transactivation domain of PDX-1, we investigated whether this phosphorylation affects the transcriptional activity of PDX-1. In contrast to what has been found for Oct-1, we failed to detect a change in PDX-1 activity in beta cells in response to DNA-PK-activating stimuli such as UV treatment. Consistent with this finding, we also demonstrate that UV light-induced DNA damage does not change the ability of PDX-1 to bind to the insulin promoter or to the glucokinase promoter.



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  		FIGURE 7.
Radiation decreases PDX-1 target genes expression. A, the mRNA expression levels of PDX-1 and some of its target genes (glut2 and glucokinase) were studied by quantitative PCR in response to UV radiation. As a negative control, we also quantified the mRNA expression level of non-PDX-1-specific target genes, ywhah and mafk. 2 and 5.5 h after UV treatment, total RNAs were extracted, and quantitative PCR was performed on the cDNAs by using the primers described under "Materials and Methods." Values are mean ± S.E., p < 0.05. *, p = 0.14. B, the mRNA expression level of Glut2 was studied by quantitative PCR in response to UV treatment and in the presence or not of transfected exogenous PDX-1-T11A. Values are mean ± S.E., p < 0.05, n = three individual experiments. C, to check the MIN6 cellular function after radiation, we tested their ability to respond to forskolin. Cells were exposed to 12.5 or 50 J/m2 of UV light. 5 or 10 h later, cells were treated with 10 µM of forskolin for 30 min. A Western blot using an anti-phospho-CREB is shown.

 
Our results bring new insights into the regulation of PDX-1 expression. Indeed, we demonstrate that expression of PDX-1 and its target genes is reduced in response to DNA-damaging stimuli. Our results suggest that the phosphorylated form of PDX-1 may be degraded by the proteosome in response to DNA damage. Most interestingly, aside from the effect on the activity of several transcription factors, previous studies have investigated the role of DNA-PK on protein stability. It has been demonstrated that activation of DNA-PK stabilizes the IRF-3 protein (23). In response to incubation with virus, IRF-3 is phosphorylated on threonine by DNA-PK. This phosphorylation acts to retain IRF-3 in the nucleus and thus reduces its degradation by the proteosome in the cytoplasm. More recently, another study demonstrates that DNA damage, induced by {gamma}-radiation, increases the stability of the homeodomain protein Oct-1 (24). However, most surprisingly, the same authors found that DNA damage simultaneously decreases the transcriptional activity of Oct-1.

Previous data from Kawamori et al. (43) showed that PDX-1 function can be negatively regulated by JNK. Indeed, in response to oxidative stress, PDX-1 translocates from the nucleus to the cytoplasm, and this process implicates JNK. Most interestingly, it has been demonstrated that oxidative stress leads to beta cell dysfunction and concomitantly increases DNA-PK expression. Moreover, it is well established that radiations activate the JNK signaling pathway. In this context the novel regulating pathway we described here and the one suggested by Kawamori et al. (43) represent major pathways, which by acting in concert lead to the negative regulation of PDX-1 and then to functional alterations in beta cells.

PDX-1 appears to be the main switch for the expression of genes crucial for both exocrine and endocrine pancreatic development. Indeed, disruption of the pdx-1 gene in mice leads to pancreatic agenesis (6). Moreover, it has been described that PDX-1 plays a major role in pancreas regeneration from the ducts in which the expression of PDX-1 is strongly increased after a 90% pancreatectomy (44). In this context, PDX-1 is finely regulated, and it is crucial to define the molecular mechanisms controlling not only its activity but also its expression level. The transcription of PDX-1 has been studied by many groups, and it appears that other transcription factors such as HNF-3{beta}, HNF-1{alpha}, NEURO-D, and also PDX-1 itself can regulate this transcription during pancreatic development (reviewed in Ref. 45). However, very little is known about the regulation of PDX-1 protein expression. Our study enlarges our understanding of the mechanisms implicated in PDX-1 protein expression. We propose that DNA-PK interacting with PDX-1 via the Ku proteins induces its phosphorylation on threonine 11, and this leads to its degradation by the proteosome. Our findings may help in identifying ways to prevent the loss of PDX-1 protein not only in diabetic patients but also in patients undergoing radiation therapy for diseases such as cancer.


   FOOTNOTES
 
* This work was supported in part by the Association pour la Recherche Contre le Cancer, the Catherina Foundation, and the Ligue Nationale Contre le Cancer Grant GL/VP-4457. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by the Philippe Foundation. To whom correspondence may be addressed. Fax: 33-4-93-81-54-32; E-mail: plebrun{at}unice.fr. 2 To whom correspondence may be addressed. Fax: 33-4-93-81-54-32; E-mail: vanobbeg{at}unice.fr.

3 The abbreviations used are: DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; NHEJ, nonhomologous end-joining; GST, glutathione S-transferase; FL, full length; JNK, c-Jun N-terminal kinase; CREB, cAMP-responsive element-binding protein; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia Rad3-related; IRF-3, interferon regulatory factor-3; CIP, calf intestinal alkaline phosphatase. Back


   ACKNOWLEDGMENTS
 
We are very grateful to U. Jhala and P. L. Puri (San Diego) for helpful discussions and to S. Hedrick (San Diego) for data gathering. We thank S. Longnus (Nice, France) for careful reading of this manuscript.



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