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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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GSK-3 is maintained inactive at the leading edge of migratory cells and colocalizes with F-actin. Distribution of P-(Ser9)-GSK-3β immunoreactivity in MDA-MB-231 breast carcinoma cells (A, left) and in primary chick fibroblast (B, left). A parallel-performed phalloidin staining (middle) reveals a great overlap of inactive serine-phosphorylated GSK-3 with F-actin (right, merge). Likewise, in DRG growth cones the signals detected using an anti–P-(Ser21)-GSK-3α antibody (C) and an anti–P-(Ser9)-GSK-3β (D) are found colocalized with F-actin in the filopodia and at the leading edge of the lamellipodia. Stainings performed using a pan GSK-3α (E) and a P-(Y)-GSK-3 (F) antibody reveal that GSK-3 in present throughout the entire growth cone structure. Bars, 15 μm. (G) Western blots probed with indicated P-(Ser)–specific antibodies: lanes 1 and 3 show chick brain lysate, and lanes 2 and 4 show Cos-7 cell lysate that have been transfected with GSK-3α and GSK-3β, respectively.
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fig1: GSK-3 is maintained inactive at the leading edge of migratory cells and colocalizes with F-actin. Distribution of P-(Ser9)-GSK-3β immunoreactivity in MDA-MB-231 breast carcinoma cells (A, left) and in primary chick fibroblast (B, left). A parallel-performed phalloidin staining (middle) reveals a great overlap of inactive serine-phosphorylated GSK-3 with F-actin (right, merge). Likewise, in DRG growth cones the signals detected using an anti–P-(Ser21)-GSK-3α antibody (C) and an anti–P-(Ser9)-GSK-3β (D) are found colocalized with F-actin in the filopodia and at the leading edge of the lamellipodia. Stainings performed using a pan GSK-3α (E) and a P-(Y)-GSK-3 (F) antibody reveal that GSK-3 in present throughout the entire growth cone structure. Bars, 15 μm. (G) Western blots probed with indicated P-(Ser)–specific antibodies: lanes 1 and 3 show chick brain lysate, and lanes 2 and 4 show Cos-7 cell lysate that have been transfected with GSK-3α and GSK-3β, respectively.

Mentions: We have used antibodies that specifically recognize phosphorylated Ser9 and phosphorylated Ser21 in GSK-3β and GSK-3α to examine the distribution of the inactive pools of GSK-3 in cells. In highly migratory MDA-MB-231 breast cancer cells, an inactive pool of GSK-3β is found localized at the leading edge of the cells alongside F-actin (Fig. 1 A). Likewise, inactive GSK-3β colocalizes with F-actin in primary chick fibroblasts (Fig. 1 B). In both cases, inactive GSK-3α showed a similar distribution (unpublished data). In the developing embryo, GSK-3 is highly enriched in the nervous system. Interestingly, inactive pools of both GSK-3α and GSK-3β are found enriched in the filopodia and at the leading edge of the lamellipodia of growth cone extending from an E7 chick dorsal root ganglion (DRG) explants where they again colocalize with F-actin (Fig. 1, C and D). These compartmentalizations were apparent in essentially all growth cones (92 + 2.8% and 94.8 + 2% of growth cones for P-GSK-3α and P-GSK-3β, respectively, n = 4 explants, mean + SEM) and demonstrate that inactive pools of GSK-3 are preferentially enriched in motile regions of growth cones and possibly other 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)

GSK-3 is maintained inactive at the leading edge of migratory cells and colocalizes with F-actin. Distribution of P-(Ser9)-GSK-3β immunoreactivity in MDA-MB-231 breast carcinoma cells (A, left) and in primary chick fibroblast (B, left). A parallel-performed phalloidin staining (middle) reveals a great overlap of inactive serine-phosphorylated GSK-3 with F-actin (right, merge). Likewise, in DRG growth cones the signals detected using an anti–P-(Ser21)-GSK-3α antibody (C) and an anti–P-(Ser9)-GSK-3β (D) are found colocalized with F-actin in the filopodia and at the leading edge of the lamellipodia. Stainings performed using a pan GSK-3α (E) and a P-(Y)-GSK-3 (F) antibody reveal that GSK-3 in present throughout the entire growth cone structure. Bars, 15 μm. (G) Western blots probed with indicated P-(Ser)–specific antibodies: lanes 1 and 3 show chick brain lysate, and lanes 2 and 4 show Cos-7 cell lysate that have been transfected with GSK-3α and GSK-3β, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199247&req=5

fig1: GSK-3 is maintained inactive at the leading edge of migratory cells and colocalizes with F-actin. Distribution of P-(Ser9)-GSK-3β immunoreactivity in MDA-MB-231 breast carcinoma cells (A, left) and in primary chick fibroblast (B, left). A parallel-performed phalloidin staining (middle) reveals a great overlap of inactive serine-phosphorylated GSK-3 with F-actin (right, merge). Likewise, in DRG growth cones the signals detected using an anti–P-(Ser21)-GSK-3α antibody (C) and an anti–P-(Ser9)-GSK-3β (D) are found colocalized with F-actin in the filopodia and at the leading edge of the lamellipodia. Stainings performed using a pan GSK-3α (E) and a P-(Y)-GSK-3 (F) antibody reveal that GSK-3 in present throughout the entire growth cone structure. Bars, 15 μm. (G) Western blots probed with indicated P-(Ser)–specific antibodies: lanes 1 and 3 show chick brain lysate, and lanes 2 and 4 show Cos-7 cell lysate that have been transfected with GSK-3α and GSK-3β, respectively.
Mentions: We have used antibodies that specifically recognize phosphorylated Ser9 and phosphorylated Ser21 in GSK-3β and GSK-3α to examine the distribution of the inactive pools of GSK-3 in cells. In highly migratory MDA-MB-231 breast cancer cells, an inactive pool of GSK-3β is found localized at the leading edge of the cells alongside F-actin (Fig. 1 A). Likewise, inactive GSK-3β colocalizes with F-actin in primary chick fibroblasts (Fig. 1 B). In both cases, inactive GSK-3α showed a similar distribution (unpublished data). In the developing embryo, GSK-3 is highly enriched in the nervous system. Interestingly, inactive pools of both GSK-3α and GSK-3β are found enriched in the filopodia and at the leading edge of the lamellipodia of growth cone extending from an E7 chick dorsal root ganglion (DRG) explants where they again colocalize with F-actin (Fig. 1, C and D). These compartmentalizations were apparent in essentially all growth cones (92 + 2.8% and 94.8 + 2% of growth cones for P-GSK-3α and P-GSK-3β, respectively, n = 4 explants, mean + SEM) and demonstrate that inactive pools of GSK-3 are preferentially enriched in motile regions of growth cones and possibly other 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