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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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Phosphorylation on Ser-269 is regulated by glucose concentrations in pancreatic islets and MIN6 β-cells. (A) Mouse pancreatic islets of Langerhans or (B) MIN6 β-cells cultured in medium containing 3 mM glucose for 1 and 16 h, respectively. Mouse islets were further cultured for 1 h at three or 16.7 mM glucose. MIN6 β-cells were further cultured at the indicated glucose concentrations for the indicated time. Nuclear lysates were separated on SDS–PAGE gels and analyzed by Western blot using anti-phospho-Ser-269 PDX1 and anti-PDX1 antibodies. The blots shown are representative of 3 independent experiments. The ratio of phospho-Ser-269 PDX1 to total PDX1 level are shown graphically (A, B lower panels). ∗p < 0.05, ∗∗p < 0.01, ns: non-significant; samples were prepared in triplicate. Mean values from three independent experiments are shown.
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fig1: Phosphorylation on Ser-269 is regulated by glucose concentrations in pancreatic islets and MIN6 β-cells. (A) Mouse pancreatic islets of Langerhans or (B) MIN6 β-cells cultured in medium containing 3 mM glucose for 1 and 16 h, respectively. Mouse islets were further cultured for 1 h at three or 16.7 mM glucose. MIN6 β-cells were further cultured at the indicated glucose concentrations for the indicated time. Nuclear lysates were separated on SDS–PAGE gels and analyzed by Western blot using anti-phospho-Ser-269 PDX1 and anti-PDX1 antibodies. The blots shown are representative of 3 independent experiments. The ratio of phospho-Ser-269 PDX1 to total PDX1 level are shown graphically (A, B lower panels). ∗p < 0.05, ∗∗p < 0.01, ns: non-significant; samples were prepared in triplicate. Mean values from three independent experiments are shown.

Mentions: PDX1 Ser-269 was identified as a potential phosphorylation site by mass spectrometry (Supplementary Fig. 1). We, therefore, explored the possibility that phosphorylation of PDX1 at Ser-269 may be regulated by glucose in living β-cells and, thus, may, at least potentially, contribute to the regulation of PDX1 function by the sugar. After treatment at low glucose concentrations (3 mM) for 16 (MIN6 β-cells) or 1 h (islets), we further incubated islets (Fig. 1A) or MIN6 β-cells (Fig. 1B) in medium containing 16.7 or 30 mM glucose, respectively, for varying times.


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)

Phosphorylation on Ser-269 is regulated by glucose concentrations in pancreatic islets and MIN6 β-cells. (A) Mouse pancreatic islets of Langerhans or (B) MIN6 β-cells cultured in medium containing 3 mM glucose for 1 and 16 h, respectively. Mouse islets were further cultured for 1 h at three or 16.7 mM glucose. MIN6 β-cells were further cultured at the indicated glucose concentrations for the indicated time. Nuclear lysates were separated on SDS–PAGE gels and analyzed by Western blot using anti-phospho-Ser-269 PDX1 and anti-PDX1 antibodies. The blots shown are representative of 3 independent experiments. The ratio of phospho-Ser-269 PDX1 to total PDX1 level are shown graphically (A, B lower panels). ∗p < 0.05, ∗∗p < 0.01, ns: non-significant; samples were prepared in triplicate. Mean values from three independent experiments are shown.
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Related In: Results  -  Collection

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fig1: Phosphorylation on Ser-269 is regulated by glucose concentrations in pancreatic islets and MIN6 β-cells. (A) Mouse pancreatic islets of Langerhans or (B) MIN6 β-cells cultured in medium containing 3 mM glucose for 1 and 16 h, respectively. Mouse islets were further cultured for 1 h at three or 16.7 mM glucose. MIN6 β-cells were further cultured at the indicated glucose concentrations for the indicated time. Nuclear lysates were separated on SDS–PAGE gels and analyzed by Western blot using anti-phospho-Ser-269 PDX1 and anti-PDX1 antibodies. The blots shown are representative of 3 independent experiments. The ratio of phospho-Ser-269 PDX1 to total PDX1 level are shown graphically (A, B lower panels). ∗p < 0.05, ∗∗p < 0.01, ns: non-significant; samples were prepared in triplicate. Mean values from three independent experiments are shown.
Mentions: PDX1 Ser-269 was identified as a potential phosphorylation site by mass spectrometry (Supplementary Fig. 1). We, therefore, explored the possibility that phosphorylation of PDX1 at Ser-269 may be regulated by glucose in living β-cells and, thus, may, at least potentially, contribute to the regulation of PDX1 function by the sugar. After treatment at low glucose concentrations (3 mM) for 16 (MIN6 β-cells) or 1 h (islets), we further incubated islets (Fig. 1A) or MIN6 β-cells (Fig. 1B) in medium containing 16.7 or 30 mM glucose, respectively, for varying times.

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