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. 2010 Jul;67(14):2481-9.
doi: 10.1007/s00018-010-0348-0. Epub 2010 Mar 26.

CK2 phosphorylation of Pdx-1 regulates its transcription factor activity

Rui Meng  1 , Faizeh Al-Quobaili, Isabelle Müller, Claudia Götz, Gerald Thiel, Mathias Montenarh
Affiliations

CK2 phosphorylation of Pdx-1 regulates its transcription factor activity

Rui Meng et al. Cell Mol Life Sci. 2010 Jul.

Abstract

The duodenal homeobox-1 protein Pdx-1 is one of the regulators for the transcription of the insulin gene. Pdx-1 is a phosphoprotein, and there is increasing evidence for the regulation of some of its functions by phosphorylation. Here, we asked whether protein kinase CK2 might phosphorylate Pdx-1 and how this phosphorylation could be implicated in the functional regulation of Pdx-1. We used fragments of Pdx-1 as well as phosphorylation mutants for experiments with protein kinase CK2. Transactivation was measured by reporter assays using the insulin promoter. Our data showed that Pdx-1 is phosphorylated by protein kinase CK2 at amino acids thr(231) and ser(232), and this phosphorylation was implicated in the regulation of the transcription factor activity of Pdx-1. Furthermore, inhibition of protein kinase CK2 by specific inhibitors led to an elevated release of insulin from pancreatic beta-cells. Thus, these findings identify CK2 as a novel mediator of the insulin metabolism.

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Figures

Fig. 1
Fig. 1
Putative CK2 phosphorylation sites on Pdx-1. a Schematic representation of the domain structure of mouse Pdx-1. Transactivation indicates the N-terminal transactivation domain with three highly conserved sub-domains, spanning amino acids 13–22, 32–38, and 60–73. Homeobox signifies the homeodomain, and NLS indicates the nuclear localization signal. Arrows show the putative CK2 phosphorylation sites. b Alignment of Pdx-1 amino acid sequences of different species. Putative consensus sites for CK2 phosphorylation are indicated in italics
Fig. 2
Fig. 2
In vitro phosphorylation of Pdx-1 by the CK2 holoenzyme and the α- and α′-subunits. a Bacterially expressed and purified GST-Pdx-1 (1.5 μg) was analyzed on a 12.5% SDS-polyacrylamide gel followed by Coomassie blue staining. b In vitro CK2 phosphorylation assay was performed in the presence of GST-Pdx-1 (1.5 μg) wild type by using a purified CK2 holoenzyme (lane 3) or CK2α (lane 7) or MBP-CK2α′ (lane 9) and CK2 holoenzyme in the presence of 10 μM TBB (lane 4). The reaction was carried out in the presence of 32PγATP for 30 min at 37°C. The CK2 holoenzyme (lane 1) or CK2α (lane 6) or MBP-CK2α’ (lane 8) was incubated with 32PγATP in the absence of Pdx-1 as a control. Phosphorylated proteins were separated in a 12.5% SDS polyacrylamide gel, and the gel was subjected to autoradiography
Fig. 3
Fig. 3
In vitro phosphorylation of Pdx-1 fragments by the CK2 holoenzyme. a Schematic representation of GST-tagged Pdx-1 fragments. The GST moiety is shown as a black bar; the Pdx-1 part is shown as a white bar. Numbers above the Pdx-1 part indicate the respective Pdx-1 amino acids according to mouse Pdx-1. Arrows show the localization of putative CK2 phosphorylation sites. b Bacterially expressed and purified Pdx-1N-terminus (1.5 μg) and Pdx-1 C-terminus (1.5 μg) were analyzed on a 12.5% SDS-polyacrylamide gel. The gel was stained by Coomassie blue. c Recombinant Pdx-1 fragments were phosphorylated by CK2 holoenzyme. Phosphorylation was conducted in the presence of GST-Pdx-1 N-terminus (lane 1) or GST-PDX-1 C-terminus (lane 3) with CK2 holoenzyme and 32PγATP. To serve as control, the GST-Pdx-1 N-terminus (lane 2) or the GST-Pdx-1 C-terminus (lane 4) or CK2 holoenzyme (lane 5) alone was also incubated with 32PγATP. Phosphorylated proteins were separated in a 12.5% SDS polyacrylamide gel. Proteins were visualized by autoradiography. The lower band shows the autophosphorylation of CK2β subunit. Molecular weight markers (in KDa) are indicated on the left
Fig. 4
Fig. 4
In vitro phosphorylation of the Pdx-1 wild type and phosphorylation inactive mutants by the CK2 holoenzyme. a Pdx-1 wild type and mutant proteins were analyzed on a 12.5% SDS polyacrylamide gel. Proteins were stained with Coomassie Brilliant Blue. b Pdx-1 is phosphorylated by CK2 on both ser232 and thr231. Bacterially expressed and purified Pdx-1 wild type and alanine mutants were incubated with CK2 holoenzyme in the presence of 32PγATP. Phosphorylated proteins were run on a 12.5% SDS polyacrylamide gel and then subjected to autoradiography. The autoradiogram shows labeled GST-Pdx-1 wild type (lane 2), S232A (lane 4), T231A (lane 6), S232A/T231A (lane 8), and the autophosphorylation of CK2 β subunit. The CK2 holoenzyme (α2β2) (lane 1) or GST-Pdx-1 wild type (lane 3) or S232A (lane 5) or T231A (lane 7) or S232A/T231A (lane 9) protein alone was included as a negative control
Fig. 5
Fig. 5
In vivo labeling of βTC-3 cells with 33P-phosphate. βTC-3 cells were labeled with 33P-phosphate for 5 h. Pdx-1 was immunoprecipitated from the cell extract and analyzed on a 12.5% SDS polyacrylamide gel. Phosphate-labeled proteins were identified by autoradiography. For control, the blot was incubated with an Pdx-1 antibody. Protein bands were visualized by the ECL method
Fig. 6
Fig. 6
Ser232 and thr231 phosphorylation modulate Pdx-1 transcriptional activity. HEK293T cells seeded in a six-well plate were transfected with 2 μg of empty vector (pcDNA3) or pcDNA3-derived vectors encoding either wild type or mutant Pdx-1, 1.5 μg of RIP luciferase, and 0.5 μg of β-galactosidase by calcium phosphate transfection methods. The cells were lysed, and luciferase activity was measured 48 h after transfection. The data between the control cells (empty vector group) and the Pdx-1 wild type-expressing cells (a) are expressed as the ratio of the RIP luciferase activity/β-galactosidase activity. On the contrary, results between Pdx-1 WT and different mutant-expressing cells (b) are shown as RIP luciferase activity divided by the amount of Pdx-1 proteins expressed in the cells. In both cases, the relative activity in Pdx-1 wild type-expressing cells was set at 100%. The results are the means ± SE of three separate experiments done in triplicate
Fig. 7
Fig. 7
Effect of CK2 inhibitors on the transcription of the insulin promotor. Min6 cells were infected with a recombinant lentivirus coding for a luciferase construct under the control of the insulin promotor and incubated with 2 mM glucose (LG) or 25 mM glucose (HG) or with HG in the presence of either 50 μM TBB or 50 μM quinalizarin. Luciferase activity measured at LG was set at 100%, and the activity of the other sample was calculated in relation to this value. Standard deviation was calculated from three independent experiments
Fig. 8
Fig. 8
Overexpression of CK2α and CK2β decreased the transcription of the insulin promotor in pancreatic β-cells. βTC-3 cells were transfected with control or CK2 encoding vectors together with RIP-luciferase and β-galactosidase. Forty-eight hours after transfection luciferase activity was determined. The luciferase activity in the cells transfected with the empty vector (−) was set as 100%, and the activity of cells transfected with CK2α and CK2β (+) was calculated in relation to this 100% value. Standard deviation was calculated from three independent experiments
Fig. 9
Fig. 9
Effect of CK2 inhibitors on insulin release from pancreatic β-cells. Min6 cells were treated with TBB or left untreated. The culture medium was collected, and the insulin content was measured by a rat/mouse insulin Elisa Kit (Millipore). The experiment was performed in triplicate, and the data are presented as percentage of insulin release in the absence of inhibitors
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