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Leptin-dependent phosphorylation of PTEN mediates actin restructuring and activation of ATP-sensitive K+ channels.

Ning K, Miller LC, Laidlaw HA, Watterson KR, Gallagher J, Sutherland C, Ashford ML - J. Biol. Chem. (2009)

Bottom Line: Both hormones increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells this action of leptin is transient.Leptin, not insulin, increases GSK3 tyrosine phosphorylation in both cell types.These results demonstrate a significant role for PTEN in leptin signal transmission and identify GSK3 as a potential important signaling node contributing to divergent outputs for these hormones.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom.

ABSTRACT
Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with insulin signaling. The exact mechanisms by which leptin alters this signaling pathway and how it relates to functional outputs are unclear at present. A previous study has established that leptin inhibits the activity of the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important tumor suppressor and modifier of phosphoinositide signaling. In this study we demonstrate that leptin phosphorylates multiple sites on the C-terminal tail of PTEN in hypothalamic and pancreatic beta-cells, an action not replicated by insulin. Inhibitors of the protein kinases CK2 and glycogen synthase kinase 3 (GSK3) block leptin-mediated PTEN phosphorylation. PTEN phosphorylation mutants reveal the critical role these sites play in transmission of the leptin signal to F-actin depolymerization. CK2 and GSK3 inhibitors also prevent leptin-mediated F-actin depolymerization and consequent ATP-sensitive K(+) channel opening. GSK3 kinase activity is inhibited by insulin but not leptin in hypothalamic cells. Both hormones increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells this action of leptin is transient. Leptin, not insulin, increases GSK3 tyrosine phosphorylation in both cell types. These results demonstrate a significant role for PTEN in leptin signal transmission and identify GSK3 as a potential important signaling node contributing to divergent outputs for these hormones.

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Leptin increases PTEN phosphorylation in MIN6 pancreatic β-cells. A, phosphorylated PTEN levels in MIN6 cells using the phospho-specific PTEN antibodies indicated (cluster site, Ser-370 and Thr-366), in response to 10 nm leptin or 10 nm insulin for the indicated times (minutes). B, the bar graph shows mean normalized levels of phosphorylated PTEN at the cluster phosphorylation site (380/2/3) for untreated cells and for cells stimulated with leptin (filled bars; n = 6) and insulin (open bars; n = 8) up to 30 min (*, p < 0.05, versus control (C) or insulin). C, bar graph shows the mean normalized levels of phosphorylated Thr-366 PTEN after stimulation with 10 nm insulin for 5 and 30 min. D, bar graphs showing mean normalized levels of phosphorylated Thr-366 PTEN and Ser-370 PTEN under non-stimulated conditions (C) and after stimulation with 10 nm leptin in the absence (L) and presence of DMAT (L+D) and CT99021 (L+CT; n = 7 for each). *, p < 0.05.
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fig3: Leptin increases PTEN phosphorylation in MIN6 pancreatic β-cells. A, phosphorylated PTEN levels in MIN6 cells using the phospho-specific PTEN antibodies indicated (cluster site, Ser-370 and Thr-366), in response to 10 nm leptin or 10 nm insulin for the indicated times (minutes). B, the bar graph shows mean normalized levels of phosphorylated PTEN at the cluster phosphorylation site (380/2/3) for untreated cells and for cells stimulated with leptin (filled bars; n = 6) and insulin (open bars; n = 8) up to 30 min (*, p < 0.05, versus control (C) or insulin). C, bar graph shows the mean normalized levels of phosphorylated Thr-366 PTEN after stimulation with 10 nm insulin for 5 and 30 min. D, bar graphs showing mean normalized levels of phosphorylated Thr-366 PTEN and Ser-370 PTEN under non-stimulated conditions (C) and after stimulation with 10 nm leptin in the absence (L) and presence of DMAT (L+D) and CT99021 (L+CT; n = 7 for each). *, p < 0.05.

Mentions: Phosphorylation of PTEN in β-Cells and KATP Activation—It was previously demonstrated that KATP channel opening and hyperpolarization of mouse pancreatic β-cells by leptin could be inhibited by the lipid phosphatase dead G129E PTEN mutant and mimicked by the dual lipid and protein phosphatase dead C124S PTEN mutant (12). This is consistent with inhibition of PTEN lipid and protein phosphatase activity for the successful transmission of the leptin signal to KATP channels in β-cells. As leptin has not been shown to alter p-PTEN status in β-cells, we used MIN6 β-cells to explore this action of leptin. Although the effects of leptin on p-PTEN were not as robust as observed for N29/4 cells, leptin (10 nm) rapidly (≤1 min) increased phosphorylation of the cluster site, Ser-370 and Thr-366, actions sustained for at least 30 min in each case (Fig. 3, A and B). Insulin (10 nm) had no effect on PTEN phosphorylation levels at any of these sites over this time period (Fig. 3, A–C). The CK2 inhibitor DMAT (2 μm) inhibited leptin-mediated PTEN phosphorylation at Ser-370 and Thr-366, whereas the GSK3 inhibitor CT99021 (10 μm) inhibited phosphorylation at Thr-366 only (Fig. 3D).


Leptin-dependent phosphorylation of PTEN mediates actin restructuring and activation of ATP-sensitive K+ channels.

Ning K, Miller LC, Laidlaw HA, Watterson KR, Gallagher J, Sutherland C, Ashford ML - J. Biol. Chem. (2009)

Leptin increases PTEN phosphorylation in MIN6 pancreatic β-cells. A, phosphorylated PTEN levels in MIN6 cells using the phospho-specific PTEN antibodies indicated (cluster site, Ser-370 and Thr-366), in response to 10 nm leptin or 10 nm insulin for the indicated times (minutes). B, the bar graph shows mean normalized levels of phosphorylated PTEN at the cluster phosphorylation site (380/2/3) for untreated cells and for cells stimulated with leptin (filled bars; n = 6) and insulin (open bars; n = 8) up to 30 min (*, p < 0.05, versus control (C) or insulin). C, bar graph shows the mean normalized levels of phosphorylated Thr-366 PTEN after stimulation with 10 nm insulin for 5 and 30 min. D, bar graphs showing mean normalized levels of phosphorylated Thr-366 PTEN and Ser-370 PTEN under non-stimulated conditions (C) and after stimulation with 10 nm leptin in the absence (L) and presence of DMAT (L+D) and CT99021 (L+CT; n = 7 for each). *, p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666585&req=5

fig3: Leptin increases PTEN phosphorylation in MIN6 pancreatic β-cells. A, phosphorylated PTEN levels in MIN6 cells using the phospho-specific PTEN antibodies indicated (cluster site, Ser-370 and Thr-366), in response to 10 nm leptin or 10 nm insulin for the indicated times (minutes). B, the bar graph shows mean normalized levels of phosphorylated PTEN at the cluster phosphorylation site (380/2/3) for untreated cells and for cells stimulated with leptin (filled bars; n = 6) and insulin (open bars; n = 8) up to 30 min (*, p < 0.05, versus control (C) or insulin). C, bar graph shows the mean normalized levels of phosphorylated Thr-366 PTEN after stimulation with 10 nm insulin for 5 and 30 min. D, bar graphs showing mean normalized levels of phosphorylated Thr-366 PTEN and Ser-370 PTEN under non-stimulated conditions (C) and after stimulation with 10 nm leptin in the absence (L) and presence of DMAT (L+D) and CT99021 (L+CT; n = 7 for each). *, p < 0.05.
Mentions: Phosphorylation of PTEN in β-Cells and KATP Activation—It was previously demonstrated that KATP channel opening and hyperpolarization of mouse pancreatic β-cells by leptin could be inhibited by the lipid phosphatase dead G129E PTEN mutant and mimicked by the dual lipid and protein phosphatase dead C124S PTEN mutant (12). This is consistent with inhibition of PTEN lipid and protein phosphatase activity for the successful transmission of the leptin signal to KATP channels in β-cells. As leptin has not been shown to alter p-PTEN status in β-cells, we used MIN6 β-cells to explore this action of leptin. Although the effects of leptin on p-PTEN were not as robust as observed for N29/4 cells, leptin (10 nm) rapidly (≤1 min) increased phosphorylation of the cluster site, Ser-370 and Thr-366, actions sustained for at least 30 min in each case (Fig. 3, A and B). Insulin (10 nm) had no effect on PTEN phosphorylation levels at any of these sites over this time period (Fig. 3, A–C). The CK2 inhibitor DMAT (2 μm) inhibited leptin-mediated PTEN phosphorylation at Ser-370 and Thr-366, whereas the GSK3 inhibitor CT99021 (10 μm) inhibited phosphorylation at Thr-366 only (Fig. 3D).

Bottom Line: Both hormones increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells this action of leptin is transient.Leptin, not insulin, increases GSK3 tyrosine phosphorylation in both cell types.These results demonstrate a significant role for PTEN in leptin signal transmission and identify GSK3 as a potential important signaling node contributing to divergent outputs for these hormones.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom.

ABSTRACT
Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with insulin signaling. The exact mechanisms by which leptin alters this signaling pathway and how it relates to functional outputs are unclear at present. A previous study has established that leptin inhibits the activity of the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important tumor suppressor and modifier of phosphoinositide signaling. In this study we demonstrate that leptin phosphorylates multiple sites on the C-terminal tail of PTEN in hypothalamic and pancreatic beta-cells, an action not replicated by insulin. Inhibitors of the protein kinases CK2 and glycogen synthase kinase 3 (GSK3) block leptin-mediated PTEN phosphorylation. PTEN phosphorylation mutants reveal the critical role these sites play in transmission of the leptin signal to F-actin depolymerization. CK2 and GSK3 inhibitors also prevent leptin-mediated F-actin depolymerization and consequent ATP-sensitive K(+) channel opening. GSK3 kinase activity is inhibited by insulin but not leptin in hypothalamic cells. Both hormones increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells this action of leptin is transient. Leptin, not insulin, increases GSK3 tyrosine phosphorylation in both cell types. These results demonstrate a significant role for PTEN in leptin signal transmission and identify GSK3 as a potential important signaling node contributing to divergent outputs for these hormones.

Show MeSH
Related in: MedlinePlus