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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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Inhibition of GSK-3 prevents Sema 3A–induced growth cone collapse. (A) Addition of Sema 3A–Fc to DRG explant cultures for 30 min induces a growth collapse response. The graphs show the percentage of collapsed growth cone ± SEM (n ≥ 4 independent experiments). In each experiment ≥100 growth cones were counted. In the presence of 20 mM LiCl, the Sema 3A response is substantially inhibited, whereas NaCl at 20 mM did not alter the Sema 3A–induced growth cone collapse. Likewise, the two specific GSK-3 inhibitors, SB-216763 and SB-415286 (used at μM concentrations as stated), inhibited the Sema 3A–induced growth cone collapse in a dose-dependent manner. (B) Examples of phalloidin-stained DRG growth cones in order to visualize the distribution of F-actin. First micrograph shows a control untreated (−) growth cone; all subsequent pictures show growth cones that have been treated with Sema 3A (+) in the absence (control) and presence of LiCl (20 mM), SB-216763 (10 μM), and SB-415286 (30 μM). In the presence of the GSK-3 antagonist, growth cone collapse is clearly inhibited. Bar, 15 μm.
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fig5: Inhibition of GSK-3 prevents Sema 3A–induced growth cone collapse. (A) Addition of Sema 3A–Fc to DRG explant cultures for 30 min induces a growth collapse response. The graphs show the percentage of collapsed growth cone ± SEM (n ≥ 4 independent experiments). In each experiment ≥100 growth cones were counted. In the presence of 20 mM LiCl, the Sema 3A response is substantially inhibited, whereas NaCl at 20 mM did not alter the Sema 3A–induced growth cone collapse. Likewise, the two specific GSK-3 inhibitors, SB-216763 and SB-415286 (used at μM concentrations as stated), inhibited the Sema 3A–induced growth cone collapse in a dose-dependent manner. (B) Examples of phalloidin-stained DRG growth cones in order to visualize the distribution of F-actin. First micrograph shows a control untreated (−) growth cone; all subsequent pictures show growth cones that have been treated with Sema 3A (+) in the absence (control) and presence of LiCl (20 mM), SB-216763 (10 μM), and SB-415286 (30 μM). In the presence of the GSK-3 antagonist, growth cone collapse is clearly inhibited. Bar, 15 μm.

Mentions: A full collapse response to Sema 3A is much more dramatic than the partial collapse response (Fig. 5). To test whether GSK-3 activity is required for this response, we tested the effect of LiCl in the “collapse assay” since it is an established inhibitor of both GSK-3α and GSK-3β (Stambolic et al., 1996) and can prevent the Sema 3A activation of GSK-3 (Fig. 4). Treatment with LiCl at 20 mM had no effect on the basal level of collapsed growth cones (unpublished data); however, it inhibited the growth cone collapse response induced by Sema 3A from 75.3 ± 4.1% to 22.7 ± 3.2% (n = 8 explants; Fig. 5, A and B). In control experiments, we found that NaCl at 20 mM had no effect on the Sema 3A response (78.5 ± 5.8%, n = 6). LiCl is not a selective inhibitor of GSK-3 and has several additional targets (Sherman et al., 1981; Davies et al., 2000). Therefore, we tested two more specific GSK-3 inhibitors for their ability to inhibit the collapse response. SB-216763 and SB-415286 are structurally distinct maleimides that are potent inhibitors of GSK-3α and GSK-3β in an ATP competitive manner, and the specificity of these antagonists has been established in assays against 25 kinases (Coghlan et al., 2000). In the collapse assay, both compounds were able to inhibit the biological activity of Sema 3A in a dose-dependent fashion (Fig. 5) with SB-216763 being more effective than SB-415286, which correlates with their established efficacy as GSK-3 inhibitors (Coghlan et al., 2000; Cross et al., 2001). These data establish that GSK-3 activity is required for the biological activity of Sema 3A in the collapse assay.


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)

Inhibition of GSK-3 prevents Sema 3A–induced growth cone collapse. (A) Addition of Sema 3A–Fc to DRG explant cultures for 30 min induces a growth collapse response. The graphs show the percentage of collapsed growth cone ± SEM (n ≥ 4 independent experiments). In each experiment ≥100 growth cones were counted. In the presence of 20 mM LiCl, the Sema 3A response is substantially inhibited, whereas NaCl at 20 mM did not alter the Sema 3A–induced growth cone collapse. Likewise, the two specific GSK-3 inhibitors, SB-216763 and SB-415286 (used at μM concentrations as stated), inhibited the Sema 3A–induced growth cone collapse in a dose-dependent manner. (B) Examples of phalloidin-stained DRG growth cones in order to visualize the distribution of F-actin. First micrograph shows a control untreated (−) growth cone; all subsequent pictures show growth cones that have been treated with Sema 3A (+) in the absence (control) and presence of LiCl (20 mM), SB-216763 (10 μM), and SB-415286 (30 μM). In the presence of the GSK-3 antagonist, growth cone collapse is clearly inhibited. Bar, 15 μm.
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fig5: Inhibition of GSK-3 prevents Sema 3A–induced growth cone collapse. (A) Addition of Sema 3A–Fc to DRG explant cultures for 30 min induces a growth collapse response. The graphs show the percentage of collapsed growth cone ± SEM (n ≥ 4 independent experiments). In each experiment ≥100 growth cones were counted. In the presence of 20 mM LiCl, the Sema 3A response is substantially inhibited, whereas NaCl at 20 mM did not alter the Sema 3A–induced growth cone collapse. Likewise, the two specific GSK-3 inhibitors, SB-216763 and SB-415286 (used at μM concentrations as stated), inhibited the Sema 3A–induced growth cone collapse in a dose-dependent manner. (B) Examples of phalloidin-stained DRG growth cones in order to visualize the distribution of F-actin. First micrograph shows a control untreated (−) growth cone; all subsequent pictures show growth cones that have been treated with Sema 3A (+) in the absence (control) and presence of LiCl (20 mM), SB-216763 (10 μM), and SB-415286 (30 μM). In the presence of the GSK-3 antagonist, growth cone collapse is clearly inhibited. Bar, 15 μm.
Mentions: A full collapse response to Sema 3A is much more dramatic than the partial collapse response (Fig. 5). To test whether GSK-3 activity is required for this response, we tested the effect of LiCl in the “collapse assay” since it is an established inhibitor of both GSK-3α and GSK-3β (Stambolic et al., 1996) and can prevent the Sema 3A activation of GSK-3 (Fig. 4). Treatment with LiCl at 20 mM had no effect on the basal level of collapsed growth cones (unpublished data); however, it inhibited the growth cone collapse response induced by Sema 3A from 75.3 ± 4.1% to 22.7 ± 3.2% (n = 8 explants; Fig. 5, A and B). In control experiments, we found that NaCl at 20 mM had no effect on the Sema 3A response (78.5 ± 5.8%, n = 6). LiCl is not a selective inhibitor of GSK-3 and has several additional targets (Sherman et al., 1981; Davies et al., 2000). Therefore, we tested two more specific GSK-3 inhibitors for their ability to inhibit the collapse response. SB-216763 and SB-415286 are structurally distinct maleimides that are potent inhibitors of GSK-3α and GSK-3β in an ATP competitive manner, and the specificity of these antagonists has been established in assays against 25 kinases (Coghlan et al., 2000). In the collapse assay, both compounds were able to inhibit the biological activity of Sema 3A in a dose-dependent fashion (Fig. 5) with SB-216763 being more effective than SB-415286, which correlates with their established efficacy as GSK-3 inhibitors (Coghlan et al., 2000; Cross et al., 2001). These data establish that GSK-3 activity is required for the biological activity of Sema 3A in the collapse assay.

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