Dock3 regulates BDNF-TrkB signaling for neurite outgrowth by forming a ternary complex with Elmo and RhoG

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1 Dock3 regulates BDNF-TrkB signaling for neurite outgrowth by forming a ternary complex with Elmo and RhoG Kazuhiko Namekata, Hayaki Watanabe, Xiaoli Guo, Daiji Kittaka, Kazuto Kawamura, Atsuko Kimura, Chikako Harada and Takayuki Harada* Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo , Japan Dock3, a new member of the guanine nucleotide exchange factor family, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes neurite outgrowth through the formation of a protein complex with Fyn and WAVE downstream of brain-derived neurotrophic factor (BDNF) signaling. Here, we report a novel Dock3-mediated BDNF pathway for neurite outgrowth. We show that Dock3 forms a complex with Elmo and activated RhoG downstream of BDNF-TrkB signaling and induces neurite outgrowth via Rac1 activation in PC12 cells. We also show the importance of Dock3 phosphorylation in Rac1 activation and show two key events that are necessary for efficient Dock3 phosphorylation: membrane recruitment of Dock3 and interaction of Dock3 with Elmo. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the central nervous system where it stimulates actin polymerization by multiple pathways. Introduction The Rho-family GTPases (Rho GTPases, including Rac1, Cdc42 and RhoA), which are best known for their roles in regulating the actin cytoskeleton, have been implicated in a broad spectrum of biological functions, such as cell motility and invasion, cell growth, cell polarity and axonal guidance (Schmidt & Hall 2002). Activation signals from Rac1 and Cdc42 are relayed to the actin-nucleating complex Arp2/3 by a family of proteins that includes Wiskott Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) (Takenawa & Suetsugu 2007). However, overexpression of a constitutively active form of Rac1 or Cdc42 inhibits neurite outgrowth and cell migration, reflecting the need for Rho GTPases to cycle between GTP bound and GDP bound states to properly regulate neuritogenesis (Zipkin et al. 1997; Kaufmann et al. 1998; Hing et al. 1999). Rho-GTPase activation is mediated by guanine nucleotide exchange factors (GEFs), which share Communicated by: Kozo Kaibuchi *Correspondence: harada-tk@igakuken.or.jp These authors contributed equally to this work. 688 common motifs: the Dbl-homology (DH) domain and the pleckstrin homology (PH) domain (Cerione & Zheng 1996; Lemmon & Ferguson 2000). Dock1 (Dock180)-related proteins are a new family of RhoGEFs that lack the DH/PH domains. Instead, Dock family proteins are characterized by two evolutionarily conserved protein domains, termed Dock homology regions 1 and 2 (DHR-1 and DHR-2) (Meller et al. 2005; Cote & Vuori 2007). We recently detected a common active center of Dock1~4 within the DHR-2 domain and reported that the DHR-1 domain is necessary for the direct binding between Dock1~4 and WAVE1~3 (Namekata et al. 2010). We also found that overexpression of Dock3 induced axonal regeneration after optic nerve injury in vivo (Namekata et al. 2010). We determined a mechanism underlying Dock3-induced axonal outgrowth, in which Dock3 activates Rac1 and stimulates spatially restricted actin dynamics through formation of a protein complex with Fyn and WAVE at the plasma membrane downstream of brain-derived neurotrophic factor (BDNF) signaling. It has been reported that Dock1 forms a ternary complex with Elmo and RhoG at the plasma membrane, which induces RhoG-dependent Rac1 DOI: /j x

2 Dock3-Elmo-RhoG complex in BDNF signaling activation (Katoh & Negishi 2003). Dock2~4, like Dock1, interacts with Elmo in the cytoplasm, and the interaction has been shown to be required for the biological function of Dock family members (Grimsley et al. 2004). Given that Dock3 shares a similar sequence with Dock1 (Namekata et al. 2010), this mechanism may also apply to Dock3, suggesting BDNF-Dock3 signaling may activate Rac1 via multiple pathways. In this study, we show that BDNF stimulates the formation of a ternary complex of Dock3-Elmo-activated RhoG and subsequent Dock3 phosphorylation at the plasma membrane induces neurite outgrowth via Rac1 activation. Furthermore, we show that membrane recruitment of Dock3 is necessary for Dock3 phosphorylation and the rate of phosphorylation is markedly enhanced by the interaction of Dock3 with Elmo. Our findings indicate that Dock3 plays several roles downstream of BDNF- TrkB signaling for neurite outgrowth and that Dock3 may be a therapeutic target for neural regeneration. Results Elmo mediates interaction between Dock3 and activated RhoG to form a ternary complex Elmo belongs to a family of scaffold proteins and it binds to members of the Dock family such as Dock1. Therefore, we first examined whether Elmo also interacts with Dock3. For this purpose, we used Cos- 7 cells transfected with His-tagged wild-type Dock3 (WT Dock3) or truncated mutants lacking the N-terminus region (D1-160, D1-260, D1-360 and D1-460) (Fig. 1A). We found that these truncated mutants failed to bind to Elmo (Fig. 1B). We also showed that Elmo binds to WT Dock3 and DDHR2 Dock3, which lacks the Rac1 binding domain (Fig. 1A), at similar degrees (Fig. 1C). These results confirmed that Elmo binds to Dock3 and that the Elmo binding domain is likely to be located within the 160 amino acid sequence at the N-terminal of Dock3. These findings in Dock3 are in agreement with those of Dock1 and Dock4 (Grimsley et al. 2004; Komander et al. 2008). We next examined whether Elmo interacts with RhoG using transfectants of wild-type RhoG (RhoG- WT) and a constitutively active form of RhoG (RhoG-V12) in a pull-down assay with a GST-Elmo fusion protein (Fig. 1D). The quantity of RhoG-V12 pulled down by GST-Elmo was much greater than that of RhoG-WT, suggesting that the effective interaction between Elmo and RhoG is GTP-dependent (Katoh & Negishi 2003). As WT Dock3 failed to bind to RhoG-V12 without Elmo (Fig. 1E), we further examined whether Dock3 binds to the Elmo-RhoG complex. The Elmo-RhoG complex binds to WT Dock3 and to DDHR2 Dock3 forming a ternary complex, but it does not bind to D1-160 Dock3, which lacks the Elmo binding domain (Fig. 1F). In addition, the ternary complex formed more effectively with RhoG-V12 than with RhoG-WT (Fig. 1F). Thus, these results indicate that Elmo mediates interaction between Dock3 and RhoG to form a ternary complex in a GTP-dependent manner. Dock3 is translocated to the membranes in the presence of Elmo and activated RhoG As Rho-family proteins including RhoG are activated at the cytoplasmic membrane (Clarke 1992; Katoh & Negishi 2003), RhoG may recruit Dock3-Elmo complex to the plasma membrane. To examine this possibility, we transfected PC12 cells with Dock3 and Elmo and/or RhoG-V12, and subjected these cells to immunocytochemical analysis. Dock3 was localized within the cytoplasm in combination with either Elmo or RhoG-V12, but it was clearly enriched at the plasma membrane when cotransfected with both Elmo and RhoG-V12 (Fig. 2A). We next examined endogenous Rac1 activity in these cells. The Rac1 activity was markedly enhanced by Dock3 in the presence of Elmo and RhoG-V12 (Fig. 2B). To examine whether BDNF is upstream of this signaling cascade, hippocampal neurons prepared from WT and TrkB knockout (KO) mice were transfected with WT RhoG and stimulated by BDNF. We found that exogenous BDNF significantly increased the activity of RhoG in hippocampal neurons from WT mice, but not from TrkB KO mice (Fig. 2C). Together, these results suggest that BDNF stimulates RhoG activity via activation of TrkB receptors and translocates Dock3-Elmo complex to the plasma membrane, where it activates Rac1. Elmo promotes Dock3 phosphorylation and Rac1 activation We recently reported that Dock3 is phosphorylated at the plasma membrane and that phosphorylated Dock3 is detected as a higher-molecular-weight species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Namekata et al. 2010, 2012). To examine whether Dock3 is phosphorylated in a ternary complex with Elmo and activated RhoG, 689

3 K Namekata et al. Figure 1 Dock3 forms a ternary complex with Elmo and activated RhoG. (A) Schematic representation showing the WT and mutant Dock3 constructs used in this pull-down assay. (B) Lysates from Cos-7 cells cotransfected with Elmo and His-tagged Dock3 mutants were subjected to a His-tag pull-down assay with an antibody against Elmo. (C) Lysates from Cos-7 cells cotransfected with Elmo and His-tagged Dock3 mutants lacking the binding site for Elmo and Rac1 were subjected to a His-tag pulldown assay with an antibody against Elmo. (D) Lysates from Cos-7 cells transfected with myc-tagged RhoG-WT, RhoG-N17 (inactive form) or RhoG-V12 (active form) were incubated with GST-Elmo. Bound RhoG proteins were detected by immunoblot analysis with an antibody against myc-tag. (E) Lysates from Cos-7 cells cotransfected as labeled above the immunoblot images were subjected to a His-tag pull-down assay with antibodies against RhoG, Elmo and Dock3. (F) Lysates from Cos-7 cells cotransfected as labeled above the immunoblot images were subjected to a His-tag pull-down assay with antibodies against RhoG, Elmo and Dock3. Relative amounts of bound proteins were plotted in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < we cotransfected Dock3, Elmo and RhoG-V12 in Cos-7 cells and subjected the cell lysates to immunoblot analyses. SDS-PAGE analyses showed the apparent presence of a higher-molecular-weight Dock3 species in cells transfected with Dock3, Elmo and RhoG-V12, but the presence of such species was almost undetectable with any other conditions tested in this study (arrow in Fig. 3A). Treatment with phosphatase eradicated the higher-molecular-weight band, thus confirming the larger protein band as a phosphorylated form of Dock3. When Cos-7 cells were transfected with D1-160 Dock3, no size variant was detected (Fig. 3B), indicating the necessity of the Dock3-Elmo interaction for Dock3 phosphorylation. We also examined the effect of BDNF on endogenous Dock3 phosphorylation using cultured hippocampal neurons; however, BDNF induced only a weak higher-molecular-weight band (Fig. 3C). This may be owing to the low expression levels of Dock3 and Elmo in hippocampal neurons compared with Cos-7 cells containing overexpressed proteins. Trk receptor-specific inhibitor K252a completely suppressed Dock3 phosphorylation. To further assess the role of Elmo in Dock3 phosphorylation, we cotransfected Cos-7 cells with Elmo and WT Dock3 or F-Dock3, which contains a farnesylation signal sequence for plasma membrane localization. Immunoblot analyses showed that the 690

4 Dock3-Elmo-RhoG complex in BDNF signaling Figure 2 Dock3 is translocated to the plasma membrane in the presence of Elmo and activated RhoG and induces Rac1 activation. (A) PC12 cells transfected as labeled above the images were probed with an antibody against Dock3. Arrowheads indicate Dock3 at the peripheral region. Scale bar, 20 lm. (B) Cos-7 cells were transfected with indicated plasmids. Lysates were subjected to a GST-CRIB assay. The relative activity of Rac1 is plotted in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < 0.05 and **P < 0.01 versus WT Dock3 alone. (C) RhoG activity was measured in cultured hippocampal neurons from WT or TrkB-deficient (KO) mice with or without BDNF treatment for 15 min. The relative activity of RhoG is plotted in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < presence of Elmo significantly increased Dock3 phosphorylation in F-Dock3-transfected cells (Fig. 3D). We next examined the effect of Dock3 phosphorylation on its GEF activity. For this purpose, lysates transfected with F-Dock3 and Elmo were subjected to a GST-CRIB assay. We found that F-Dock3, together with Elmo, activates Rac1 more effectively than F-Dock3 alone (Fig. 3E). We also prepared two mutant forms of Elmo constructs lacking the Dock3 binding domain (Elmo-DC) and lacking the RhoG binding domain (Elmo-DN), and cotransfected them with F-Dock3. Immunoblot analysis showed that the Dock3-binding domain in Elmo is required for Dock3 phosphorylation and Rac1 activation (Fig. 3F, G). These results suggest that two events are required for efficient Dock3 phosphorylation that results in increased Rac1 activity. First is the membrane recruitment of Dock3 by activated RhoG, which forms a ternary complex with Elmo and Dock3. Second is the interaction of Dock3 with Elmo, which enhances the rate of Dock3 phosphorylation at the plasma membrane. We next examined the effect of Dock3 phosphorylation on its ability to bind Elmo. Cos-7 cells were transfected with F-Dock3 and His-tagged or untagged Elmo, and were subjected to a pull-down assay. Immunoblot analysis showed that phosphorylated Dock3 is still able to bind with Elmo (Fig. 3H). We previously showed that Dock3 forms a protein complex with Fyn and WAVE at the plasma membrane (Namekata et al. 2010). To determine the possibility that Dock3 is phosphorylated by Fyn, we cotransfected Cos-7 cells with F-Dock3 and Fyn or WT Elmo. Immunoblot analysis showed that Fyn has little effect on Dock3 phosphorylation compared with WT Elmo (Fig. 3I). 691

5 K Namekata et al. Figure 3 Elmo promotes Dock3 phosphorylation and Rac1 activation. (A) Cos-7 cells were transfected with the indicated plasmids. Lysates treated with or without phosphatase were subjected to immunoblot analyses with an antibody against Dock3. Phosphorylated form of Dock3 was detected as a higher molecular size (arrow). Phosphorylated Dock3 levels were quantified and shown in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < (B) Lysates from Cos-7 cells transfected as indicated above the images were subjected to immunoblot analysis with antibodies against Dock3. Arrow indicates phosphorylated Dock3. (C) Immunoblot analysis of Dock3 in hippocampal neurons treated with or without BDNF for 15 min. K252a was applied 30 min before BDNF stimulation. Arrow indicates phosphorylated Dock3. (D) Lysates from Cos-7 cells transfected as indicated above the images were subjected to immunoblot analysis with antibodies against Dock3 and Elmo. Arrow indicates phosphorylated Dock3. Phosphorylated Dock3 levels were quantified and are shown in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < (E) Cos- 7 cells were transfected with the indicated plasmids. Lysates were subjected to a GST-CRIB assay for detecting Rac1 activity. Rac1 activities were quantified and are shown in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < (F) Immunoblot analysis of Dock3 in lysates from Cos-7 cells cotransfected with F-Dock3 and indicated myc-tagged Elmo mutants. Arrow indicates phosphorylated Dock3. Lysates were subjected to a GST-CRIB assay for detecting Rac1 activity. (G) Phosphorylated Dock3 levels and Rac1 activities were quantified and are shown in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < (H) Lysates from Cos-7 cells transfected as shown at the bottom of the images were subjected to a His-tag pull-down assay. Bound proteins were subjected to immunoblot analysis with antibodies against Dock3 and Elmo. Arrow indicates phosphorylated Dock3. Phosphorylated Dock3 levels were quantified and are shown in the histogram. Data are presented as means ± standard errors for each group from three independent cultures. *P < (I) Lysates from Cos-7 cells transfected with F-Dock3 and constitutive active form of Fyn (CA Fyn) or WT Elmo were subjected to immunoblot analysis with an antibody against Dock3. Arrow indicates phosphorylated Dock3. Effect of mutated forms of Dock3, Elmo or RhoG on BDNF-induced neurite outgrowth To determine whether the formation of the Dock3- Elmo-RhoG complex is involved in BDNF-induced neurite outgrowth, PC12 cells were transfected with TrkB, and the morphology of transfected cells was examined after stimulation with BDNF. Consistent with previous studies (Hollis et al. 2009), TrkB is required for BDNF-mediated neurite outgrowth in PC12 cells (Fig. 4A). Overexpression of a dominantnegative form of Dock3 lacking GEF activity (Dock3 Y1373A; Namekata et al. 2010), Elmo-DC, Elmo-DN and a dominant-negative variant of RhoG (RhoG- N17) strongly suppressed the number of cells with neurites (Fig. 4A). In addition, overexpression of 692

6 Dock3-Elmo-RhoG complex in BDNF signaling Figure 4 Effect of the mutated forms of Dock3, Elmo or RhoG on BDNF-induced neurite outgrowth. (A) PC12 cells transfected with GFP and the indicated plasmids were stimulated with or without BDNF (50 ng/ml) for 48 h. (B) PC12 cells transfected with GFP and the indicated plasmids were cultured in growth medium for 48 h. Cells with neurites were scored as a percentage of the total number of transfected cells, and the results are summarized in the histogram. Data are presented as the means ± standard errors of three independent experiments in which 50 cells were counted. *P < Scale bars, 20 lm. 693

7 K Namekata et al. F-Dock3 and RhoG-V12 failed to stimulate neurite outgrowth. Overexpression of both F-Dock3 and Elmo-DN also suppressed BDNF-induced neurite outgrowth. These findings may be partly owing to the continuous overstimulation of Rac1 (Zipkin et al. 1997; Kaufmann et al. 1998; Hing et al. 1999). We also found that Dock3 Y1373A, Elmo-DN and Elmo- DC blocked RhoG-induced neurite outgrowth in PC12 cells (Fig. 4B). Finally, we carried out similar experiments using primary cultured hippocampal neurons. Overexpression of D1-160 Dock3 or Elmo-DN significantly inhibited the BDNF-induced neurite outgrowth (Fig. 5). Figure 5 Effect of the mutated forms of Dock3 or Elmo on BDNF-induced neurite outgrowth in hippocampal neurons. Hippocampal neurons transfected with GFP and the indicated plasmids were cultured in the presence of BDNF for 3 days and the axon length was measured. n = 30 per experimental condition. Data are mean ± standard errors of three independent experiments. *P < Scale bar, 20 lm. Discussion Here, we report a novel Dock3-mediated BDNF pathway for neurite outgrowth. We showed that Elmo binds to Dock3, and forms a ternary complex with activated RhoG upon BDNF stimulation. The formation of this ternary complex allows the Dock3- Elmo complex to be translocated to the plasma membrane where Dock3 can be phosphorylated and thus Rac1 is efficiently activated, leading to neurite outgrowth. Our findings indicate the importance of Dock3 phosphorylation that occurs as a result of membrane recruitment of Dock3 and also of the interaction of Dock3 with Elmo in RhoG-mediated BDNF signaling for neurite outgrowth. We previously reported that BDNF also induces the formation of a Fyn-Dock3-WAVE complex at the plasma membrane and stimulates axonal outgrowth (Namekata et al. 2010). These observations suggest that BDNF stimulation recruits Dock3 to the plasma membrane by at least two pathways, indicating the significance of Dock3 localization in cellular dynamics during neurite outgrowth. In addition, we recently found that Dock3 binds to and inactivates glycogen synthase kinase-3b (GSK-3b) at the plasma membrane, thereby increasing the nonphosphorylated active form of collapsin response mediator protein-2 (CRMP-2), which promotes axonal outgrowth by microtubule assembly (Namekata et al. 2012). Taken together, it is likely that overexpression of D1-160 Dock3 and Elmo-DC mutants did not completely inhibit neurite outgrowth (Fig. 5) owing to the multiple Dock3- dependent pathways for neurite outgrowth. It has been reported that a Dock family member Dock1 binds to Elmo, leading to activation of Rac1 at the cell front (Gumienny et al. 2001; Santy et al. 2005). In addition to migration, Elmo proteins are important mediators of engulfment of apoptotic cells and bacterial invasion (Gumienny et al. 2001; Katoh & Negishi 2003; Handa et al. 2007). Here, we showed that Dock3 binds to Elmo and that Dock3 phosphorylation depends on this interaction, suggesting that Elmo is a key regulator of Dock3 phosphorylation. Our previous study showed that one of the essential events in Fyn-Dock3-WAVE signaling is Dock3 phosphorylation, which dissociates WAVE from the Dock3 complex near the plasma membrane, leading to the stimulation of actin rearrangement. We speculate that Elmo also stimulates Dock3 phosphorylation in the Fyn-Dock3-WAVE complex and thus regulates the interaction between Dock3 and WAVE protein, enabling the tightly controlled Rac1-WAVE-Arp2/3 694

8 Dock3-Elmo-RhoG complex in BDNF signaling signaling. To further investigate the mechanism underlying the formation of this complex, we are currently working to determine the specific site of BDNF-induced Dock3 phosphorylation and to create a site-specific phosphorylation antibody. In this study, we have established that BDNF- TrkB signaling acts upstream of a Dock3-mediated pathway for neurite outgrowth. BDNF-TrkB signaling is important for the formation of both inhibitory and excitatory synapses in the central nervous system including the hippocampus (Aguado et al. 2003; Luikart et al. 2005). Consistently, BDNF-deficient mice exhibit a marked suppression of long-term potentiation (LTP) (Korte et al. 1995; Patterson et al. 1996). Previous studies have reported that cytoskeletal regulation is involved in synaptogenesis. For example, Rac1 regulates dendritic spine density and shape in rat and mouse pyramidal neurons (Nakayama et al. 2000; Tashiro & Yuste 2004). Rac1 regulation of cell motility and neurite outgrowth is mediated, in part, by interaction with p21-activated kinases (PAK) (Kumar et al. 2006; Wu et al. 2009). Dominant-negative PAK transgenic mice show enhanced LTP and suppressed long-term depression, as well as specific deficits in the consolidation phase of hippocampusdependent memory (Hayashi et al. 2003). As BDNF activates Rac1, at least partly, through Dock3 phosphorylation (Namekata et al. 2010), Dock3 may participate in mediating multiple BDNF-TrkB functions including the establishment of LTP. In addition to having physiological functions, BDNF has recently been associated with neurological diseases. For example, Val66Met functional polymorphism in human BDNF is involved in the pathogenesis of attentiondeficit hyperactivity disorder (ADHD) (Egan et al. 2003; Friedel et al. 2005; Kent et al. 2005). Interestingly, a pericentric inversion breakpoint in the DOCK3 gene has been described in ADHD patients (de Silva et al. 2003). Thus, disruption of the BDNF- Dock3 signaling may have therapeutic benefits to various neurodevelopmental disorders. Further studies are required to fully elucidate the precise roles played by Dock family members during neural development, neurodegeneration and neural regeneration. Experimental procedures Animals All procedures involving animals were approved by the Institutional Animal Care and Use Committees of the Tokyo Metropolitan Institute of Medical Science in accordance with the Standards Relating to the Care and Management of Experimental Animals in Japan. C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan) and TrkB KO mice used in this study have previously been generated and characterized (Luikart et al. 2005; Namekata et al. 2010; Harada et al. 2011). Expression plasmids Plasmids encoding wild-type RhoG (RhoG-WT), constitutively active form of RhoG (RhoG-V12), inactive form of RhoG (RhoG-N17) tagged with myc and wild-type Elmo tagged with hemagglutinin (HA) were generous gifts from Drs. H. Katoh and M. Negishi (Kyoto University, Kyoto, Japan) (Katoh & Negishi 2003). Deletion mutants of Elmo lacking RhoG binding domain (Elmo-DN; amino acid ) or Dock3 binding domain (Elmo-DC; amino acid 1-531) were generated by PCR. Farnesylation-signal-attached Dock3 (F-Dock3), inactive form of Dock3 (Dock3 Y1373A) and active form of Fyn (CA Fyn) were generated as described previously (Namekata et al. 2010). His-tagged WT Dock3, Dock3 deletion mutants and TrkB were generated by PCR and subcloned into the pcmv expression vector. The cdna fragment of Elmo (amino acids 1-80) was generated by PCR and subcloned into the pgex-4t-2 vector. Cell culture and transfection Transient transfection was carried out using Lipofectamine Plus for COS-7 cells and Lipofectamine 2000 for PC12 cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer s instructions. His-tagged WT Dock3 or Dock3 deletion mutants were coexpressed with myc-tagged RhoG or HAtagged Elmo. Primary cultured hippocampal neurons were prepared as previously reported, and stimulated with or without BDNF (50 ng/ml) and K252a (200 ng/ml; Alomone Labs, Jerusalem, Israel). Immunoblotting and immunoprecipitations After 24 h of transfection, the cell lysate was incubated with TALON resin (BD Biosciences Pharmingen, San Jose, CA, USA) for 20 min at 4 C with gentle agitation. After washing, precipitated samples were subjected to SDS-PAGE followed by immunoblot analysis as previously described (Namekata et al. 2010). The following antibodies were used: anti-dock3 polyclonal antibody (Namekata et al. 2010); anti-ha polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-myc monoclonal antibody (Santa Cruz); and anti- Rac1 monoclonal antibody (BD Biosciences). Measurement of small GTPase activity The activities of Rac1 and RhoG were measured as described previously (Hiramoto et al. 2006). Briefly, glutathione S-transferase (GST)-fused CRIB (Cdc42/Rac interacting 695

9 K Namekata et al. binding)-bound Rac1 and GST-fused Elmo (amino acid 1 80)-bound RhoG were purified from bacterial lysate using glutathione-agarose (GE Healthcare, Buckinghamshire, UK). After 24 h of transfection, Cos-7 cells were washed twice with PBS, lysed with a lysis buffer (25 mm Tris ph7.4, 150 mm NaCl, 1% Triton X-100, 20 mm MgCl 2, 10% glycerol containing protease inhibitor mixture) and centrifuged for 15 min at 10,000 g. Rac1 bound to GST-CRIB and RhoG bound to GST-Elmo were resolved by SDS-PAGE, and assessed by immunoblot analysis. Immunostaining PC12 cells were fixed and incubated with anti-dock3 polyclonal antibody (1 : 1000) (Namekata et al. 2010). Cy2-conjugated donkey anti-rabbit IgG or Cy3-conjugated donkey anti-mouse IgG were used as secondary antibodies (Invitrogen). Statistics Data are presented as mean ± standard errors unless noted otherwise. Statistical analyses were carried out using a twotailed Student s t-test. Results were considered statistically significant if P < Acknowledgements We are grateful to H. Kato and M. Negishi (Kyoto University) for plasmids and antibodies, and to Dr L.F. Parada (University of Texas Southwestern Medical Center) for providing TrkB-deficient mice. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (K.N., H.W., X.G., D.K., A.K., C.H.), Takeda Science Foundation (K.N.), and the Funding Program for Next Generation World-Leading Researchers (NEXT Program) (T.H.). References Aguado, F., Carmona, M.A., Pozas, E., Aguiló, A., Martínez- Guijarro, F.J., Alcantara, S., Borrell, V., Yuste, R., Ibañez, C.F. & Soriano, E. (2003) BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K + /Cl co-transporter KCC2. Development 130, Cerione, R.A. & Zheng, Y. (1996) The Dbl family of oncogenes. Curr. Opin. Cell Biol. 8, Clarke, S. 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