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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 and insulin increase PKB and GSK3 serine phosphorylation in N29/4 and MIN6 cells. PKB (Ser-473 (S)) and GSK3 (Ser-9/Ser-21) phosphorylation was determined in N29/4 (A and B) and MIN6 (C and D) cells, stimulated with 10 nm leptin (A and C), or 10 nm insulin (B and D) for the indicated times (minutes). The bar graphs below each set of blots show mean normalized p-PKB and p-GSK3 levels for untreated cells and cells stimulated with leptin (N29/4 (A, n = 9), MIN6 (C, n = 6)) or with insulin (N29/4 (B, n = 9), MIN6 (D, n = 6)). *, p < 0.05, compared with control level for each treatment. Cont, control.
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fig5: Leptin and insulin increase PKB and GSK3 serine phosphorylation in N29/4 and MIN6 cells. PKB (Ser-473 (S)) and GSK3 (Ser-9/Ser-21) phosphorylation was determined in N29/4 (A and B) and MIN6 (C and D) cells, stimulated with 10 nm leptin (A and C), or 10 nm insulin (B and D) for the indicated times (minutes). The bar graphs below each set of blots show mean normalized p-PKB and p-GSK3 levels for untreated cells and cells stimulated with leptin (N29/4 (A, n = 9), MIN6 (C, n = 6)) or with insulin (N29/4 (B, n = 9), MIN6 (D, n = 6)). *, p < 0.05, compared with control level for each treatment. Cont, control.

Mentions: This outcome is inconsistent with the requirement for active GSK3 causing phosphorylation of PTEN Thr-366 allowing subsequent downstream signaling. Indeed, leptin (10 nm) increases the phosphorylation of PKB (Ser-473) and GSK3 (Ser-9/Ser-21) in N29/4 hypothalamic cells (Fig. 5A), although this is not well sustained in the hypothalamic cells, as previously reported for rat arcuate (13) and mouse muscle (32, 33). Insulin (10 nm) induced a sustained rise in PKB (Ser-473) and GSK3 (Ser-9/Ser-21) phosphorylation in N29/4 cells (Fig. 5B). Similar results were obtained for MIN6 β-cells, although in this case both leptin (10 nm) and insulin (10 nm) produced sustained increases (Fig. 5, C and D) in the levels of phosphorylated PKB (Ser-473) and GSK3 (Ser-9/Ser-21). These results are ostensibly inconsistent with the view that leptin signaling to actin and KATP channels requires GSK3-dependent phosphorylation and inhibition of PTEN activity. However, GSK3 also has a tyrosine residue in the activation loop of the enzyme (Tyr-216 in GSK3β and Tyr-279 in GSK3α), the phosphorylation of which is important for GSK3 activity (38, 39). Most studies conclude that this site is not physiologically regulated by growth factors or cytokines (40). Insulin (10 nm) did not affect GSK3 Tyr-216/Tyr-279 phosphorylation levels in either N29/4 cells (Fig. 6A) or MIN6 β-cells (Fig. 6C). In contrast, leptin (10 nm) rapidly increased GSK3 phosphorylation at Tyr-216/Tyr-279 in N29/4 (Fig. 6B), and MIN6 (Fig. 6D) cells, which was sustained for at least 1 h. Thus, in contrast to insulin, which increases phosphorylation of GSK3 serine sites only (consistent with kinase inhibition), leptin increases the phosphorylation level of both serine and tyrosine sites on GSK3. To define the outcome of this dual phosphorylation of GSK3, we examined the effects of leptin (10 nm) and insulin (50 nm) on GSK3 activity in N29/4 cells. Stimulation of N29/4 cells by insulin for 30 min results in a substantial inhibition of GSK3 activity, as expected from increased PKB activity causing GSK3 Ser-9/Ser-21 phosphorylation. In contrast, stimulation of N29/4 cells with leptin (10 nm) for 30 min, a time when PTEN phosphorylation is increased, PtdIns(3,4,5)P3 levels are high, F-actin is depolymerized, and β-cells are hyperpolarized, results in maintained GSK3 activity (Fig. 6E). This outcome is consistent with the finding that, although both hormones increase GSK3 Ser-9/Ser-21 phosphorylation levels, leptin, but not insulin, also increases GSK3 Tyr-216/Tyr-279 phosphorylation.


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 and insulin increase PKB and GSK3 serine phosphorylation in N29/4 and MIN6 cells. PKB (Ser-473 (S)) and GSK3 (Ser-9/Ser-21) phosphorylation was determined in N29/4 (A and B) and MIN6 (C and D) cells, stimulated with 10 nm leptin (A and C), or 10 nm insulin (B and D) for the indicated times (minutes). The bar graphs below each set of blots show mean normalized p-PKB and p-GSK3 levels for untreated cells and cells stimulated with leptin (N29/4 (A, n = 9), MIN6 (C, n = 6)) or with insulin (N29/4 (B, n = 9), MIN6 (D, n = 6)). *, p < 0.05, compared with control level for each treatment. Cont, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Leptin and insulin increase PKB and GSK3 serine phosphorylation in N29/4 and MIN6 cells. PKB (Ser-473 (S)) and GSK3 (Ser-9/Ser-21) phosphorylation was determined in N29/4 (A and B) and MIN6 (C and D) cells, stimulated with 10 nm leptin (A and C), or 10 nm insulin (B and D) for the indicated times (minutes). The bar graphs below each set of blots show mean normalized p-PKB and p-GSK3 levels for untreated cells and cells stimulated with leptin (N29/4 (A, n = 9), MIN6 (C, n = 6)) or with insulin (N29/4 (B, n = 9), MIN6 (D, n = 6)). *, p < 0.05, compared with control level for each treatment. Cont, control.
Mentions: This outcome is inconsistent with the requirement for active GSK3 causing phosphorylation of PTEN Thr-366 allowing subsequent downstream signaling. Indeed, leptin (10 nm) increases the phosphorylation of PKB (Ser-473) and GSK3 (Ser-9/Ser-21) in N29/4 hypothalamic cells (Fig. 5A), although this is not well sustained in the hypothalamic cells, as previously reported for rat arcuate (13) and mouse muscle (32, 33). Insulin (10 nm) induced a sustained rise in PKB (Ser-473) and GSK3 (Ser-9/Ser-21) phosphorylation in N29/4 cells (Fig. 5B). Similar results were obtained for MIN6 β-cells, although in this case both leptin (10 nm) and insulin (10 nm) produced sustained increases (Fig. 5, C and D) in the levels of phosphorylated PKB (Ser-473) and GSK3 (Ser-9/Ser-21). These results are ostensibly inconsistent with the view that leptin signaling to actin and KATP channels requires GSK3-dependent phosphorylation and inhibition of PTEN activity. However, GSK3 also has a tyrosine residue in the activation loop of the enzyme (Tyr-216 in GSK3β and Tyr-279 in GSK3α), the phosphorylation of which is important for GSK3 activity (38, 39). Most studies conclude that this site is not physiologically regulated by growth factors or cytokines (40). Insulin (10 nm) did not affect GSK3 Tyr-216/Tyr-279 phosphorylation levels in either N29/4 cells (Fig. 6A) or MIN6 β-cells (Fig. 6C). In contrast, leptin (10 nm) rapidly increased GSK3 phosphorylation at Tyr-216/Tyr-279 in N29/4 (Fig. 6B), and MIN6 (Fig. 6D) cells, which was sustained for at least 1 h. Thus, in contrast to insulin, which increases phosphorylation of GSK3 serine sites only (consistent with kinase inhibition), leptin increases the phosphorylation level of both serine and tyrosine sites on GSK3. To define the outcome of this dual phosphorylation of GSK3, we examined the effects of leptin (10 nm) and insulin (50 nm) on GSK3 activity in N29/4 cells. Stimulation of N29/4 cells by insulin for 30 min results in a substantial inhibition of GSK3 activity, as expected from increased PKB activity causing GSK3 Ser-9/Ser-21 phosphorylation. In contrast, stimulation of N29/4 cells with leptin (10 nm) for 30 min, a time when PTEN phosphorylation is increased, PtdIns(3,4,5)P3 levels are high, F-actin is depolymerized, and β-cells are hyperpolarized, results in maintained GSK3 activity (Fig. 6E). This outcome is consistent with the finding that, although both hormones increase GSK3 Ser-9/Ser-21 phosphorylation levels, leptin, but not insulin, also increases GSK3 Tyr-216/Tyr-279 phosphorylation.

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