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An inactive pool of GSK-3 at the leading edge of growth cones is implicated in Semaphorin 3A signaling.

Eickholt BJ, Walsh FS, Doherty P - J. Cell Biol. (2002)

Bottom Line: Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades.We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A.These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology Group, Medical Research Council Centre for Developmental Biology, King's College London, London SE1 1UL, United Kingdom. Britta.J.Eickholt@kcl.ac.uk

ABSTRACT
Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades. We now report that an inactive phosphorylated pool of the enzyme colocalizes with F-actin in both neuronal and nonneuronal cells. Semaphorin 3A (Sema 3A), a molecule that inhibits axonal growth, activates GSK-3 at the leading edge of neuronal growth cones and in Sema 3A-responsive human breast cancer cells, suggesting that GSK-3 activity might play a role in coupling Sema 3A signaling to changes in cell motility. We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A. These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.

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Decrease in phosphorylation of GSK-3 after Sema 3A treatment in MDA-MB-231 breast carcinoma cell lines. Western blots of MDA-MB 231 cell extracts using anti–P-(Ser9)-GSK-3β, anti–GSK-3β, and antiactin antibodies. Cell lysates were prepared from PBS-treated control and Sema 3A–Fc–treated (1 μg/ml for 1 h) MDA-MB 231 cells in the presence or absence of LiCl (at 20 mM). All experiments were performed in duplicates. Sema 3A treatment notably decreases Ser9 phosphorylation of GSK-3. Treatment with the GSK-3 inhibitor LiCl enhances GSK-3 phosphorylation slightly and antagonizes the Sema 3A–mediated decrease in Ser9 phosphorylation. Blots were reprobed with an anti–GSK-3β antibody to confirm equal loading of samples.
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fig4: Decrease in phosphorylation of GSK-3 after Sema 3A treatment in MDA-MB-231 breast carcinoma cell lines. Western blots of MDA-MB 231 cell extracts using anti–P-(Ser9)-GSK-3β, anti–GSK-3β, and antiactin antibodies. Cell lysates were prepared from PBS-treated control and Sema 3A–Fc–treated (1 μg/ml for 1 h) MDA-MB 231 cells in the presence or absence of LiCl (at 20 mM). All experiments were performed in duplicates. Sema 3A treatment notably decreases Ser9 phosphorylation of GSK-3. Treatment with the GSK-3 inhibitor LiCl enhances GSK-3 phosphorylation slightly and antagonizes the Sema 3A–mediated decrease in Ser9 phosphorylation. Blots were reprobed with an anti–GSK-3β antibody to confirm equal loading of samples.

Mentions: Sema 3A can also “collapse” neural crest cells (Eickholt et al., 1999), and it inhibits the migration of several other cell types (Miao et al., 1999; Bagnard et al., 2001). We have observed recently that MDA-MB-231 breast cancer cells show similar responsiveness to Sema 3A (unpublished data). Since inactive GSK-3 is found almost exclusively at the leading edge of these cells (Fig. 1), we reasoned that they might be a good model for examining the effects of Sema 3A on GSK-3 phosphorylation. In these cells, Western blotting consistently revealed that treatment with Sema 3A (applied for 1 h at 1 μg/ml) induces a substantial reduction in serine phosphorylation on GSK-3β in the absence of any detectable change in the level of GSK-3 protein (Fig. 4). As expected, this activation was not apparent when LiCl was included in the cultures (20 mM) (Fig. 4). These data not only confirm that Sema 3A can activate GSK-3, they also extend the observation to nonneuronal cells.


An inactive pool of GSK-3 at the leading edge of growth cones is implicated in Semaphorin 3A signaling.

Eickholt BJ, Walsh FS, Doherty P - J. Cell Biol. (2002)

Decrease in phosphorylation of GSK-3 after Sema 3A treatment in MDA-MB-231 breast carcinoma cell lines. Western blots of MDA-MB 231 cell extracts using anti–P-(Ser9)-GSK-3β, anti–GSK-3β, and antiactin antibodies. Cell lysates were prepared from PBS-treated control and Sema 3A–Fc–treated (1 μg/ml for 1 h) MDA-MB 231 cells in the presence or absence of LiCl (at 20 mM). All experiments were performed in duplicates. Sema 3A treatment notably decreases Ser9 phosphorylation of GSK-3. Treatment with the GSK-3 inhibitor LiCl enhances GSK-3 phosphorylation slightly and antagonizes the Sema 3A–mediated decrease in Ser9 phosphorylation. Blots were reprobed with an anti–GSK-3β antibody to confirm equal loading of samples.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199247&req=5

fig4: Decrease in phosphorylation of GSK-3 after Sema 3A treatment in MDA-MB-231 breast carcinoma cell lines. Western blots of MDA-MB 231 cell extracts using anti–P-(Ser9)-GSK-3β, anti–GSK-3β, and antiactin antibodies. Cell lysates were prepared from PBS-treated control and Sema 3A–Fc–treated (1 μg/ml for 1 h) MDA-MB 231 cells in the presence or absence of LiCl (at 20 mM). All experiments were performed in duplicates. Sema 3A treatment notably decreases Ser9 phosphorylation of GSK-3. Treatment with the GSK-3 inhibitor LiCl enhances GSK-3 phosphorylation slightly and antagonizes the Sema 3A–mediated decrease in Ser9 phosphorylation. Blots were reprobed with an anti–GSK-3β antibody to confirm equal loading of samples.
Mentions: Sema 3A can also “collapse” neural crest cells (Eickholt et al., 1999), and it inhibits the migration of several other cell types (Miao et al., 1999; Bagnard et al., 2001). We have observed recently that MDA-MB-231 breast cancer cells show similar responsiveness to Sema 3A (unpublished data). Since inactive GSK-3 is found almost exclusively at the leading edge of these cells (Fig. 1), we reasoned that they might be a good model for examining the effects of Sema 3A on GSK-3 phosphorylation. In these cells, Western blotting consistently revealed that treatment with Sema 3A (applied for 1 h at 1 μg/ml) induces a substantial reduction in serine phosphorylation on GSK-3β in the absence of any detectable change in the level of GSK-3 protein (Fig. 4). As expected, this activation was not apparent when LiCl was included in the cultures (20 mM) (Fig. 4). These data not only confirm that Sema 3A can activate GSK-3, they also extend the observation to nonneuronal cells.

Bottom Line: Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades.We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A.These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology Group, Medical Research Council Centre for Developmental Biology, King's College London, London SE1 1UL, United Kingdom. Britta.J.Eickholt@kcl.ac.uk

ABSTRACT
Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades. We now report that an inactive phosphorylated pool of the enzyme colocalizes with F-actin in both neuronal and nonneuronal cells. Semaphorin 3A (Sema 3A), a molecule that inhibits axonal growth, activates GSK-3 at the leading edge of neuronal growth cones and in Sema 3A-responsive human breast cancer cells, suggesting that GSK-3 activity might play a role in coupling Sema 3A signaling to changes in cell motility. We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A. These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.

Show MeSH
Related in: MedlinePlus