Asef2 and Neurabin2 Cooperatively Regulate Actin Cytoskeletal Organization and Are Involved in HGF-Induced Cell Migration

Oncogene (2009) 28, 1357–1365 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Asef2 and Neurabin2 cooperatively regulate actin cytoskeletal organization and are involved in HGF-induced cell migration

M Sagara1, Y Kawasaki1, S-i Iemura2, T Natsume2, Y Takai3 and T Akiyama1

1Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan; 2Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan and 3Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan

The tumor suppressor adenomatous polyposis coli (APC) polyps in the colon (Kinzler and Vogelstein, 1996; is mutated in sporadic and familial colorectal tumors. Fodde et al., 2001). The product of the APC gene APC interacts with the Rac1- and Cdc42-specific guanine- negatively regulates the Wnt signaling pathway by nucleotide exchange factors (GEF), Asef and Asef2, which inducing degradation of b-catenin (Cadigan and Nusse, contain an APC-binding region (ABR) in addition to Dbl 1997; Bienz and Clevers, 2000; Polakis, 2000). Mutant homology, Pleckstrin homology (PH) and Src homology 3 APCs identified in colon cancers are defective in these (SH3) domains. APC stimulates the GEF activity of Asef activities and, as a result, b-catenin levels are elevated and Asef2, and thereby regulates cell adhesion and and Wnt signaling is constitutively activated in color- migration. Here we show that Asef2, but not Asef, ectal cancer cells. Thus, the ability of APC to negatively interacts with Neurabin2/Spinophilin, a scaffold protein regulate Wnt signaling is believed to be essential for its that binds to Filamentous actin (F-actin). In response to tumor suppressor function (Fodde et al., 2001). In hepatocyte growth factor (HGF) treatment of HeLa addition, it has been reported that APC also regulates cells, Asef2, Neurabin2 and APC were induced to cytoskeletal networks through interacting with various accumulate and colocalize in lamellipodia and membrane proteins, including Asef, kinesin-2 and IQGAP1 ruffles. Neurabin2 did not affect the GEF activity of (Kawasaki et al., 2000, 2003; Jimbo et al., 2002; Asef2. RNA interference experiments showed that Asef2, Watanabe et al., 2004; Akiyama and Kawasaki, 2006). Neurabin2 and APC are involved in HGF-induced cell APC is reported to regulate cell polarization under the migration. Furthermore, knockdown of Neurabin2 re- control of Cdc42, Par6 and aPKC in primary astrocytes sulted in the suppression of Asef2-induced filopodia (Etienne-Manneville and Hall, 2003). APC and formation. These results suggest that Asef2, Neurabin2 IQGAP1 localize interdependently to the leading edge and APC cooperatively regulate actin cytoskeletal in migrating Vero cells, and link the actin cytoskeleton organization and are required for HGF-induced cell and microtubule dynamics, and are required for cell migration. polarization and directional cell migration (Watanabe Oncogene (2009) 28, 1357–1365; doi:10.1038/onc.2008.478; et al., 2004). published online 19 January 2009 Asef was identified as a Rac1- and Cdc42-specific guanine-nucleotide exchange factor (GEF) that interacts Keywords: APC; Asef2; Neurabin2/Spinophilin; HGF; with APC (Kawasaki et al., 2000, 2007; Hamann et al., filopodia; migration 2007). Asef contains Dbl homology, Pleckstrin homology (PH) and Src homology 3 (SH3) domains. The crystal structure of Asef has revealed that the SH3 and Dbl homology domains interact intramolecularly, thereby blocking the GTPase-binding site (Mitin et al., 2007; Introduction Murayama et al., 2007). APC binds to the amino- terminal APC-binding region (ABR) of Asef through its The tumor suppressor adenomatous polyposis coli armadillo repeat domain and enhances its GEF activity, (APC) gene is mutated in sporadic colorectal tumors and thereby regulates cell morphology, adhesion and and in familial adenomatous polyposis, a dominantly migration. A mutant form of Asef lacking the amino- inherited disease characterized by multiple adenomatous terminal ABR shows strong GEF activity even in the absence of APC, suggesting that APC activates the GEF Correspondence: Professor T Akiyama, Laboratory of Molecular and activity of Asef by binding to ABR and relieving the Genetic Information, Institute for Molecular and Cellular Biosciences, negative regulation by the SH3 domain. Furthermore, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, truncated mutant APCs present in colorectal tumor cells Japan. E-mail: [email protected] activate Asef constitutively, and cause decreased cell-cell Received 11 September 2008; revised 8 December 2008; accepted 13 adhesion and aberrant migratory properties (Kawasaki December 2008; published online 19 January 2009 et al., 2003). These findings suggest that truncated Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1358 mutant APCs and Asef are important for adenoma formation and tumor progression to invasive malig- nancy. In addition, Asef is phosphorylated by Src-family protein kinases, and required for epidermal growth factor-induced Rac1 and Cdc42 activation (Itoh et al., 2008). Asef2 was identified as a homolog of Asef, and shows significant structural and functional similarities to Asef (Hamann et al., 2007; Kawasaki et al., 2007). Asef2 also acts as a GEF specific for Rac1 and Cdc42. APC interacts with the ABR and SH3 domains of Asef2, and enhances its GEF activity. Furthermore, Asef2 is required for migration of colorectal tumor cells expressing truncated mutant APC. To better understand the function of Asef2, we have searched for proteins that interact with Asef2, and identified Neurabin2/Spinophilin, a scaffold protein that binds to filamentous actin (F-actin) (Allen et al., 1997; Satoh et al., 1998). Neurabin2 is a multifunctional protein that regulates the cytoskeleton and signal transduction (Sarrouilhe et al., 2006). For example, Neurabin2 is known to interact with several GEFs, including Tiam-1 and Lfc, and regulate their activity (Buchsbaum et al., 2003; Ryan et al., 2005). We show here that Asef2, but not Asef, interacts with Neurabin2. In response to hepatocyte growth factor (HGF) treatment of HeLa cells, Asef2, Neurabin2 and APC were induced to colocalize in lamellipodia and membrane ruffles. Furthermore, we show that Neurabin2 is required for Asef2-induced filopodia formation and that Asef2, Neurabin2 and APC are required for HGF- induced cell migration.

Results

We attempted to identify Asef2-associated proteins using liquid chromatography-based electrospray tandem Figure 1 Association of Asef2 with Neurabin2 in vivo. mass spectrometry (Natsume et al., 2002). Human (a) Association of endogenous Asef2 with Neurabin2 in vivo. Lysates embryonic kidney (HEK293T) cells were transfected prepared from Human embryonic kidney (HEK293T) cells were with Flag-tagged Asef2, and lysates from these cells subjected to immunoprecipitation with the antibodies indicated, were subjected to immunoprecipitation with anti-Flag fractionated by 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the anti- antibody. Immunoprecipitated proteins were subjected bodies indicated. Pep þ , antibodies were pre-incubated with to proteolysis followed by liquid chromatography-based antigen before use in immunoprecipitation. (b) Association of electrospray tandem mass spectrometry. We found that Asef2 with Neurabin2 in HeLa cells. Cells were co-transfected with Neurabin2 co-precipitates with Asef2. Flag-tagged Asef or Flag-tagged Asef2 along with HA-tagged We therefore performed pull-down assays to examine Neurabin2. Lysates were subjected to immunoprecipitation with the antibodies indicated, fractionated by SDS-PAGE and immu- whether endogenous Asef2 is indeed associated with noblotted with the antibodies indicated. (c) Neither Asef nor Asef2 Neurabin2 in HEK293T cells. When a cell lysate was interacts with Neurabin. Cells were co-transfected with Flag-tagged subjected to immunoprecipitation with anti-Asef2 Asef or Flag-tagged Asef2 along with Myc-tagged Neurabin. antibody followed by immunoblotting with anti-Neur- Lysates were subjected to immunoprecipitation with the antibodies indicated, fractionated by SDS–PAGE and immunoblotted with abin2 antibody, Asef2 was found to co-immunoprecipi- the antibodies indicated. APC, adenomatous polyposis coli; tate with Neurabin2 as well as APC (Figure 1a). In IB, immunoblotting; IP, immunoprecipitation. these experiments, co-precipitation of Asef2 and Neurabin2 was inhibited by pre-incubation of the antibody with the antigen used for immunization. These results suggest that Asef2 and Neurabin2 are We next examined whether Asef2 could interact with contained in the same complex in vivo. On the other Neurabin2 in vitro. We found that Asef2 produced by hand, Asef did not co-precipitate with Neurabin2 in vitro translation speciﬁcally interacts with Neurabin2 (Figure 1b). Also, neither Asef2 nor Asef interacted fused to glutathione S-transferase (GST) but not with with Neurabin, which is closely related to Neurabin2 GST alone (Figure 2a). Likewise, in vitro-translated (Figure 1c). Neurabin2 interacted with GST-Asef2, but not with

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1359 GST alone. To identify the region of Asef2 and Neurabin2 responsible for their interaction, we performed in vitro pull-down assays using various deletion fragments of Asef2 and Neurabin2. We found that

GST-Neurabin2 GST+Asef2 GST Asef2-DABR/SH3, a mutant lacking the amino-terminal 140kD ABR and SH3 domains, interacts with Neurabin2 95kD (Figures 2b and c). By contrast, Asef2-DC, a mutant Asef2 input 10% +GST +GST-Neurabin2 Neurabin2 input 10% +GST +GST+Asef2 * 55kD Neurabin2 lacking the carboxy-terminal region, showed no interaction with Neurabin2 (Figures 2b and c). These results Asef2 28kD suggest that the carboxy-terminal region of Asef2 is required for its interaction with Neurabin2, although + GST-Neurabin2 + GST-Asef2 this region of Asef2 alone showed weak interaction. In addition, Asef2 interacted with Neurabin2-DN,

N C a mutant lacking the amino-terminal F-actin-binding ∆ ∆ domain, and Neurabin2-Cter, a carboxy-terminal ABR/SH3 C

∆ ∆ fragment of Neurabin2, but not Neurabin2-DC, a mutant lacking the carboxy-terminal region. These

Asef2-full Asef2- Asef2- Asef2-Cter Neurabin2-full Neurabin2- Neurabin2- Neurabin2-Cter results suggest that the carboxy-terminal region of Neurabin2 is required and sufficient for its interaction with Asef2. pull-down We previously found that expression of Asef along with APC induces abundant lamellipodia in MDCK cells and filopodia in HeLa cells (Kawasaki et al., 2000, 2007). Furthermore, we showed that Asef has the potential to promote the migration of MDCK cells in input 5% a GEF activity-dependent manner (Kawasaki et al., 2003). As these effects of APC-activated Asef appear to be similar to those of HGF, we assumed that APC and Asef function downstream of HGF. Indeed, we recently F-actin binding PDZ coiled-coil binding 1 817 found that APC and Asef are colocalized in lamellipodia Neurabin2-full + and membrane ruffles in HeLa cells treated with HGF Neurabin2-∆N + and are required for HGF-induced cell migration (Kawasaki et al., unpublished observation). Thus, we Neurabin2-∆C - examined whether Asef2 and Neurabin2 also function Neurabin2-Cter + downstream of HGF. First, we examined whether the subcellular localization of these proteins is altered when 1ABR SH3 DH PH 652 Asef2-full + HeLa cells are treated with HGF. Immunostaining analysis using anti-Asef2 and anti-Neurabin2 antibodies Asef2-∆ABR/SH3 + revealed that Asef2 is localized mainly in the cytoplasm Asef2-∆C - and Neurabin2 is localized in the cytoplasm and plasma membrane in the absence of HGF stimulation Asef2-Cter ± (Figure 3a). When HeLa cells were treated with HGF, Figure 2 Mapping of the regions in Asef2 and Neurabin2 required Asef2 and Neurabin2 were found to translocate and for their interaction. (a) Association of Asef2 with Neurabin2 accumulate in lamellipodia and membrane ruffles. We in vitro. In vitro translated [35S]methionine-labeled Asef2 was incubated with glutathione S-transferase (GST)-Neurabin2 or GST also found that in response to HGF treatment, APC was bound to Sepharose. In vitro translated Neurabin2 was incubated induced to colocalize with Asef2 in lamellipodia and with GST-Asef2 or GST bound to Sepharose. Bound proteins were membrane ruffles (Figure 3b). Consistent with these analysed by sodium dodecyl sulfate polyacrylamide gel electro- results, exogenously expressed APC and Asef2 accumu- phoresis (SDS–PAGE) followed by autoradiography. The right lated in membrane ruffles and lamellipodia in response panel shows Coomassie blue staining of GST and GST-fusion proteins used in pull-down assays. (b) In vitro translated to HGF treatment (Figure 3c). Although exogenous [35S]methionine-labeled deletion constructs of Asef2 or Neurabin2 expression of Neurabin2 caused morphological changes, were incubated with deletion constructs of Neurabin2 or Asef2 including filopodia formation, exogenously expressed fused to GST, respectively. Bound proteins were analysed by SDS- Neurabin2 also accumulated in membrane ruffles and PAGE followed by autoradiography. (c) Domain organization of Asef2 and Neurabin2. The schematic structure of deletion mutants lamellipodia, as well as in filopodia. These results raise of Asef2 and Neurabin2 are shown. Positive binding activity the possibility that Asef2, Neurabin2 and APC function is indicated as þ . Weak binding activity is indicated as ±. downstream of HGF. *, degradation products of GST-Neurabin2; ABR, APC-binding To examine whether Asef2, Neurabin2 and APC are region; DH, Dbl homology; F-actin, Filamentous actin; required for HGF-induced cell migration, we performed PH, Pleckstrin homology; SH3, Src homology 3. knockdown experiments using small interfering RNAs (siRNAs). Immunoblotting analysis revealed that expression of these proteins was suppressed in cells

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1360

Figure 3 Subcellular localization of Asef2, Neurabin2 and adenomatous polyposis coli (APC). (a) Colocalization of endogenous Asef2 and Neurabin2 in HeLa cells treated with Hepatocyte growth factor (HGF). After stimulation with HGF (20 ng/ml) for 10 min, cells were double stained with anti-Asef2 and anti-Neurabin2 antibodies. Scale bar, 10 mm. (b) Colocalization of endogenous Asef2 and APC in HeLa cells treated with HGF. After stimulation with HGF (20 ng/ml) for 10 min, cells were double stained with anti-Asef2 and anti-APC antibodies. Scale bar, 10 mm. (c) Subcellular localization of exogenously expressed APC, Asef2 and Neurabin2 in HGF-stimulated HeLa cells. HeLa cells were transfected with expression plasmids encoding Myc-tagged APC, HA-tagged Asef2 or HA-tagged Neurabin2 and were treated with HGF (20 ng/ml) for 10 min. Cells were stained with antibodies against Myc or HA. Scale bar, 10 mm.

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1361 transfected with specific siRNA but not with control (Figures 6c and d). These results suggest that Neurabin2 siRNA (Figure 4a). When siRNA-transfected cells were does not affect the GEF activity of Asef2. subjected to migration assays using transwell chambers, HGF-induced migratory activity of these cells were reduced compared with cells transfected with control Discussion siRNA (Figure 4b). These results suggest that Neur- abin2, Asef2 and APC play important roles in HGF- In this study, we showed that Asef2 interacts with induced cell migration. Neurabin2. Neurabin2 is known to interact with several Truncated mutant APCs present in colorectal tumor GEFs, including Tiam-1 and Lfc (Buchsbaum et al., cells activate Asef and Asef2 constitutively and cause 2003; Ryan et al., 2005). Tiam-1, a Rac1-specific GEF, aberrant migratory properties (Kawasaki et al., 2003, interacts with the region containing PDZ and coiled-coil 2007). We therefore examined whether Neurabin2 is also domains of Neurabin2 through its coiled-coil domain. involved in migration of colorectal tumor SW480 cells, Interaction between Neurabin2 and Tiam-1 enhances which express truncated mutant APC. We found that p70 S6 kinase activity by affecting the signaling cells expressing short hairpin RNA (shRNA) against specificity of Rac1. We therefore examined whether Neurabin2 showed decreased motility compared with the Asef2–Neurabin2 complex is involved in the cells expressing mutant shRNA (Figures 4c and d). regulation of p70 S6 kinase activity. However, we could These results suggest that Neurabin2 is also important not detect the ternary complex containing Asef2, for the migration of colorectal tumor cells expressing Neurabin2 and p70 S6 kinase (data not shown). Lfc, a truncated mutant APC. Rho-specific GEF, interacts with the coiled-coil domain When HeLa cells were transfected with Asef2-DABR/ of Neurabin2. Neurabin2 functions as a scaffold to SH3, a mutant Asef2 that shows strong GEF activity in target Lfc to dendritic spines, in which Lfc can activate the absence of APC, the cells showed abundant Rho and organize spine morphology. Both Tiam1 and filopodia (Kawasaki et al., 2007). It is known that Lfc interact with Neurabin2 through the region contain- Neurabin2 has crosslinking and capping activity, and ing the coiled-coil domain. Thus, Tiam-1/Lfc and induces filopodia when overexpressed (Satoh et al., Neurabin2 may form coiled-coil structures. As Asef2 is 1998; Futter et al., 2005; Schuler and Peti, 2008). not predicted to have a coiled-coil domain, the mode of Furthermore, some F-actin-binding proteins are re- the interaction between Asef2 and Neurabin2 may be ported to interact with GEFs and cooperatively regulate different from those between Tiam-1/Lfc and actin cytoskeletal organization (Bellanger et al., 2000). Neurabin2. We therefore hypothesized that Neurabin2 is involved in Although the function and structure of Asef2 are very Asef2-induced rearrangement of the actin cytoskeleton. similar to those of Asef, only Asef2, but not Asef, To test this possibility, we examined whether Neurabin2 interacts with Neurabin2. These results suggest that Asef is involved in filopodia formation in HeLa cells and Asef2 may regulate the actin cytoskeleton in a overexpressing Asef2-DABR/SH3. Cells were trans- different manner. We found that the carboxy-terminal fected with Neurabin2-specific or control siRNA along region of Asef2 is required for its interaction with with HA-tagged Asef2-DABR/SH3, subjected to im- Neurabin2. It was reported that the carboxy-terminal munostaining with antibody against HA, and the region of Asef2 interacts with the ABR and SH3 numbers of filopodia per transfected cell were counted domains intramolecularly and is required for the GEF (Figures 5a and c). We found that cells transfected with activity of Asef2 (Hamann et al., 2007). On the other siRNA specific for Neurabin2 showed fewer filopodia hand, another group reported that the carboxy-terminal compared with cells transfected with control siRNA region does not influence the GEF activity (Mitin et al., (Figure 5b). Thus, Neurabin2 may be important for 2007). The carboxy-terminal region of Asef2 is well Asef2-induced filopodia formation. conserved among animals, including monkey and To investigate whether the effect of Neurabin2 on mouse, but its similarity to that of Asef is relatively Asef2-induced filopodia formation depends on Rac1/ low compared with the ABR, SH3, PH and Dbl Cdc42 activity, we examined whether Neurabin2 plays a homology domains. Thus, this region may be involved role in the regulation of Asef2 GEF activity. HeLa cells in the Asef2-specific functions. were transfected with HA-tagged Rac1 or Cdc42 along We previously found that Asef2 is involved in with Flag-tagged Asef2-DABR/SH3, and Neurabin2- filopodia formation in HeLa cells (Kawasaki et al., specific or control siRNA. As we reported earlier 2007). Neurabin2 is also known to be involved in (Kawasaki et al., 2007), expression of Asef2-DABR/ filopodia formation. Epidermal growth factor stimula- SH3 significantly increased the amounts of GTP-bound tion induces ERK2-mediated phosphorylation of form of Rac1 and Cdc42 (Figures 6a and b). Knock- Neurabin2 in Cos-7 cells, and a phospho-Neurabin2 down of Neurabin2 expression had no effects on the mimic showed decreased ability to bind to F-actin amounts of GTP-bound form of Rac1 and Cdc42. To and increased potential to induce filopodia in confirm these results, HeLa cells were transfected with HEK293T cells and hippocampal neurons (Futter HA-tagged Rac1 or Cdc42 along with Flag-tagged et al., 2005). Neurons from Neurabin2-deficient mice Asef2, Neurabin2 or Asef2-DABR/SH3. Asef2 showed exhibit a large increase in filopodial protrusions when weaker effects than Asef2-DABR/SH3 and its effects cultured at low density (Feng et al., 2000). Thus, were not enhanced by co-expression of Neurabin2 Neurabin2 may facilitate spine retraction during

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1362 maturation of neurons or suppress the initial outgrowth spatiotemporally by phosphorylation and/or binding of spine from dendrite. These observations suggest that partners. Hence, it would be interesting to examine the Neurabin2 regulates ﬁlopodial initiation, extension and role of the Asef2–Neurabin2 complex in spine morpho- retraction, and these activities might be regulated genesis in neurons.

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1363

Figure 6 Effects of Neurabin2 on the guanine-nucleotide ex- Figure 5 Neurabin2 is required for Asef2-induced filopodia change factor (GEF) activity of Asef2. Cells were transfected with formation. (a) Cells were transfected with Neurabin2-specific or the indicated constructs along with HA-tagged Rac1 (a and c)or control small interfering RNA (siRNA) along with expression Cdc42 (b and d). GTPase activity was assessed using GST-PAK- plasmids encoding HA-tagged Asef2-DABR/SH3. Lysates pre- CRIB immobilized onto glutathione (GSH)-Sepharose beads. The pared from transfected cells were analysed by immunoblotting with GTP-bound forms of GTPases were detected by immunoblotting anti-Neurabin2 antibody or anti-HA antibody. Anti-a-tubulin with anti-HA antibody. The total amounts of HA-tagged GTPases antibody was used as a control. (b and c) Role of Neurabin2 in in the lysates and the expression levels of Flag-tagged Asef2, Asef2- Asef2-induced filopodia formation. Cells were transfected with DABR/SH3 and Neurabin2 are shown. ABR, APC-binding region; Neurabin2-specific or control siRNA along with expression GST, glutathione S-transferase; SH3, Src homology 3. plasmids encoding HA-tagged Asef2-DABR/SH3. Cells were stained with anti-HA antibody, and (b) the numbers of filopodia per transfected cell were counted. The bar graph shows the mean number of filopodia per cell ±s.e.m. 150 cells were counted in three Neurabin2 could regulate HGF-induced translocation independent experiments. ABR, APC-binding region; SH3, Src homology 3. of Asef2 and APC to lamellipodia and membrane ruffles. We found that knockdown of Neurabin2 does not induce detectable changes in Asef2 and APC Some F-actin binding proteins are known to interact localization (data not shown). We previously reported with GEFs and cooperatively regulate actin cytoskeletal that Asef is targeted to the site of cell-cell adhesion organization. For example, Filamin, a scaffold protein through its PH domain in MDCK II cells (Muroya that has the potential to crosslink F-actin, interacts with et al., 2007). As the PH domains of Asef and Asef2 are Trio, a GEF specific for Rac1, RhoG and RhoA highly conserved, Asef2 localization may be mediated (Bellanger et al., 2000). Filamin is required for actin through its PH domain. These results, together with our cytoskeletal modification induced by Trio. In this study, observation that Neurabin2 does not affect the GEF we showed that Neurabin2 is required for Asef2-induced activity of Asef2, suggest that Neurabin2 functions as a filopodia formation. In addition, we examined whether scaffold, which links Asef2 with actin cytoskeletal

Figure 4 Asef2, Neurabin2 and adenomatous polyposis coli (APC) are required for cell migration. (a) Suppression of Neurabin2, Asef2 and APC expression by small interfering RNAs (siRNAs). HeLa cells were transfected with Neurabin2-, Asef2-, APC-speciﬁc or control siRNA, respectively. Lysates prepared from transfected cells were subjected to immunoblot analysis with anti-Neurabin2, anti-Asef2 or anti-APC antibody. Anti-a- tubulin antibody was used as a control. (b) Role of Neurabin2, Asef2 and APC in migration of HeLa cells. Cells were transfected with Neurabin2-, Asef2-, APC-speciﬁc or control siRNA, respectively, and subjected to migration assays using Transwell migration chambers. Cells were allowed to migrate for 8 h in the presence or absence of Hepatocyte growth factor (HGF) (20 ng/ml). Results are expressed as the means±s.e.m. of three independent experiments. (c) Suppression of Neurabin2 expression by short hairpin RNA (shRNAs) in SW480 cells. SW480 cells were transfected with expression vectors containing oligonucleotides encoding shRNA-Neurabin2 or mut-shRNA-Neurabin2. Lysates prepared from transfected cells were analysed by immunoblotting with anti-Neurabin2 antibody. Anti-a-tublin antibody was used as a control. (d) Migration of SW480 cells expressing shRNAs. Cells were transfected with vectors containing oligonucleotides encoding shRNA-Neurabin2 or mut-shRNA-Neurabin2 and subjected to migration assays. Error bar represents the means±s.e.m. of three independent experiments.

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1364 networks. As APC interacts with microtubules directly reticulocyte lysate system (Promega corporation Madison, WI, or indirectly through EB1 protein (Su et al., 1995), Asef2 USA). Proteins fused to GST were synthesized in Escherichia may function as a link between microtubules and the coli and isolated by absorption to glutathione (GSH)- actin cytoskeleton. Sepharose (GE Healthcare UK Ltd, Buckinghamshire, UK). The roles of Neurabin2 in non-neuronal cells are not GST and GST-fusion proteins (1 mg) immobilized to beads were incubated with in vitro translation products in binding well studied compared with those in neurons, despite the buffer (0.1% NP-40, 50 mM HEPES (pH 7.0), 150 mM NaCl, fact that Neurabin2 is expressed ubiquitously. In this 50mM NaF, 5m M EDTA, 1 mM dithiothreitol (DTT)) and study, we showed that Neurabin2 is required for HGF- then washed thrice with binding buffer. Proteins adhering to induced migration of HeLa cells. Furthermore, we the beads were analysed by sodium dodecyl sulfate poly- found that Neurabin2 is also required for migration of acrylamide gel electrophoresis followed by autoradiography. SW480 cells, which express the truncated mutant APC. These results suggest that Neurabin2, together with Immunoprecipitation and immunoblotting Asef2 and APC, contributes to aberrant migratory Immunoprecipitation and immunoblotting analysis were properties of tumor cells. HGF is known to be involved performed as described earlier (Kawasaki et al., 2007). in invasion and metastasis of cancer cells (Birchmeier et al., 2003; Boccaccio and Comoglio, 2006). Moreover, GTPase activation assays HGF is known to play important roles in angiogenesis, After 24 or 48 h of transfection, cells were lysed in buffer a process important for embryonic development and containing 2% IGEPAL, 50 mM Tris–HCl (pH 7.5), 10 mM tumorigenesis (Ferrara and Kerbel, 2005). As Asef2 and MgCl2 and 0.3 M NaCl. To detect the active GTP-bound form of GTPases in the cell lysates, the supernatants were mixed Neurabin2 are both expressed in endothelial cells (data with 30 mg of recombinant GST-PAK-CRIB bound to GSH- not shown), it would be interesting to examine the role Sepharose beads for 30 min. The beads and the proteins bound of the Asef2–Neurabin2 complex in endothelial cell to the fusion protein were washed thrice with wash buffer migration, angiogenesis, invasion and metastasis. containing 25m M Tris–HCl (pH 7.5), 30 mM MgCl2 and 40 mM NaCl, and then the bound proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis Materials and methods followed by immunoblotting analysis. Expression vectors and antibodies Mutant Asef2s and Neurabin2s were generated by PCR and Immunostaining subcloned into the mammalian expression vector Cells were fixed with 3.7% formaldehyde in phosphate pcDNA3.1( þ ) (Invitrogen, Carlsbad, CA, USA). Mouse buffered saline for 5min, permeabilized with 0.2% Triton monoclonal antibody (mAb) against APC was raised against X-100/phosphate buffered saline for 5min and stained with a peptide containing 119–250 amino acids of APC. Polyclonal indicated antibody for 60 min at room temperature. Staining antibody to Asef2 was prepared by immunizing rabbits with patterns obtained with these antibodies were visualized by peptide containing 1–137 amino acids of Asef2-b as described incubation with Alexa Fluor 594-conjugated goat anti-mouse earlier (Kawasaki et al., 2007). Antibodies were purified by IgG or Alexa Fluor 488-conjugated goat anti-rabbit affinity chromatography using columns, to which the antigens IgG (Invitrogen, Carlsbad, CA, USA) for 40 min at room used for immunization had been linked. Mouse polyclonal temperature. Cells were photographed with a Carl Zeiss LSM antibody to Neurabin2 (PPP1R9B polyclonal antibody; A01) 510 laser microscope (Carl Zeiss, Oberkochen, Germany). was obtained from Abnova (Walnut, CA,USA). Mouse mAb to the Myc tag (9E10) and rat mAb to the HA tag (3F10) were RNA interference experiments from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and DNA oligonucleotides encoding shRNAs were subcloned into Roche (Palo Alto, CA, USA), respectively. A mouse mAb pSuper-retro-puro (OligoEngine, Seattle, WA, USA). The against Flag tag and a mAb to a-tubulin were from Sigma (St sequence of the region targeted for shRNAs in the human Louis, MO, USA) and Oncogene Research (San Diego, CA, Neurabin2 cDNA was 50-CTGAˇ GGAATTCCAATTCTACT-30. USA), respectively. The sequence of mut-shRNA-Neurabin2 was 50-CTGATGAAT TACAATTATACT-30. Stealth siRNA duplexes against Asef2, Cell culture and transfection Neurabin2 and APC were purchased from Invitrogen. The HEK293T cells were cultured in Dulbecco’s modified Eagle’s Sequences of the regions in the human Asef2, Neurabin2 and medium supplemented with 10% fetal calf serum. HeLa cells APC cDNAs targeted for siRNAs were: Asef2-1, 50-CCAGACC were cultured in minimum essential medium (MEM) supple- CAGGAACTGGACAATCTT-30; Asef2-2, 50-CGGCAGATG mented with 10% fetal calf serum and 0.1 mM non-essential AGAGCATCCAACGTTT-30;Neurabin2-1,50-GATGGCC amino acids. SW480 cells were cultured in Leibovitzs L-15 ATCGAGGTGTTTGAGCTA-30; Neurabin2-2, 50-AGGTCA medium supplemented with 10% fetal calf serum. Plasmids ACTCGAAGCTGGTCAGCAA-30; APC-1, 50-TCGTCTG were transfected into these cells using Lipofectamine 2000 ATTCAGATTCCATCCTTT-30; and APC-2, 50-CCCACC (Invitrogen). TAATCTCAGTCCCACTATA-30. Validated Stealth negative Identification of interacting proteins control siRNA duplex with low GC content (Invitrogen) was HEK293T cells were transfected with pcDNA3.1( þ )-Flag- used as a control. Asef2-b and binding proteins were analysed by direct nano-flow liquid chromatography/electrospray tandem mass spectrometry, Cell migration assays as described earlier (Natsume et al., 2002). Cell migration assays were performed in Transwell migration chambers (diameter 6.5mm; pore size 8 mm; Costar Corpora- In vitro binding assay tion, Cambridge, MA, USA) as described earlier (Kawasaki [35S]Methionine-labeled Asef2 and Neurabin2 were synthesized et al., 2007). For HeLa cells, the top chamber was coated with by in vitro transcription–translation using the TNT-coupled 10 mg/ml fibronectin onto the underside of the filter membrane

Oncogene Role of Asef2 and Neurabin2 in HGF-induced cell migration M Sagara et al 1365 and allowed to air-dry. Both sides of the membrane were then puromycin resistance gene plasmid. The transfected cells were coated with 10 ml/ml collagen type I (Koken, Toshima-ku, cultured for 72 h in the presence of puromycin (4 mg/ml) and Tokyo, Japan) overnight. After 72 h of transfection, HeLa cells the surviving cells were stained with anti-HA antibody. The (2.0 Â 104 cells per well) were added to the upper compartment numbers of filopodia per transfected cell were counted, and the of the Transwell chamber and allowed to migrate to the mean numbers of filopodia were indicated. underside of the top chamber for 8 h. For SW480 cells, the underside of the membrane was coated with 10 mg/ml fibronectin. SW480 cells transfected with shRNA-Neurabin2 Acknowledgements were cultured for 96 h in the presence of puromycin (1.5 mg/ml) and the surviving cells were used for migration assays. Cell This work was supported by Grants-in-Aid for Scientific migration was determined by counting the cells that migrate to Research on Priority Areas and the Organization for the lower side of the polycarbonate filters. Pharmaceutical Safety and Research, Research Fellowships of the Japan Society for the Promotion of Science for Young Quantification of filopodia formation Scientists, and in part by Global COE Program (Integrative HeLa cells were transfected with Neurabin2-specific or control Life Science Based on the Study of Biosignaling Mechanisms), siRNA along with pcDNA-HA-Asef2-DABR/SH3 and a MEXT, Japan.

References

Akiyama T, Kawasaki Y. (2006). Wnt signalling and the actin Kawasaki Y, Sagara M, Shibata Y, Shirouzu M, Yokoyama S, cytoskeleton. Oncogene 25: 7538–7544. Akiyama T. (2007). Identification and characterization of Asef2, a Allen PB, Ouimet CC, Greengard P. (1997). Spinophilin, a novel guanine-nucleotide exchange factor specific for Rac1 and Cdc42. protein phosphatase 1 binding protein localized to dendritic spines. Oncogene 26: 7620–7627. Proc Natl Acad Sci USA 94: 9956–9961. Kawasaki Y, Sato R, Akiyama T. (2003). Mutated APC and Asef are Bellanger JM, Astier C, Sardet C, Ohta Y, Stossel TP, Debant A. involved in the migration of colorectal tumour cells. Nat Cell Biol 5: (2000). The Rac1- and RhoG-specific GEF domain of Trio targets 211–215. filamin to remodel cytoskeletal actin. Nat Cell Biol 2: 888–892. Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Bienz M, Clevers H. (2000). Linking colorectal cancer to Wnt Y et al. (2000). Asef, a link between the tumor suppressor APC and signaling. Cell 103: 311–320. G-protein signaling. Science 289: 1194–1197. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. (2003). Kinzler KW, Vogelstein B. (1996). Lessons from hereditary colorectal Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4: cancer. Cell 87: 159–170. 915–925. Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. (2007). Boccaccio C, Comoglio PM. (2006). Invasive growth: a MET-driven Release of autoinhibition of ASEF by APC leads to CDC42 genetic programme for cancer and stem cells. Nat Rev Cancer 6: activation and tumor suppression. Nat Struct Mol Biol 14: 814–823. 637–645. Murayama K, Shirouzu M, Kawasaki Y, Kato-Murayama M, Buchsbaum RJ, Connolly BA, Feig LA. (2003). Regulation of p70 S6 Hanawa-Suetsugu K, Sakamoto A et al. (2007). Crystal structure kinase by complex formation between the Rac guanine nucleotide of the rac activator, Asef, reveals its autoinhibitory mechanism. exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin. J J Biol Chem 282: 4238–4242. Biol Chem 278: 18833–18841. Muroya K, Kawasaki Y, Hayashi T, Ohwada S, Akiyama T. (2007). Cadigan KM, Nusse R. (1997). Wnt signaling: a common theme in PH domain-mediated membrane targeting of Asef. Biochem Biophys animal development. Genes Dev 11: 3286–3305. Res Commun 355: 85–88. Etienne-Manneville S, Hall A. (2003). Cdc42 regulates GSK-3beta and Natsume T, Yamauchi Y, Nakayama H, Shinkawa T, Yanagida M, adenomatous polyposis coli to control cell polarity. Nature 421: Takahashi N et al. (2002). A direct nanoflow liquid chromatogra- 753–756. phy-tandem mass spectrometry system for interaction proteomics. Feng J, Yan Z, Ferreira A, Tomizawa K, Liauw JA, Zhuo M et al. Anal Chem 74: 4725–4733. (2000). Spinophilin regulates the formation and function of dendritic Polakis P. (2000). Wnt signaling and cancer. Genes Dev 14: 1837–1851. spines. Proc Natl Acad Sci USA 97: 9287–9292. Ryan XP, Alldritt J, Svenningsson P, Allen PB, Wu GY, Nairn AC Ferrara N, Kerbel RS. (2005). Angiogenesis as a therapeutic target. et al. (2005). The Rho-specific GEF Lfc interacts with neurabin and Nature 438: 967–974. spinophilin to regulate dendritic spine morphology. Neuron 47: 85–100. Fodde R, Smits R, Clevers H. (2001). APC, signal transduction and Sarrouilhe D, di Tommaso A, Metaye T, Ladeveze V. (2006). genetic instability in colorectal cancer. Nat Rev Cancer 1: 55–67. Spinophilin: from partners to functions. Biochimie 88: 1099–1113. Futter M, Uematsu K, Bullock SA, Kim Y, Hemmings Jr HC, Nishi A Satoh A, Nakanishi H, Obaishi H, Wada M, Takahashi K, Satoh K et al. (2005). Phosphorylation of spinophilin by ERK and cyclin- et al. (1998). Neurabin-II/spinophilin. An actin filament-binding dependent PK 5(Cdk5). Proc Natl Acad Sci USA 102: 3489–3494. protein with one pdz domain localized at cadherin-based cell-cell Hamann MJ, Lubking CM, Luchini DN, Billadeau DD. (2007). Asef2 adhesion sites. J Biol Chem 273: 3470–3475. functions as a Cdc42 exchange factor and is stimulated by the release Schuler H, Peti W. (2008). Structure-function analysis of the of an autoinhibitory module from a concealed C-terminal activation filamentous actin binding domain of the neuronal scaffolding element. Mol Cell Biol 27: 1380–1393. protein spinophilin. FEBS J 275: 59–68. Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M. Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R et al. (2008). Phosphorylation and activation of the Rac1 and Cdc42 GEF (1995). APC binds to the novel protein EB1. Cancer Res 55: Asef in A431 cells stimulated by EGF. J Cell Sci 121(Part 16): 2972–2977. 635–642. Watanabe T, Wang S, Noritake J, Sato K, Fukata M, Takefuji M et al. Jimbo T, Kawasaki Y, Koyama R, Sato R, Takada S, Haraguchi K (2004). Interaction with IQGAP1 links APC to Rac1, Cdc42, and et al. (2002). Identification of a link between the tumour suppressor actin filaments during cell polarization and migration. Dev Cell 7: APC and the kinesin superfamily. Nat Cell Biol 4: 323–327. 871–883.

Oncogene