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First published online 29 July 2008
doi: 10.1242/jcs.029660

Journal of Cell Science 121, 2718-2730 (2008)
Published by The Company of Biologists 2008
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Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential β-catenin phosphorylation

Monica D. David1,*, Andrée Yeramian1,*, Mireia Duñach2, Marta Llovera1, Carles Cantí1, Antonio García de Herreros3, Joan X. Comella1,{ddagger} and Judit Herreros1,§

1 Laboratori d'Investigació, Hospital Universitari Arnau de Vilanova, Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, IRBLleida, Spain
2 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain
3 Programa de Recerca en Càncer, IMIM-Hospital del Mar, Parc de Recerca Biomèdica de Barcelona, Spain


Figure 1
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  		Fig. 1. β-catenin interacts with TrkA in PC12 6/15 cells, and with TrkB and TrkC in hippocampal neurons. (A) Control (Myc) or β-catenin immunoprecipitation from PC12 6/15 cells starved or stimulated with NGF (100 ng/ml) for different times (as indicated) shows that TrkA (detected with anti-pan-Trk antibodies: 140-kDa and 110-kDa forms) co-immunoprecipitates with β-catenin, independent of its phosphorylation state. (B) Control (GFP) or pan-Trk immunoprecipitation from hippocampal neurons untreated or treated with BDNF or NT-3 (50 ng/ml; 5 minutes) shows that TrkB and TrkC co-immunoprecipitate with β-catenin.

 

Figure 2
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  		Fig. 2. Trk phosphorylates β-catenin at Y654. (A) In vitro kinase assay using recombinant TrkA kinase and WT or mutant GST-β-catenin. Anti-phospho-Tyr (anti-Tyr-P) western blot shows phosphorylated TrkA kinase and GST–β-catenin. WT GST–β-catenin is phosphorylated by TrkA kinase upon addition of ATP, whereas K252a inhibits TrkA-induced β-catenin phosphorylation. Phosphorylation of mutant Y654F and Y142F GST–β-catenin by TrkA kinase is ~45% and 15% lower than that of WT GST–β-catenin, respectively. The panel below shows the anti-β-catenin western blot for the GST–β-catenin input in the kinase assay (0.8 µg). (B) In vitro kinase assay using TrkA immunoprecipitated from PC12 6/15 cells, and recombinant WT and Y654F β-catenin. Anti-Tyr-P western blot shows TrkA (140-kDa and 110-kDa bands) and the tyrosine phosphorylation of β-catenin. Phosphorylation of WT β-catenin is decreased by preincubation with K252a (100 nM), whereas phosphorylation of Y654F β-catenin is lower than that of WT β-catenin and is not inhibited by K252a. In the first lane, no recombinant β-catenin was added to control for phosphorylation of any endogenous β-catenin that might have been immunoprecipitated with TrkA. Quantification corresponds to % of the increase in the intensity of Tyr-P β-catenin relative to –ATP divided by total β-catenin. (C) Control (Myc) or β-catenin immunoprecipitation from PC12 6/15 cells untreated or treated with NGF (100 ng/ml) and pervanadate for the last 10 minutes of stimulation. Phosphorylation of β-catenin Y654 increases after NGF treatment in parallel with TrkA stimulation as detected by anti-Tyr-P western blot. (D) Control (His) or β-catenin immunoprecipitation from untreated hippocampal neurons or those treated for 10 minutes with 50 ng/ml BDNF or NT-3 in the presence of pervanadate. Upon NT stimulation, phosphorylation of β-catenin at Y654 increases parallel with phosphorylation of TrkB and/or TrkC detected by anti-phospho-Tyr western blot. (E) Control or β-catenin immunoprecipitation from hippocampal neurons untreated or treated for 10 minutes with 50 ng/ml BDNF with or without pervanadate. Level of Y654-P β-catenin increases with BDNF treatment both with and without pervanadate, whereas phosphorylation at Y142 is maintained at the basal level, increasing only slightly with pervanadate. TrkB and N-cadherin co-immunoprecipitate with β-catenin, and N-cadherin association with β-catenin shows a small decrease after pervanadate treatment. (F) β-catenin immunoprecipitation from untreated neurons or those treated with 50 ng/ml BDNF for the indicated times in the presence of pervanadate for the last 10 minutes of stimulation. The level of Y654-P β-catenin peaks at 1 hour and is reduced after overnight (o/n) stimulation. Quantifications in C-F correspond to % of the increase in the intensity of the phosphospecific β-catenin (Y654-P or Y142-P) band relative to the control normalized to total β-catenin.

 

Figure 3
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  		Fig. 3. Met phosphorylates β-catenin at Y142. (A) In vitro kinase assay with recombinant Met kinase and WT or mutant GST–β-catenin. Anti-phospho-Tyr (anti-Tyr-P) western blot shows phosphorylated Met kinase upon addition of ATP and its basal activation (–ATP condition). Anti-Y142-P-β-catenin western blot (top) indicates that WT and Y654F GST–β-catenin are phosphorylated by Met kinase at Y142, whereas phosphorylation of Y142F GST–β-catenin is as low as in basal condition (–ATP). (B) Control (His) and β-catenin immunoprecipitation from untreated hippocampal neurons or those treated with 50 ng/ml HGF for 10 minutes with or without pervanadate. The level of Y142-P β-catenin increases with HGF treatments, whereas phosphorylation at Y654 is not stimulated by HGF signalling. Met and {alpha}-catenin co-immunoprecipitate with β-catenin, and the recovery of both proteins decreases upon phosphorylation of β-catenin at Y142. (C) β-catenin immunoprecipitation from untreated neurons or those treated with 50 ng/ml HGF for the indicated times. The level of Y142-P β-catenin peaks at 10 minutes, is maintained at similar levels up to 1 hour and starts decaying slightly after overnight (o/n) stimulation. Quantifications in B and C correspond to % of the increase in the intensity of the phosphospecific β-catenin band relative to the control normalized to total β-catenin.

 

Figure 4
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  		Fig. 4. β-catenin is involved in axon growth downstream of BDNF- and HGF-signalling. (A) Western blot from hippocampal neurons infected with lentiviral-driven scrambled shRNA or β-catenin shRNA#1 or #2 shows the decrease in β-catenin expression following expression of β-catenin shRNAs compared with scrambled shRNA (scr) or non-infected (ni) neurons. Actin was used as a loading control. Plot represents quantification of β-catenin corrected against the loading control (n≥4 experiments). Panels show confocal images for anti-β-catenin and anti-GFP (indicating transduced neurons) immunostainings in neurons expressing scrambled shRNA or β-catenin shRNA#1. Note the lower levels of β-catenin especially along the axon in β-catenin-shRNA#1-expressing neurons. Arrows indicate colocalization between GFP and β-catenin immunostainings in neurons expressing scrambled shRNA that is reduced in neurons expressing β-catenin shRNA#1. Scale bar: 20 µm. (B) Hippocampal neurons expressing scrambled shRNA or β-catenin shRNA#1 fixed and immunostained against GFP (driven by the lentiviral vector) at 3 DIV show the decrease in axon length obtained by expressing β-catenin shRNA#1. Scale bar: 40 µm. (C) Quantification of axon length of neurons expressing scrambled or β-catenin shRNA, untreated or treated with BDNF or HGF (normalized to untreated neurons expressing scrambled shRNA). Neurons expressing β-catenin shRNA#1 or #2 display axons that are shorter than controls. Stimulation with BDNF or HGF (50 ng/ml) promotes axon growth in neurons expressing scrambled shRNA, but not in neurons expressing β-catenin shRNA#1 or #2 (n=4-8 experiments). **P≤0.01, ***P≤0.001 when compared with the untreated neurons expressing scrambled shRNA; #, P≤0.05; ##, P≤0.01 when compared with the BDNF- or HGF-treated neurons expressing scrambled shRNA. (D) Quantification of axon length in neurons expressing intracellular N-cadherin or β-catenin, either untreated or stimulated with BDNF or HGF (normalized to the respective untreated neurons). Neurons expressing β-catenin display axons longer than controls upon BDNF or HGF (50 ng/ml) stimulation. By contrast, in neurons expressing intracellular N-cadherin, BDNF or HGF cannot stimulate axon growth (n=4 experiments). *P≤0.05, ***P≤0.001 when compared with the corresponding untreated control; #, P≤0.05, ##, P≤0.01 when compared with the BDNF- or HGF-stimulated β-catenin-expressing neurons.

 

Figure 5
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  		Fig. 5. β-catenin phosphorylation at Y654 regulates axon growth and branching induced by NTs. (A) Hippocampal neurons transfected with EYFP alone (control) or with EYFP and WT or mutant (Y654F or Y142F) β-catenin. BDNF and NT-3 (50 ng/ml) induce axon growth and branching in control neurons and in neurons overexpressing WT or Y142F β-catenin, but NT-induced axon growth is inhibited in neurons expressing the mutation Y654F. Treatment with BDNF plus K252a results in axons with lengths and branching values similar to those of untreated neurons and of Y654F-expressing cells. Scale bar: 70 µm. (B) Quantification of axon length normalized to the respective control. BDNF and NT-3 increase axon length in control EYFP neurons and in neurons expressing WT or Y142F β-catenin, but not in neurons expressing the Y654F mutant. Treatment with BDNF together with NT-3 (B+NT3) does not result in a significant further increase. Treatment with K252a (100 nM) and BDNF (B+K252a) abolishes the increase in axon length induced by BDNF. (C) Quantification of axon branching (TABTN) shows that BDNF and NT-3 increase branching of axons expressing WT and Y142F β-catenin, but expression of Y654F β-catenin abolishes the branching induced by NTs. K252a (100 nM) together with BDNF abolishes BDNF-promoted branching (*P≤0.05, **P≤0.01, ***P≤0.001; n=3-8 experiments).

 

Figure 6
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  		Fig. 6. β-catenin phosphorylation at Y142 regulates axon growth induced by HGF. (A) Hippocampal neurons transfected with EYFP alone (control) or with EYFP and WT or mutant (Y654F or Y142F) β-catenin. HGF (50 ng/ml) induces axon growth in neurons expressing WT or Y654F β-catenin, but HGF-induced axon growth is inhibited in neurons expressing the mutation Y142F. Scale bar: 40 µm. (B) Quantification of axon length normalized to the respective control shows that HGF increases axon length in neurons expressing WT or Y654F β-catenin, but not in neurons expressing Y142F. (C) Quantification of axon branching shows that HGF treatment induces axon branching that is blocked by the Y142F mutation (*P≤0.05, **P≤0.01; n=4-7 experiments).

 

Figure 7
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  		Fig. 7. Y654-P β-catenin colocalizes with the neuronal cytoskeleton and Y142-P β-catenin is found in the nucleus. (A) Double immunostaining for endogenous β-catenin and F-actin (phalloidin) in hippocampal neurons shows that β-catenin and F-actin partially colocalize (arrows) at neurite tips and lamella (inset). (B) Confocal image of hippocampal neurons fixed using detergent fixation shows the double immunostaining for Y654-P β-catenin and βIII-tubulin that colocalize at enlarged growth cones where microtubules unbundle and along the axon (arrows). Y654-P β-catenin immunostaining can also be observed along filopodial-like processes and axonal-spread areas devoid of βIII-tubulin (arrowheads). (C) Confocal image of neurons fixed by detergent fixation shows the double immunostaining for Y654-P β-catenin and F-actin (phalloidin). Similar to anti-β-catenin antibodies, anti-Y654-P-β-catenin antibodies stain the cell body and neurite tips, where Y654-P β-catenin and F-actin colocalize (arrows). (D) β-catenin and Y142-P β-catenin colocalize within the cytoplasm of neurons (arrows), whereas colocalization of Hoestch and anti-Y142-P-β-catenin immunostaining shows that Y142-P β-catenin is found in the nucleus (arrowheads). (E) Anti-Y654-P-β-catenin antibodies stain the cell membrane at cell contacts and the cytoplasm, but the nucleus (stained by Hoestch) is negative. Scale bars: 15 µm (A), 20 µm (B,C) and 45 µm (D,E).

 

Figure 8
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  		Fig. 8. {Delta}N-TCF4 abolishes the effect of HGF in axon growth and branching. (A) Hippocampal neurons transfected with EYFP and {Delta}N-TCF4, untreated or treated with BDNF, NT-3 and HGF (50 ng/ml). {Delta}N-TCF4 abolishes the axon growth induced by HGF, but not by BDNF or NT-3. Scale bar: 40 µm. (B) Quantification of axon length in neurons expressing EYFP or EYFP plus {Delta}N-TCF4 normalized to the respective untreated control shows that HGF and Wnt3a (used as positive control; 50 ng/ml) do not increase axon length in neurons expressing {Delta}N-TCF4, whereas NTs similarly stimulate axon growth in {Delta}N-TCF4-expressing or control neurons. (C) Quantification of axon branching in neurons expressing EYFP alone or EYFP and {Delta}N-TCF4 shows that HGF-induced branching is inhibited by {Delta}N-TCF4 expression, whereas the BDNF- and NT-3-induced effects are not affected (n=5-7 experiments). *P≤0.05, **P≤0.01, ***P≤0.001, when compared with the corresponding untreated controls. #, P≤0.05; ##, P≤0.01; ###, P≤0.001, when compared with the corresponding stimulated control.

 

Figure 9
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  		Fig. 9. Model for the role of β-catenin tyrosine phosphorylation in axon growth and branching. Adhesion complexes are assembled at the plasma membrane of the cell body, axon and growth cone. β-catenin residue Y654 is found at the region that binds to N-cadherin, whereas β-catenin Y142 is in the {alpha}-catenin-binding region (Aberle et al., 1996Go; Lilien and Balsamo, 2005Go). {alpha}-catenin also binds to actin (Drees et al., 2005Go; Yamada et al., 2005Go). Trk and Met associate with a pool of β-catenin that is bound to N-cadherin and {alpha}-catenin. β-catenin phosphorylation by HGF-Met signalling at β-catenin Y142 targets β-catenin to the nucleus, where it activates TCF4-mediated transcription to increase axon growth and branching. The detachment of Y142-P β-catenin from the adhesion complex might imply dynamic regulation of Y654 phosphorylation, perhaps by Src-family tyrosine kinases (Roura et al., 1999Go), and its dephosphorylation before nuclear translocation of Y142-P β-catenin (according to the lack of nuclear immunostaining for Y654-P β-catenin). β-catenin phosphorylation by NT-Trk signalling at Y654 detaches β-catenin from N-cadherin (Bamji et al., 2006Go; Huber and Weis, 2001Go; Lilien and Balsamo, 2005Go; Roura et al., 1999Go), and Y654-P β-catenin associates with actin and microtubules at the growth cone, possibly regulating the cytoskeleton to promote axon growth and branching. Note that, whereas we demonstrate that β-catenin–{alpha}-catenin dissociates upon phosphorylation of β-catenin Y142, the depicted β-catenin dissociation from N-cadherin upon Y654 phosphorylation is based on the references above. For simplicity, Met has been located at the cell body and Trk at the growth-cone plasma membrane. N, N-cadherin; β, β-catenin; {alpha}, {alpha}-catenin; ––, F-actin; ....., microtubules.

 

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© The Company of Biologists Ltd 2008