Report |
Address correspondence to Britta J. Eickholt, Molecular Neurobiology Group, MRC Centre for Developmental Biology, King's College London, London SE1 1UL, UK. Tel.: 44-20-7848-6812. Fax: 44-20-7848-6816. E-mail: Britta.J.Eickholt{at}kcl.ac.uk
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Abstract |
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Key Words: GSK-3; axon guidance; actin; Semaphorin 3A; growth cone
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Introduction |
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Glycogen synthase kinase (GSK)*-3 is an evolutionary conserved serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades (Siegfried et al., 1992; He et al., 1995; Pierce and Kimelman, 1995; Pap and Cooper, 1998). Two genes encode the closely related proteins GSK-3 and GSK-3ß, and both forms are widely distributed with highest levels found in the developing brain (Woodgett, 1990; Lau et al., 1999). A function for GSK-3 in the establishment of functional circuits in the nervous system can be postulated based on its developmental expression in axonal tracts. The steady increase in expression from embryonic stages to early postnatal development in rat brains with a decrease in expression at P20 to low levels broadly correlates with the period of axonal extension and dendritic plasticity (Takahashi et al., 1994, 2000; Leroy and Brion, 1999).
Our understanding of the function of GSK-3 in relationship to axonal growth and guidance is poor and has been limited by the lack of specific antagonists of the enzyme. In the present study, we have used phosphospecific antibodies to reveal the presence of an inactive pool of GSK-3 at the leading edge of the navigating growth cone and migratory cells. We have used LiCl and two specific GSK-3 inhibitors to investigate the function of the enzyme in the growth cone (Coghlan et al., 2000; Cross et al., 2001). Our results provide evidence that GSK-3 plays a crucial role in allowing the growth cone to respond to an inhibitory guidance cue. We show that after treatment with the inhibitory guidance molecule Semaphorin 3A (Sema 3A; Luo et al., 1993) the inactive pool of GSK-3 is rapidly lost from the leading edge of the growth cone, and that the three different GSK-3 antagonists 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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Results and discussion |
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We have used antibodies that specifically recognize phosphorylated Ser9 and phosphorylated Ser21 in GSK-3ß and GSK-3 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-3ß is found localized at the leading edge of the cells alongside F-actin (Fig. 1 A). Likewise, inactive GSK-3ß colocalizes with F-actin in primary chick fibroblasts (Fig. 1 B). In both cases, inactive GSK-3
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-3
and GSK-3ß 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-3
and P-GSK-3ß, 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.
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The phosphatidylinositol (PI) 3-kinase pathway is one of the major pathways that inactivates GSK-3 by stimulating a PKB/Akt-dependent phosphorylation of Ser21 and/or Ser9 (Cross et al., 1995). In primary DRG neurons, treatment with two selective PI 3-kinase inhibitors (wortmannin and LY294002) induces a dramatic reduction in the phosphorylation of GSK-3 on Ser21 and GSK-3ß on Ser9 as determined by Western blotting (Fig. 2 A) and immunocytochemistry (Fig. 2 B). These results demonstrate that under our culture conditions PI 3-kinase activity is required for inactivating GSK-3 in the growth cones of primary neurons. It is also interesting to note that although PI 3-kinase inhibition by wortmannin does not result in a collapse of the growth cone, it reduces its outspread morphology and appears to alter the appearance of the actin filaments (Fig. 2 C).
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Inhibition of GSK-3 masks the biological activity of Sema 3A
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-3 and GSK-3ß (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-3
and GSK-3ß 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.
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The presence of a pool of GSK-3 that is specifically maintained in an inactive state leads to the immediate issue as to how this phosphorylation is controlled. In principle, this could reflect a relative increase in the activity of the enzymes that phosphorylate GSK-3 within this region of the growth cone or a decrease in the activity of phosphatases. It is also possible that the inactive and active forms of GSK-3 have different binding partners and that the phosphorylated GSK-3 might selectively interact with F-actin or an F-actin binding protein. Our results show that at least in neurons the inactivation of both GSK-3 and GSK-3ß is under the control of the PI 3-kinase pathway. Since an increased activity of PI 3-kinase at the leading edge in several cell types has been linked to chemotactic migratory responses (Meili et al., 1999; Parent and Devreotes, 1999; Servant et al., 2000), inactive GSK-3 may be the common link by which PI 3-kinase keeps the growth cone in a "motile" state.
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Materials and methods |
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Cell culture and collapse assays
Fertile eggs were obtained from a local supplier (Needle farm). DRG explants cultures from E7 chick embryos were prepared as described previously (Eickholt et al., 1997). For immunohistochemistry, DRG explants were plated onto poly-L-Lysine (20 µg/ml)/laminin (20 µg/ml)coated glass coverslips, and cultures were incubated for 20 h in DME/10% FCS/penicillin/streptomycin supplemented with 20 ng/ml NGF before fixation in 4% paraformaldhyde/10% sucrose. For collapse assays, DRGs were cultured for 24 h on 20 µg/ml laminin-coated Labtec chamberslides (Nunc). LiCl (Sigma-Aldrich) and specific GSK-3 inhibitors were applied at given concentrations and incubated for 1 h before the Sema 3AFc was applied (at 1 µg/ml). After 30 min, the cultures were carefully fixed in 4% paraformaldehyde/10% sucrose.
For cultures of isolated neurons, E7 DRGs were incubated in 1 mg/ml trypsin (Worthington) diluted in Hanks' buffer for 10 min at 37°C. The trypsin solution was removed, and explants were triturated in DME/10% FCS using a fire-polished Pasteur pipette. Isolated cells were preplated in DME/10% FCS for 2 h on tissue culture plastic in order to enrich for neurons, which were cultured on laminin (20 µg/ml) as before. MDA-MB-231 cells (American Type Culture Collection) were maintained in DME/10% FCS/PenStrep.
Immunocytochemical procedures
PFA-fixed DRG explants were washed twice with PBS, permeablized in PBS/1% Triton X-100, and blocked in blocking buffer (PBS, 0.5% Triton, 2% BSA). Primary antibody was applied (all antibodies were diluted 1:50 in blocking buffer except anti-P(Y279/216)-GSK-3 antibody that was diluted 1:100) and incubated overnight at 4°C with agitation. Bound antibody was visualized using FITC-conjugated secondary antibodies (Sigma-Aldrich; antisheep antibody was from Dako). The distribution of F-actin was visualized using Texas redconjugated phalloidin (Molecular Probes). All samples were analyzed using volume deconvolution.
Cell/tissue lysates and Western blot analysis
Cos-7 cells were transfected using Lipofectamine Plus reagents according to the manufacturer's protocol. Transfected cells were washed with ice-cold PBS and lysed in lysis buffer (20 mM Hepes, 150 mM NaCl, 1% Triton, 5 mM CaCl2, 1 mM MgCl2, protease and phosphatase inhibitors). Brain lysate from E9 chick brains and lysates of primary DRG neurons were prepared using the same lysis conditions. MDA-MB-231 cells were stimulated with Sema 3AFc (1 µg/ml) for 1 h and washed on ice with ice-cold PBS. LiCl was applied at 20 mM for 30 min before the Sema 3AFc was added. Cells were lysed in lysis buffer (10 mM Tris, pH 8, 0.25 sucrose, 0.5% NP-40, 10 mM MgCl2, 0.1 mM DTT, protease and phosphatase inhibitors). After removing insoluble material by centrifugation, protein concentration was determined using Bio-Rad Laboratories protein assay, and protein extracts (40 µg per lane) were separated by SDS-PAGE (10%) and transferred onto nitrocellulose. Bound proteins were detected by Western blotting. All primary antibodies were used at 1:1,000. Secondary antibodies were purchased from Vector Laboratories and used at 1:5,000.
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Footnotes |
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Acknowledgments |
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The work was supported in part by a King's College London Inter-Disciplinary Research Group fellowship to B.J. Eickholt and the Welcome Trust.
Submitted: 22 January 2002
Revised: 13 March 2002
Accepted: 13 March 2002
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