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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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Sema 3A activates GSK-3 at the leading edge of sensory growth cones. DRG cultures grown overnight were treated with a Sema 3A–Fc chimera for 2 min before fixation. Growth cones were stained with phalloidin–Texas red (left) and anti–GSK-3α (A, right) and with phalloidin–Texas red (left) and anti–P-(Ser21)-GSK-3α (B, right). After exposure to Sema 3A, the entire GSK-3α pool remains homogeneously distributed throughout the growth cone and is still detectable in filopodia, whereas the inactive P-(Ser21)-GSK-3α is depleted at the leading edge of the growth cone and filopodia (arrowheads). The insert shows the parallel performed P-(Ser21)-GSK-3α staining in untreated control cultures. Bars, 15 μm. (C) Percentage of growth cones showing enriched staining at the leading edge before and after treatment with Sema 3A–Fc. Bars show ± SEM.
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fig3: Sema 3A activates GSK-3 at the leading edge of sensory growth cones. DRG cultures grown overnight were treated with a Sema 3A–Fc chimera for 2 min before fixation. Growth cones were stained with phalloidin–Texas red (left) and anti–GSK-3α (A, right) and with phalloidin–Texas red (left) and anti–P-(Ser21)-GSK-3α (B, right). After exposure to Sema 3A, the entire GSK-3α pool remains homogeneously distributed throughout the growth cone and is still detectable in filopodia, whereas the inactive P-(Ser21)-GSK-3α is depleted at the leading edge of the growth cone and filopodia (arrowheads). The insert shows the parallel performed P-(Ser21)-GSK-3α staining in untreated control cultures. Bars, 15 μm. (C) Percentage of growth cones showing enriched staining at the leading edge before and after treatment with Sema 3A–Fc. Bars show ± SEM.

Mentions: The specific localization of an inactive pool of GSK-3 at the leading edge of the growth cone suggests a function in the control of growth cones motility. However it seems highly unlikely GSK-3 activity is required for responsiveness to guidance cues that promote growth. For example, several factors that promote axonal growth (e.g., the neurotrophins and the fibroblast growth factors) do so by activating tyrosine kinase receptors that have been shown to couple to PI 3-kinase–dependent pathways (Torres et al., 1999; Hadari et al., 2001; Huang and Reichardt, 2001; Ong et al., 2001) and would thereby be expected to inhibit GSK-3 activity. An alternative possibility is that growth cone responsiveness to inhibitory guidance cues might depend on GSK-3 activity. Sema 3A is an inhibitory guidance cue that restricts axonal extension to permissive areas by demarcating inhibitory territories (Luo et al., 1993; Messersmith et al., 1995). A “hallmark” of Sema 3A activity is its ability to induce a very rapid collapse of growth cones, a response that initially involves depolymerization and/or redistribution of F-actin at the leading edge of the growth cone (Fan et al., 1993; Fournier et al., 2000). However, if growth cones are treated with Sema 3A for a relatively short time (2 min) the redistribution of signaling components within growth cones can be assessed in the absence of a full collapse response (Fournier et al., 2000). Treatment with a Sema 3A–Fc chimera (Eickholt et al., 1997) under these conditions has no obvious effect on the distribution of GSK-3 protein within the partially collapsed growth cones (Fig. 3 A). In contrast, the short treatment with Sema 3A induces a dramatic loss of phosphorylation of both GSK-3α and GSK-β at the periphery of the growth cone (Fig. 3, B and C). Western blotting using the phospho–GSK-3 antibodies on cell lysates obtained from untreated primary DRG cultures and Sema 3A–treated cultures revealed that the total level of P–GSK-3 was in some experiments decreased (unpublished data). However, these results were not robust, and this might be due to the effects on the small pool of GSK-3 at the leading edge of the growth cone being masked by little or no change in the larger pool present in other neuronal compartments including, for example, the axon.


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)

Sema 3A activates GSK-3 at the leading edge of sensory growth cones. DRG cultures grown overnight were treated with a Sema 3A–Fc chimera for 2 min before fixation. Growth cones were stained with phalloidin–Texas red (left) and anti–GSK-3α (A, right) and with phalloidin–Texas red (left) and anti–P-(Ser21)-GSK-3α (B, right). After exposure to Sema 3A, the entire GSK-3α pool remains homogeneously distributed throughout the growth cone and is still detectable in filopodia, whereas the inactive P-(Ser21)-GSK-3α is depleted at the leading edge of the growth cone and filopodia (arrowheads). The insert shows the parallel performed P-(Ser21)-GSK-3α staining in untreated control cultures. Bars, 15 μm. (C) Percentage of growth cones showing enriched staining at the leading edge before and after treatment with Sema 3A–Fc. Bars show ± SEM.
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Related In: Results  -  Collection

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fig3: Sema 3A activates GSK-3 at the leading edge of sensory growth cones. DRG cultures grown overnight were treated with a Sema 3A–Fc chimera for 2 min before fixation. Growth cones were stained with phalloidin–Texas red (left) and anti–GSK-3α (A, right) and with phalloidin–Texas red (left) and anti–P-(Ser21)-GSK-3α (B, right). After exposure to Sema 3A, the entire GSK-3α pool remains homogeneously distributed throughout the growth cone and is still detectable in filopodia, whereas the inactive P-(Ser21)-GSK-3α is depleted at the leading edge of the growth cone and filopodia (arrowheads). The insert shows the parallel performed P-(Ser21)-GSK-3α staining in untreated control cultures. Bars, 15 μm. (C) Percentage of growth cones showing enriched staining at the leading edge before and after treatment with Sema 3A–Fc. Bars show ± SEM.
Mentions: The specific localization of an inactive pool of GSK-3 at the leading edge of the growth cone suggests a function in the control of growth cones motility. However it seems highly unlikely GSK-3 activity is required for responsiveness to guidance cues that promote growth. For example, several factors that promote axonal growth (e.g., the neurotrophins and the fibroblast growth factors) do so by activating tyrosine kinase receptors that have been shown to couple to PI 3-kinase–dependent pathways (Torres et al., 1999; Hadari et al., 2001; Huang and Reichardt, 2001; Ong et al., 2001) and would thereby be expected to inhibit GSK-3 activity. An alternative possibility is that growth cone responsiveness to inhibitory guidance cues might depend on GSK-3 activity. Sema 3A is an inhibitory guidance cue that restricts axonal extension to permissive areas by demarcating inhibitory territories (Luo et al., 1993; Messersmith et al., 1995). A “hallmark” of Sema 3A activity is its ability to induce a very rapid collapse of growth cones, a response that initially involves depolymerization and/or redistribution of F-actin at the leading edge of the growth cone (Fan et al., 1993; Fournier et al., 2000). However, if growth cones are treated with Sema 3A for a relatively short time (2 min) the redistribution of signaling components within growth cones can be assessed in the absence of a full collapse response (Fournier et al., 2000). Treatment with a Sema 3A–Fc chimera (Eickholt et al., 1997) under these conditions has no obvious effect on the distribution of GSK-3 protein within the partially collapsed growth cones (Fig. 3 A). In contrast, the short treatment with Sema 3A induces a dramatic loss of phosphorylation of both GSK-3α and GSK-β at the periphery of the growth cone (Fig. 3, B and C). Western blotting using the phospho–GSK-3 antibodies on cell lysates obtained from untreated primary DRG cultures and Sema 3A–treated cultures revealed that the total level of P–GSK-3 was in some experiments decreased (unpublished data). However, these results were not robust, and this might be due to the effects on the small pool of GSK-3 at the leading edge of the growth cone being masked by little or no change in the larger pool present in other neuronal compartments including, for example, the axon.

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