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First published online 4 July 2006
doi: 10.1242/jcs.03043

Journal of Cell Science 119, 3067-3077 (2006)
Published by The Company of Biologists 2006
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Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase

Mayumi Uchida1, Atsushi Enomoto1, Toshifumi Fukuda2, Kei Kurokawa3, Kengo Maeda4, Yoshinori Kodama5, Naoya Asai1, Taisaku Hasegawa1, Yohei Shimono1, Mayumi Jijiwa1, Masatoshi Ichihara6, Yoshiki Murakumo1 and Masahide Takahashi1,7,*

1 Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
2 Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan
3 Department of Pathology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
4 Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
5 Division of Surgical Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
6 Department of Medical Technology, Nagoya University of Health Sciences, Higashi-ku, Nagoya 461-8673, Japan
7 Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan


Figure 1
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  		Fig. 1. Knockdown of Dok-4 suppresses GDNF-dependent neurite outgrowth in TGW neuroblastoma cells. (A) (a) Expression of Dok-4 in TGW cells. Lysates from human cell lines were analyzed by western blotting with anti-Dok-4 polyclonal antibody under reducing conditions. HEK (human embryonic kidney), HeLa (human cervical adenocarcinoma), TGW (human neuroblastoma), SK-N-SH (human neuroblastoma), SK-N-MC (human primitive neuroectodermal tumor) and TT (human medullary thyroid carcinoma) cell lines were used. Lysate from HEK293 cells transfected with Dok-4 cDNA was used as a positive control. (b) Depletion of Dok-4 in TGW cells by siRNA. Total cell extracts from control siRNA- and Dok-4 siRNA-transfected TGW cells were subjected to western blot analysis with anti-Dok-4 and anti-Shc antibodies. (B) (a) TGW cells were transfected with control (left panel) or Dok-4 (right panel) siRNA, fixed 72 hours after transfection, and then stained with anti-Dok-4 antibody. Arrowheads denote the localization of Dok-4 to the plasma membrane. (b) TGW cells were serum-starved, incubated with or without GDNF (50 ng/ml) for 48 hours, and then stained with anti-Dok-4 antibody. Boxes 1 and 2 denote a shaft and a tip of an extended neurite which was positively stained with anti-Dok-4 antibody, respectively. (C) Depletion of Dok-4 attenuates neurite outgrowth by TGW cells in response to GDNF. (a) The target sequence of Dok-4 siRNA and the nucleotide substitutions used to generate siRNA-resistant (siRNAr) Dok-4 are indicated. (b) HEK293 cells were transfected with control or Dok-4 siRNA together with Dok4-V5 or siRNAr-Dok4-V5. Dok-4 expression was monitored by western blot analysis. (c) TGW cells transfected with siRNA were serum-starved, incubated with or without GDNF for 48 hours and then visualized by microscopy. TGW cells transfected with both siRNA and siRNAr-Dok4-V5 were also analyzed. (d) Mean of the longest neurite was determined for each culture from measurements of 100 neurons for three different experiments. Each data point represents the mean ± s.e.m. (*P<0.05).

 

Figure 2
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  		Fig. 2. Expression of Dok-4 induces neurite outgrowth in TGW cells. (A) Neurite outgrowth of TGW cells transfected with Dok-4. (a) Expression of Dok-4V5 was monitored by western blot analysis. (b) TGW cells stably expressing Dok-4 [TGW(Dok-4)] or parental TGW cells (TGW) were serum-starved and incubated for 48 hours with or without GDNF (50 ng/ml) in the presence or absence of DMSO (0.1%), PD98059 (18 µM), LY294002 (15 µM) or SP600125 (20 µM). (B) Mean of the longest neurite was determined for each culture from measurements of 100 neurons for three different experiments. Each data point represents the mean ± s.e.m. (*P<0.05).

 

Figure 3
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  		Fig. 3. Dok-4 mediates sustained ERK and AKT activation in the GDNF/RET signaling pathway. TGW cells, TGW cells stably expressing Dok-4 [TGW(Dok-4)], or Dok-4 knockdown cells [TGW(Dok-4 siRNA)] were serum-starved and stimulated with GDNF (50 ng/ml) for the indicated times, and cell extracts subjected to western blot analysis with the indicated antibodies.

 

Figure 4
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  		Fig. 4. Conserved tyrosine residues in Dok-4 are important for sustained ERK and AKT activation. (A) (a) Schematic representation of Dok-4, -5 and -6 structures. Y indicates tyrosine residues, and the tyrosine residues conserved among Dok-4-6 are indicated in bold. (b) Y187, Y220 and Y279 are conserved across a wide range of species. (B) (a) Each mutant was stably expressed in TGW cells as a V5 epitope-tagged protein. Dok-4 was immunoprecipitated with an anti-V5 antibody after GDNF stimulation, followed by immunoblotting with anti-V5 or anti-phosphotyrosine antibody. (b) TGW cells and TGW cells stably expressing each mutant were stimulated with GDNF (50 ng/ml) for the indicated times, and cell extracts subjected to western blot analysis with the indicated antibodies.

 

Figure 5
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  		Fig. 5. The conserved tyrosine residues in Dok-4 are important for GDNF-dependent neurite outgrowth in TGW cells. (A) TGW cells and TGW cells stably expressing each mutant were serum-starved and stimulated with GDNF (50 ng/ml) for 48 hours. (B) Mean of the longest neurite was determined for each culture from measurements of 100 neurons for three different experiments. Each data point represents the mean ± s.e.m.

 

Figure 6
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  		Fig. 6. Dok-4 activates Rap1 that promotes neurite outgrowth in TGW cells. (A) Rap1 and Ras activation in TGW cells expressing Dok-4. Subconfluent TGW cells, TGW cells expressing Dok-4 wild-type or Y220F mutant, or Dok-4 knockdown cells were stimulated with GDNF (50 ng/ml) for the indicated times, thoroughly washed, and lysed. Lysates were clarified and incubated at 4°C with GST-RalGDS RBD (a) or GST-Raf-1 RBD (b) fusion proteins for 1 hour. Beads were washed, SDS sample buffer added, and the proteins subjected to western blot analysis for Rap1 (a) or Ras (b). (B) Rap1 induces neurite outgrowth downstream of Dok-4 in TGW cells. (a) TGW cells or Dok-4 knockdown cells were transfected with either GFP or GFP-RapV12 and incubated for 48 hours. (b) Mean of the longest neurite was determined for each culture from measurements of 50 neurons for three different experiments. Each data point represents the mean ± s.e.m.

 

Figure 7
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  		Fig. 7. Dominant negative Rap1 (RapN17) or Rap1GAP impairs neurite outgrowth and sustained ERK activation (A,B) (a) TGW cells (A) or TGW(Dok-4) cells (B) were transfected with GFP, GFP-RapGAP (SPA-1) or GFP-RapN17 and incubated for 48 hours with or without GDNF. (b) Mean of the longest neurite was determined for each culture from measurements of 50 neurons for three different experiments. Each data point represents the mean ± s.e.m. (C) TGW(Dok-4) cells were transfected with GFP, GFP-RapGAP (SPA-1) or GFP-RapN17, serum-starved, and stimulated with GDNF (50 ng/ml) for the indicated times, and cell extracts subjected to western blot analysis with the anti-ERK or anti-phosphoERK antibody.

 

Figure 8
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  		Fig. 8. Dok-4 induces neurite outgrowth in rat hippocampal neurons. (A) Expression of rat Dok-4 and RET in rat hippocampal neurons. Total RNA was isolated from PC12 cells and rat hippocampal neurons, and RT-PCR performed using rat Dok-4-and RET-specific primers as described in Materials and Methods. ß-actin was amplified as a control. (B) Hippocampal neurons were transfected with GFP, GFP-Dok-4 wild-type, GFP-Dok-4 Y220F mutant, GFP-RapGAP (SPA-1) or GFP-RapN17, incubated for 72 hours, and visualized by confocal microscopy. Boxes denote dendrites of neurons identified by MAP2 expression (lower panels). (C) Hippocampal neurons transfected with the indicated constructs were incubated for 72 hours with or without GDNF (50 ng/ml), and dendrite length quantified. Mean of the longest dendrite was determined for each culture from measurements of 50 hippocampal neurons for three different experiments. Each data point represents the mean ± s.e.m.

 

Figure 9
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  		Fig. 9. A proposed model for the involvement of Dok-4 in GDNF/RET signaling. After activation of RET by GDNF, the tyrosine at position 1062 (Y1062) in RET is autophosphorylated, which then recruits Shc or Dok4 adaptor proteins. In one pathway, phosphorylated Shc stimulates the Grb2/SOS/Ras/ERK cascade, thereby inducing transient activation of ERK. Alternatively, activation of Dok-4 leads to increased GTP-bound Rap1 levels, which induces sustained activation of ERK and neuronal differentiation including neurite outgrowth.

 

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© The Company of Biologists Ltd 2006