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Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization.

An R, da Silva Xavier G, Semplici F, Vakhshouri S, Hao HX, Rutter J, Pagano MA, Meggio F, Pinna LA, Rutter GA - Biochem. Biophys. Res. Commun. (2010)

Bottom Line: Silencing of HIPK2 led to a 51+/-0.2% decrease in Ser-269 phosphorylation in MIN6 beta-cells.Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life.Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Imperial College London, London SW7 2AZ, UK.

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Ser-269 phosphorylation and PDX1 subnuclear localization (A) Immunocytochemical analysis of the subcellular distribution of PDX1-c-myc. MIN6 β-cells were transduced with adenoviruses encoding for the indicated PDX1 molecules. Cells were cultured at 3 mM glucose for 16 h and then treated with 3 or 20 mM glucose for 6 h. c-myc-tagged PDX1 was detected using anti-c-myc antibody (Roche) and Alexa-568 (Molecular Proves). Nuclear staining was achieved using DAPI. Confocal images were captured using a Leica SP2 upright laser scanning confocal microscope (x63/1.32 oil-immersion lens) equipped with a krypton/argon laser (488 and 568 nm excitation lines) and UV light. Images were analyzed off-line using VolocityTM 4.0 software. Each sample was prepared in triplicate. (B) The average number of cells displaying PDX1 localization predominantly at nuclear periphery is expressed as a percentage of the total number of cells analyzed. Mean values from three independent experiments are shown.
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fig4: Ser-269 phosphorylation and PDX1 subnuclear localization (A) Immunocytochemical analysis of the subcellular distribution of PDX1-c-myc. MIN6 β-cells were transduced with adenoviruses encoding for the indicated PDX1 molecules. Cells were cultured at 3 mM glucose for 16 h and then treated with 3 or 20 mM glucose for 6 h. c-myc-tagged PDX1 was detected using anti-c-myc antibody (Roche) and Alexa-568 (Molecular Proves). Nuclear staining was achieved using DAPI. Confocal images were captured using a Leica SP2 upright laser scanning confocal microscope (x63/1.32 oil-immersion lens) equipped with a krypton/argon laser (488 and 568 nm excitation lines) and UV light. Images were analyzed off-line using VolocityTM 4.0 software. Each sample was prepared in triplicate. (B) The average number of cells displaying PDX1 localization predominantly at nuclear periphery is expressed as a percentage of the total number of cells analyzed. Mean values from three independent experiments are shown.

Mentions: In order to determine whether de-phosphorylation at Ser-269 at elevated glucose concentrations may contribute to the nucleoplasmic accumulation of PDX1, MIN6 β-cells were transduced with adenoviruses encoding c-myc-tagged wild-type, S269A or S269E mutant forms of PDX1. Cells were then cultured at either 3 or 30 mM glucose. High glucose caused a significant redistribution both of the de-phosphomimetic mutant S269A PDX1 and wild-type PDX1 from nuclear periphery to nucleoplasm (Fig. 4), consistent with previous studies [18]. Thus, at 30 mM glucose wild-type PDX1 was predominantly present in the nuclear region, as was the de-phosphomimetic mutant, while the phospho-mimetic mutant displayed a different localization (nuclear periphery) as compared to wild-type PDX1.


Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization.

An R, da Silva Xavier G, Semplici F, Vakhshouri S, Hao HX, Rutter J, Pagano MA, Meggio F, Pinna LA, Rutter GA - Biochem. Biophys. Res. Commun. (2010)

Ser-269 phosphorylation and PDX1 subnuclear localization (A) Immunocytochemical analysis of the subcellular distribution of PDX1-c-myc. MIN6 β-cells were transduced with adenoviruses encoding for the indicated PDX1 molecules. Cells were cultured at 3 mM glucose for 16 h and then treated with 3 or 20 mM glucose for 6 h. c-myc-tagged PDX1 was detected using anti-c-myc antibody (Roche) and Alexa-568 (Molecular Proves). Nuclear staining was achieved using DAPI. Confocal images were captured using a Leica SP2 upright laser scanning confocal microscope (x63/1.32 oil-immersion lens) equipped with a krypton/argon laser (488 and 568 nm excitation lines) and UV light. Images were analyzed off-line using VolocityTM 4.0 software. Each sample was prepared in triplicate. (B) The average number of cells displaying PDX1 localization predominantly at nuclear periphery is expressed as a percentage of the total number of cells analyzed. Mean values from three independent experiments are shown.
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fig4: Ser-269 phosphorylation and PDX1 subnuclear localization (A) Immunocytochemical analysis of the subcellular distribution of PDX1-c-myc. MIN6 β-cells were transduced with adenoviruses encoding for the indicated PDX1 molecules. Cells were cultured at 3 mM glucose for 16 h and then treated with 3 or 20 mM glucose for 6 h. c-myc-tagged PDX1 was detected using anti-c-myc antibody (Roche) and Alexa-568 (Molecular Proves). Nuclear staining was achieved using DAPI. Confocal images were captured using a Leica SP2 upright laser scanning confocal microscope (x63/1.32 oil-immersion lens) equipped with a krypton/argon laser (488 and 568 nm excitation lines) and UV light. Images were analyzed off-line using VolocityTM 4.0 software. Each sample was prepared in triplicate. (B) The average number of cells displaying PDX1 localization predominantly at nuclear periphery is expressed as a percentage of the total number of cells analyzed. Mean values from three independent experiments are shown.
Mentions: In order to determine whether de-phosphorylation at Ser-269 at elevated glucose concentrations may contribute to the nucleoplasmic accumulation of PDX1, MIN6 β-cells were transduced with adenoviruses encoding c-myc-tagged wild-type, S269A or S269E mutant forms of PDX1. Cells were then cultured at either 3 or 30 mM glucose. High glucose caused a significant redistribution both of the de-phosphomimetic mutant S269A PDX1 and wild-type PDX1 from nuclear periphery to nucleoplasm (Fig. 4), consistent with previous studies [18]. Thus, at 30 mM glucose wild-type PDX1 was predominantly present in the nuclear region, as was the de-phosphomimetic mutant, while the phospho-mimetic mutant displayed a different localization (nuclear periphery) as compared to wild-type PDX1.

Bottom Line: Silencing of HIPK2 led to a 51+/-0.2% decrease in Ser-269 phosphorylation in MIN6 beta-cells.Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life.Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Imperial College London, London SW7 2AZ, UK.

Show MeSH
Related in: MedlinePlus