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Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development
Differentiation (2004) 72:48–55
r International Society of Differentiation 2004
OR IGI N A L A R T IC L E
Akira Miyakoshi . Naoto Ueno . Noriyuki Kinoshita
Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development
Received October 8, 2003; accepted in revised form January 7, 2004
Abstract During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway. . ) Akira Miyakoshi Naoto Ueno Noriyuki Kinoshita (* Department of Developmental Biology National Institute for Basic Biology 38 Nishigonaka, Myodaiji Okazaki, Aichi 444-8585, Japan Tel: 181-564-55-7573 Fax: 181-564-55-7571 e-mail: [email protected] Akira Miyakoshi Naoto Ueno Department of Molecular Biomechanics The Graduate University for Advanced Studies 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan U.S. Copyright Clearance Center Code Statement:
Key words RhoGEF NET1 RhoA Xenopus gastrulation
Introduction Rho GTPases control many actin-dependent processes such as migration, adhesion, morphogenesis, axon guidance, and phagocytosis (Hall and Nobes, 2000). In Xenopus embryos, Rho GTPases are involved in cytokinesis and cell adhesion (Kishi et al., 1993; Drechsel et al., 1997; Wunnenberg-Stapleton et al., 1999; Hens et al., 2002). In addition, Xenopus Rho GTPases play important roles in convergent extension movements during gastrulation (Habas et al., 2001, 2003; Choi and Han, 2002; Penzo-Mendez et al., 2003). In this process, cells are polarized and elongated mediolaterally, and then the cells are intercalated. This movement forms the dorsal mesodermal structure and extends the anteroposterior body axis (Wilson and Keller, 1991; Shih and Keller, 1992; Wallingford et al., 2002). The non-canonical Wnt pathway was implicated in the regulation of the convergent extension movements (Wallingford et al., 2000; Wallingford and Harland, 2001; Kuhl, 2002; Tada et al., 2002). One of the intracellular signaling components, Xenopus Dishevelled (Xdsh), plays a pivotal role in this process (Wallingford et al., 2000; Wallingford and Harland, 2001; Kuhl, 2002; Tada et al., 2002). RhoA and Rac form a complex with Xdsh and are activated by this pathway (Habas et al., 2001, 2003). RhoA and Rac also regulate cell shape, motility, and protrusive activity during this process (Tahinci and Symes, 2003). During zebrafish gastrulation, Rho kinase 2 is involved in the regulation of convergent extension movements (Marlow et al., 2002). Rho GTPases are regulated by cycling between an inactive (GDP-bound) and an active (GTP-bound) conformational state. Guanine nucleotide exchange factors (GEFs) are critical mediators of Rho GTPase
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activation (Zheng, 2001; Schmidt and Hall, 2002a). RhoGEFs stimulate the exchange of GDP for GTP to generate the active form of Rho, which is then capable of recognizing downstream targets. GEFs possess a conserved functional module that is composed of a Dbl homology (DH) domain and a pleckstrin homology (PH) domain; the PH domain is located immediately C-terminal to the DH domain. In most cases, the DH-PH module is the minimal structural unit responsible for catalyzing the GDP-GTP exchange reaction of Rho proteins (Zheng, 2001; Schmidt and Hall, 2002a). To date, numerous proteins containing the DH-PH module have been identified in many organisms. Although Rho GTPases are involved in important processes during Xenopus embryogenesis, the regulatory mechanisms of their activities are not well understood. To our knowledge, only one GEF, XGef, which activates Cdc42 and functions in oocyte maturation, has been reported in this context (Reverte et al., 2003). Because of the importance of Rho GTPases during embryogenesis, we speculated that a number of other RhoGEFs might be involved. Thus, we searched our EST database and identified several genes that encode proteins homologous to mammalian RhoGEFs. We analyzed one of these genes, designated xNET1, which is very similar to mammalian NET1. Mammalian NET1 was identified in neuroepithelioma cells as having transforming activity (Chan et al., 1996). It has also been shown to have a RhoA-specific GEF activity (Alberts and Treisman, 1998; Alberts et al., 1998). The N-terminal region of NET1 harbors a nuclear localization signal (NLS). A mutation in the NLS causes re-localization of NET1 to the cytoplasm, leading to the activation of RhoA (Schmidt and Hall, 2002b). The localization of NET1 may be important for the regulation of its function, but the regulatory mechanism is still unknown. Here, we report the identification and characterization of xNET1. Biochemical and cell biological analyses showed that xNET1 is a RhoA-specific GEF. Overexpression of wild-type or a mutant form of xNET1 impaired gastrulation movements, suggesting that it is involved in the regulation of this process.
Methods Cloning and mutagenesis of Xenopus xNET1 The clone XL015i15, containing full-length xNET1, was identified by searching our EST database (http://Xenopus.nibb.ac.jp; accessed January 9, 2004). Its coding region was amplified by polymerase chain reaction (PCR) with the following primers using Xenopus embryonic cDNA as a template: forward, 5 0 -GCCGAATTCATGGTGGCTCATGATGAAATTGGAG-3 0 ; reverse, 5 0 -GCCCTCGAGCACCAAAGTCTCTTTTTTCTGCGG-3 0 . The resulting product was cloned into the pBluescript and pCS21 vectors.
The DNLS mutants and the DH domain mutant L267E were created by a standard PCR-mediated mutagenesis method. xNET1DNLS1 is a mutant in which the amino acid sequence from residue 36 to 39 was replaced with Ala-Ile-Pro-Ala. xNET1DNLS2 is a mutant in which residues 95–97 were replaced with Ala-GlySer. xNET1DNLS112 contains both of these replacements. The DDH mutant lacks residues 252–271 in the DH domain of xNET1. To construct expression plasmids, the genes were sub-cloned into the pCS21 vector. Capped mRNA was synthesized using a Message Machine kit (Ambion, Austin, TX).
In situ hybridization and reverse transcription-PCR analysis In situ hybridization in Xenopus was carried out as described (Harland, 1991). The digoxygenin-labeled probe for xNET1 was synthesized using the coding region sub-cloned into pBluescript as a template. For reverse transcription (RT)-PCR analysis, RNA from Xenopus embryos was prepared with Trizol (Life Technologies, Carlsbad, CA). The cDNA was synthesized with reverse transcriptase (TRT-101; Toyobo, Osaka, Japan). The sequences of the primers for Histone H4 were as described (Iemura et al., 1998). The primers for xNET1 were: forward, 5 0 -GACAAATTGGAGTACCTC-3 0 ; reverse, 5 0 -CACCAAAGTCTCTTTTTTCTGCGG-3 0 .
Immunostaining of Xenopus embryos The procedure for whole-mount immunostaining was as described (Kurata and Ueno, 2003). The antibody used for staining somites was 12/101 (Development Studies Hybridoma Bank; Kintner and Brockes, 1984). Alkaline phosphatase-conjugated antibody was used as the secondary antibody. For immunocytochemistry of Xenopus embryonic cells, myctagged mRNA was injected into the animal pole of two-cell embryos. The animal caps were dissected from stage 9–10 embryos and fixed with MEMFA (0.1 M MOPS [pH 7.2], 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde; Harland, 1991), followed by immunostaining by a standard method using anti-myc 9E10 (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescencelabeled secondary antibodies. The localizations were determined by confocal laser-scanning microscopy, using a Carl Zeiss LSM510 microscope (Zeiss, Jena, Germany).
Glutathione-S-transferase-pull-down and RhoA activation assay The glutathione-S-transferase (GST)-pull-down assay was carried out basically as described (Ren et al., 1998; van Horck et al., 2001) with some modifications. Xenopus Cdc42 and RhoA were cloned by PCR using primers that were based on previously reported sequences (Wunnenberg-Stapleton et al., 1999; Choi and Han, 2002). Xenopus Rac was cloned as described (Kinoshita et al., 2003). These genes were fused with the GST gene in pGEX-6P-1 (Pharmacia, Piscataway, NJ). The fusion proteins were produced in E. coli BL21(DE3). The GST fusion proteins were immobilized with glutathione Sepharose 4B (Amersham Biosciences, Piscataway, NJ). Myc-xNET1-transfected HEK293 T cells were lysed at 41C in lysis buffer and spun at 15,000g for 10 min. The supernatant was mixed with the Sepharose beads and incubated for 4 hr at 41C. The beads were washed three times with the lysis buffer and analyzed by Western blotting using the anti-myc antibody. HEK293 T cells were transfected with the wild-type and mutant forms of xNET1 using Lipofectamine (Invitrogen, Carlsbad, CA). A pull-down assay using the RhoA-binding domain of Rhotekin (RBD; Ren et al., 1999) was carried out using a RhoA Activation
50 Assay kit (BK036; Cytoskeleton Inc., Denver, CO) following the manufacturer’s instructions.
xNET1 encodes a protein that is highly homologous to mammalian NET1 (Fig. 1A); it is 74.3% identical to human NET1. Mammalian NET1 contains two NLS
Immunoprecipitation and Western blotting HEK293 T cells were transiently transfected with the indicated constructs using Lipofectamine (Invitrogen). The myc-Xdsh was a kind gift from Dr. R.M. Harland (University of California, Berkeley). Cell lysates were prepared in PBS containing 0.1% Triton X-100, 50 mM NaF, 0.5 mM PMSF, and a 1/200 volume of protease inhibitor cocktail (P8340; Sigma, St. Louis, MO), and spun at 15,000g for 10 min. The indicated antibodies were added to the supernatants, and incubated at 41C overnight. Protein A/G agarose (SC-2003; Santa Cruz Biotechnology) was added, and the mixture was incubated for 1 hr in a tumbling mixer. The agarose beads were washed five times with the lysis buffer. The antibodies used for immunoprecipitation and Western blotting were anti-myc 9E10 (Santa Cruz Biotechnology) and anti-flag M2 (Sigma) antibodies.
Immunofluorescence microscopy of tissue culture cells Cells were fixed in PBS containing 4% paraformaldehyde for 10 min at 371C, washed with PBS, and then permeabilized with PBS containing 0.2% Triton X-100. Next, the cells were incubated with blocking buffer (PBS containing 10% goat serum) for 5 min followed by incubation in the first antibody solution (the blocking buffer containing the antibody) for 1 hr. The cells were washed with the wash buffer (PBS containing 0.2% gelatin) three times, and then incubated in the secondary antibody solution (the blocking buffer containing the secondary antibody) for 30 min. The cells were washed twice with the wash buffer, twice with PBS, and once with distilled water. They were then mounted on slides and observed with the laser-scanning confocal microscope. To stain actin filaments, FITC-phalloidin (Sigma) was added to the secondary antibody solution at a 1/500 dilution.
Results Identification of the xNET1 gene expressed in Xenopus embryos Almost all RhoGEFs contain conserved domains called Dbl homology (DH) and pleckstrin homology (PH) domains (Zheng, 2001; Schmidt and Hall, 2002a). These two domains form a module that is a minimal functional unit for the guanine nucleotide exchange reaction. We searched our EST database (http:// Xenopus.nibb.ac.jp) and identified six clones that encode proteins containing the DH-PH module. We focused on one clone because, when we expressed the DH-PH module of each clone in Xenopus embryos, we found that only one strongly inhibited gastrulation movements (data not shown; discussed below). Because gastrulation movements are also inhibited by constitutively active RhoA or Rac mutants (Habas et al., 2003; Tahinci and Symes, 2003), we thought this clone might effectively activate endogenous Rho GTPases. Thus, this clone, designated xNET1, was analyzed further, and the other genes will be reported elsewhere.
Fig. 1 Identification of xNET1 in the Xenopus EST library. (A) Sequence alignment of xNET1 with human NET1 (hNET1; Chan et al., 1996) and mouse NET1 (mNET1; Alberts and Treisman, 1998). The nuclear localization signals, NLS1 and 2, are indicated by brown lines. The DH and PH domains are indicated by blue and red lines, respectively. The star indicates the mutation site for mNET1(L321E) and xNET1(L267E). (B) Schematic illustration of the mutant forms of xNET1 that were constructed for this study. Deleted regions are shown in gray.
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Fig. 2 xNET1 expression in Xenopus embryos. (A) RT-PCR analysis of xNET1 expression during Xenopus development. H4 (Histone H4) was detected as a control. (B) Whole-mount in situ hybridization with antisense and sense xNET1 probes. A, anterior; P, posterior.
sequences in its N-terminal region (Schmidt and Hall, 2002b). The domain containing the NLSs is conserved between xNET1 and mammalian NET1. We investigated the expression pattern of xNET1 in Xenopus embryos. RT-PCR analysis revealed that it was expressed throughout embryogenesis (Fig. 2A). In situ hybridization showed that it was broadly expressed in the mesoderm and ectoderm during gastrulation (Fig. 2B). During the neurula and tailbud stages it was still broadly expressed, and the signal was more intense in the axial structures, such as neural tissues and somites. This expression pattern suggested that xNET1 might function during embryogenesis.
xNET1 specifically binds to RhoA Mouse NET1 has been shown to have a RhoA-specific GEF activity (Alberts and Treisman, 1998; Alberts et al., 1998). We tested to see whether xNET1 has the same activity. Myc-tagged xNET1 was expressed in HEK293 T cells, and a lysate was prepared from the transfected cells. We prepared bacterially produced
Fig. 3 xNET1 binds to and activates RhoA. (A) GST-RhoA, -Rac, and -Cdc42 were produced in bacteria and then bound to glutathione Sepharose beads. The cell lysate was prepared from HEK293 T cells transiently expressing xNET1. The lysates and beads were mixed, and the proteins that bound to the beads were analyzed by Western blotting. (B) HEK293 T cells were transfected with wild-type and mutant forms of xNET1. The cell lysates were mixed with the immobilized RhoA-binding domain (RBD), which specifically binds to GTP-bound RhoA. The bound proteins were analyzed by Western blotting with an anti-RhoA antibody. WT, wild-type xNET1; DNLS, xNET1 DNLS112; DDH, xNET1 lacking the DH domain.
RhoA, Rac, and Cdc42 fused with GST and immobilized them to glutathione Sepharose beads. We then tested whether xNET1 bound to the fusion proteins. As shown in Figure 3A, RhoA-GST pulled down xNET1, but Rac- and Cdc42-GST did not. We also tested the activation of RhoA in HEK293 T cells (Fig. 3B). The RBD was used to pull down the GTP-bound form of RhoA (Ren et al., 1999). When wild-type xNET1 was expressed in these cells, the amount of the active form of RhoA was increased. The mutant lacking the NLS had the same activity as wildtype xNET1. The DDH mutant, lacking a 20 amino acid sequence in the DH domain that is essential for the activity (Fig. 1B), did not activate RhoA. These results
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suggest that xNET1 may be a specific mediator of RhoA activation.
xNET1 is located in the nucleus Mouse NET1 is located in the nucleus (Schmidt and Hall, 2002b). To test whether xNET1 also shows the same localization, we expressed myc-tagged xNET1 in HeLa cells. Immunostaining with the anti-myc antibody revealed that xNET1 was located mainly in the nucleus (Fig. 4A). Mouse NET1 has two NLS sequences in the N terminus (Schmidt and Hall, 2002b), and these are conserved in xNET1 (Fig. 1A). When both of these signals were deleted (DNLS112; Fig. 1B), we found xNET1 was localized to the cytoplasm (Fig. 4A), indicating that these NLS regions are responsible for nuclear localization. In addition, xNET1DNLS112 promoted stress fiber formation, suggesting that cytoplasmic xNET1 activated RhoA (Ridley and Hall, 1992). We next tested the localization of xNET1 in Xenopus ectodermal cells. In addition to the DNLS112 mutant, we constructed DNLS1 and DNLS2 mutants that lacked only one of the two NLS sequences. All of these mutants were tagged with the myc epitope. The mRNAs encoding these proteins were injected near the animal pole of twocell embryos. Ectodermal explants (animal caps) were isolated at the blastula stage and immunostained with the anti-myc antibody. As shown in Figure 4B, the wildtype xNET1 was located in the nucleus in Xenopus embryonic cells. The DNLS1 and DNLS2 mutants were also located in the nucleus, but the DNLS112 mutant was located in the cell cortex and cytoplasm. This indicates that either of the two NLS sequences was sufficient to localize xNET1 to the nucleus. It has been shown that mouse NET1 is regulated by nuclear sequestration (Schmidt and Hall, 2002b). xNET1 may be regulated by a similar mechanism.
Overexpression of xNET1 inhibits gastrulation movements To examine the effect of xNET1 overexpression in Xenopus embryos, an mRNA encoding the full-length xNET1 was injected into the two dorsal blastomeres of four-cell embryos. Gastrulation movements were inhibited in these embryos, as was the extension of the anteroposterior body axis (Fig. 5A). The percentage of affected embryos increased with increasing doses of the mRNA. When xNET1 mRNA was injected into the ventral blastomeres, it did not inhibit gastrulation (data not shown). This phenotype suggests that overexpression of xNET1 inhibits the involution and extension of the dorsal mesodermal tissue. It has been shown that an active mutant of RhoA inhibits gastrulation movements (Habas et al., 2003; Tahinci and Symes, 2003). The
Fig. 4 xNET1 is localized to the nucleus. (A) Wild-type xNET1 (WT) and xNET1 lacking both NLS1 and 2 (DNLS112) tagged with the myc epitope were expressed in HeLa cells. Cells were stained with the anti-myc antibody and FITC-phalloidin. The scale bar represents 20 mm. (B) mRNAs encoding wild-type and mutant forms of xNET1 tagged with the myc epitope were expressed in the ectodermal cells of Xenopus embryos. Cells were immunostained with the anti-myc antibody. The scale bar represents 100 mm.
gastrulation-defective phenotype caused by the xNET1 mRNA injection may be due to the activation of RhoA in the embryos. Interestingly, overexpression of xNET1DNLS inhibited gastrulation movements more effectively than did the overexpression of wild-type xNET1 (Fig. 5A), suggesting that xNET1 might be active in the cytoplasm. A DH domain mutant of mouse NET1(L321E) loses its RhoGEF activity in vitro and in vivo (Alberts and Treisman, 1998). We made an xNET1(L267E) mutant
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Fig. 6 Flag-tagged xNET1 was co-expressed with myc-tagged Xenopus Dishevelled (myc-Xdsh) or myc-tagged green fluorescent protein (myc-GFP) in HEK293 T cells. The myc-tagged proteins were immunoprecipitated with the anti-myc antibody, and the precipitated proteins were analyzed by Western blotting.
xNET1 is co-immunoprecipitated with Dishevelled
Fig. 5 The phenotype of xNET1 mRNA-injected embryos. mRNAs encoding wild-type xNET1 (WT), DNLS112 (DNLS) mutant (A), or xNET1L267E(L267E) (B) were injected into two blastomeres of four-cell embryos. (C) Water was injected as a control and no phenotype was observed. (D) To examine mesoderm formation, the injected embryos were immunostained with the somite-specific antibody 12/101.
by replacing the corresponding leucine with glutamate in xNET1 (Figs. 1A, 1B). When this mutant was injected into the two dorsal blastomeres of four-cell embryos, it inhibited gastrulation movements and the extension of the anteroposterior axis (Fig. 5B). Because a dominantnegative mutant of RhoA inhibits gastrulation movements (Habas et al., 2003; Tahinci and Symes, 2003), xNET1(L267E) may prevent RhoA activation by inhibiting endogenous xNET1. Therefore, xNET1 may play an essential role in normal gastrulation movements. We examined the formation of somites by wholemount immunostaining with the 12/101 antibody (Kintner and Brockes, 1984; Fig. 5D). Somites differentiated in embryos that received injections of wild-type or mutant xNET1. This result suggests that the gastrulation-defective phenotype is likely to be caused by a defect, not in mesoderm induction, but in the movements of the mesodermal cells.
The non-canonical Wnt pathway has been shown to regulate gastrulation movements. RhoA is one of the downstream signaling components of this pathway. RhoA interacts with Dishevelled (Xdsh), and this interaction is mediated by Daam1 (Habas et al., 2001). The Wnt pathway activates RhoA, but RhoGEFs that function in this pathway have not yet been identified. Thus, we examined the relationship between xNET1 and the Wnt pathway and tested whether xNET1 interacts with Xdsh (Fig. 6). Flag-tagged xNET1 and myc-tagged Xdsh were expressed in HEK293 T cells and immunoprecipitated with the anti-myc antibody. When the immunoprecipitation was done with the anti-myc antibody, xNET1 was co-immunoprecipitated with myctagged Xdsh, but not with myc-tagged green fluorescent protein. This indicates that xNET1 and Xdsh can form a complex. The overexpression of xNET1 may affect this pathway, leading to the gastrulation-defective phenotype.
Discussion xNET1 is a RhoA-specific GEF In the present study, we have identified and characterized a RhoGEF, xNET1. The predicted amino acid sequence of xNET1 is highly homologous to that of mammalian NET1. In addition, it has very similar characteristics to mammalian NET1. Mouse NET1 is a RhoA-specific GEF (Alberts and Treisman, 1998; Alberts et al., 1998). xNET1 also seems
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to be RhoA specific. The in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. Using the RhoA pull-down assay, we showed that xNET1 activated endogenous RhoA in mammalian cells. Additionally, overexpression of xNET1 lacking the NLS promoted stress fiber formation in tissue culture cells, which also suggests that xNET1 activated RhoA (Ridley and Hall, 1992). NET1 contains two NLS sequences in the N-terminal region. This region is conserved in xNET1. xNET1 is localized mainly to the nucleus in mammalian tissue culture cells. When the putative NLS sequences were deleted, xNET1 localized to the cytoplasm in both mammalian tissue culture cells and in the ectodermal cells of Xenopus embryos. The xNET1DNLS mutant localized to the cytoplasm and promoted significant stress fiber formation in tissue culture cells, but wildtype xNET1 did not. In Xenopus embryos, overexpression of xNET1DNLS inhibited gastrulation movements more effectively than did the overexpression of wildtype xNET1. These results indicate that xNET1 may be active in the cytoplasm and inactive when sequestered in the nucleus. xNET1 may be regulated by a similar mechanism as mammalian NET1. It would be interesting to know how the localization of xNET1 is regulated.
Relationship between xNET1 and the non-canonical Wnt pathway RhoA is a component of the non-canonical Wnt signaling pathway. It interacts with Xdsh through a formin homology protein, Daam1 (Habas et al., 2001). The Wnt pathway has been shown to activate RhoA, which suggests that Xdsh may activate a specific RhoGEF. However, such a RhoA GEF has not been identified. We showed that xNET1 was co-immunoprecipitated with Xdsh. xNET1 is therefore a candidate molecule for the activation of RhoA by Xdsh. Xdsh is located in both the cytoplasm and nucleus (Cheyette et al., 2002). Xdsh that is located in the nucleus may interact with xNET1 in vivo. We tested to see whether activation of the non-canonical Wnt signaling pathway altered the localization of xNET1 in animal cap cells, but its location did not change in this experiment. Some other signaling pathways may regulate xNET1’s localization and cooperate with the Wnt pathway to regulate RhoA activity. It is also possible that other cytoplasmic RhoGEFs similar to xNET1 interact with Xdsh in the cytoplasm. Acknowledgments This work was supported by grants from the Ministry of Education, Science, and Culture of Japan to N.U. and N.K.
Roles of xNET1 in Xenopus development xNET1 is ubiquitously expressed throughout embryogenesis, suggesting that it functions from the early stages of development. In Xenopus development, both dominant-negative and constitutively active RhoA mutants inhibit gastrulation movements (Habas et al., 2003; Tahinci and Symes, 2003). Overexpression of xNET1 in Xenopus embryos severely impaired gastrulation movements. It is very likely that overexpression of xNET1 activated RhoA, leading to the inhibition of these movements. Interestingly, a mutant form of xNET1 that is expected to have no guanine exchange activity also inhibited gastrulation movements. The L321E mutation in the DH domain of mouse NET1 cannot activate RhoA in vitro or in vivo (Alberts and Treisman, 1998). The xNET1(L267E) mutant, in which the corresponding leucine was replaced with glutamate, inhibited gastrulation movements and the extension of the anteroposterior axis. Thus, xNET1(L267E) may inhibit the activation of endogenous xNET1 and RhoA by acting in a dominant-negative manner. It is unknown why overexpression of either wild-type or mutant forms led to the same phenotype. Not only is RhoA activity required, but also it must be tightly regulated during gastrulation movements. RhoA might be activated in a particular region of the mesodermal cell for cell polarity control and cell movements, and hyperactivation of RhoA might disrupt its polarity.
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Ridley, A.J. and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389–399. Schmidt, A. and Hall, A. (2002a) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16:1587–1609. Schmidt, A. and Hall, A. (2002b) The Rho exchange factor Net1 is regulated by nuclear sequestration. J Biol Chem 277: 14581–14588. Shih, J. and Keller, R. (1992) Cell motility driving mediolateral intercalation in explants of Xenopus laevis. Development 116: 901–914. Tada, M., Concha, M.L. and Heisenberg, C.P. (2002) Noncanonical Wnt signalling and regulation of gastrulation movements. Semin Cell Dev Biol 13:251–260. Tahinci, E. and Symes, K. (2003) Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation. Dev Biol 259:318–335. van Horck, F.P., Ahmadian, M.R., Haeusler, L.C., Moolenaar, W.H. and Kranenburg, O. (2001) Characterization of p190 RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules. J Biol Chem 276: 4948–4956. Wallingford, J.B. and Harland, R.M. (2001) Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis. Development 128:2581–2592. Wallingford, J.B., Rowning, B.A., Vogeli, K.M., Rothbacher, U., Fraser, S.E. and Harland, R.M. (2000) Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405:81–85. Wallingford, J.B., Fraser, S.E. and Harland, R.M. (2002) Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev Cell 2: 695–706. Wilson, P. and Keller, R. (1991) Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants. Development 112:289–300. Wunnenberg-Stapleton, K., Blitz, I.L., Hashimoto, C. and Cho, K.W. (1999) Involvement of the small GTPases XRhoA and XRnd1 in cell adhesion and head formation in early Xenopus development. Development 126:5339–5351. Zheng, Y. (2001) Dbl family guanine nucleotide exchange factors. Trends Biochem Sci 26:724–732.
r International Society of Differentiation 2004
OR IGI N A L A R T IC L E
Akira Miyakoshi . Naoto Ueno . Noriyuki Kinoshita
Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development
Received October 8, 2003; accepted in revised form January 7, 2004
Abstract During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway. . ) Akira Miyakoshi Naoto Ueno Noriyuki Kinoshita (* Department of Developmental Biology National Institute for Basic Biology 38 Nishigonaka, Myodaiji Okazaki, Aichi 444-8585, Japan Tel: 181-564-55-7573 Fax: 181-564-55-7571 e-mail: [email protected] Akira Miyakoshi Naoto Ueno Department of Molecular Biomechanics The Graduate University for Advanced Studies 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan U.S. Copyright Clearance Center Code Statement:
Key words RhoGEF NET1 RhoA Xenopus gastrulation
Introduction Rho GTPases control many actin-dependent processes such as migration, adhesion, morphogenesis, axon guidance, and phagocytosis (Hall and Nobes, 2000). In Xenopus embryos, Rho GTPases are involved in cytokinesis and cell adhesion (Kishi et al., 1993; Drechsel et al., 1997; Wunnenberg-Stapleton et al., 1999; Hens et al., 2002). In addition, Xenopus Rho GTPases play important roles in convergent extension movements during gastrulation (Habas et al., 2001, 2003; Choi and Han, 2002; Penzo-Mendez et al., 2003). In this process, cells are polarized and elongated mediolaterally, and then the cells are intercalated. This movement forms the dorsal mesodermal structure and extends the anteroposterior body axis (Wilson and Keller, 1991; Shih and Keller, 1992; Wallingford et al., 2002). The non-canonical Wnt pathway was implicated in the regulation of the convergent extension movements (Wallingford et al., 2000; Wallingford and Harland, 2001; Kuhl, 2002; Tada et al., 2002). One of the intracellular signaling components, Xenopus Dishevelled (Xdsh), plays a pivotal role in this process (Wallingford et al., 2000; Wallingford and Harland, 2001; Kuhl, 2002; Tada et al., 2002). RhoA and Rac form a complex with Xdsh and are activated by this pathway (Habas et al., 2001, 2003). RhoA and Rac also regulate cell shape, motility, and protrusive activity during this process (Tahinci and Symes, 2003). During zebrafish gastrulation, Rho kinase 2 is involved in the regulation of convergent extension movements (Marlow et al., 2002). Rho GTPases are regulated by cycling between an inactive (GDP-bound) and an active (GTP-bound) conformational state. Guanine nucleotide exchange factors (GEFs) are critical mediators of Rho GTPase
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activation (Zheng, 2001; Schmidt and Hall, 2002a). RhoGEFs stimulate the exchange of GDP for GTP to generate the active form of Rho, which is then capable of recognizing downstream targets. GEFs possess a conserved functional module that is composed of a Dbl homology (DH) domain and a pleckstrin homology (PH) domain; the PH domain is located immediately C-terminal to the DH domain. In most cases, the DH-PH module is the minimal structural unit responsible for catalyzing the GDP-GTP exchange reaction of Rho proteins (Zheng, 2001; Schmidt and Hall, 2002a). To date, numerous proteins containing the DH-PH module have been identified in many organisms. Although Rho GTPases are involved in important processes during Xenopus embryogenesis, the regulatory mechanisms of their activities are not well understood. To our knowledge, only one GEF, XGef, which activates Cdc42 and functions in oocyte maturation, has been reported in this context (Reverte et al., 2003). Because of the importance of Rho GTPases during embryogenesis, we speculated that a number of other RhoGEFs might be involved. Thus, we searched our EST database and identified several genes that encode proteins homologous to mammalian RhoGEFs. We analyzed one of these genes, designated xNET1, which is very similar to mammalian NET1. Mammalian NET1 was identified in neuroepithelioma cells as having transforming activity (Chan et al., 1996). It has also been shown to have a RhoA-specific GEF activity (Alberts and Treisman, 1998; Alberts et al., 1998). The N-terminal region of NET1 harbors a nuclear localization signal (NLS). A mutation in the NLS causes re-localization of NET1 to the cytoplasm, leading to the activation of RhoA (Schmidt and Hall, 2002b). The localization of NET1 may be important for the regulation of its function, but the regulatory mechanism is still unknown. Here, we report the identification and characterization of xNET1. Biochemical and cell biological analyses showed that xNET1 is a RhoA-specific GEF. Overexpression of wild-type or a mutant form of xNET1 impaired gastrulation movements, suggesting that it is involved in the regulation of this process.
Methods Cloning and mutagenesis of Xenopus xNET1 The clone XL015i15, containing full-length xNET1, was identified by searching our EST database (http://Xenopus.nibb.ac.jp; accessed January 9, 2004). Its coding region was amplified by polymerase chain reaction (PCR) with the following primers using Xenopus embryonic cDNA as a template: forward, 5 0 -GCCGAATTCATGGTGGCTCATGATGAAATTGGAG-3 0 ; reverse, 5 0 -GCCCTCGAGCACCAAAGTCTCTTTTTTCTGCGG-3 0 . The resulting product was cloned into the pBluescript and pCS21 vectors.
The DNLS mutants and the DH domain mutant L267E were created by a standard PCR-mediated mutagenesis method. xNET1DNLS1 is a mutant in which the amino acid sequence from residue 36 to 39 was replaced with Ala-Ile-Pro-Ala. xNET1DNLS2 is a mutant in which residues 95–97 were replaced with Ala-GlySer. xNET1DNLS112 contains both of these replacements. The DDH mutant lacks residues 252–271 in the DH domain of xNET1. To construct expression plasmids, the genes were sub-cloned into the pCS21 vector. Capped mRNA was synthesized using a Message Machine kit (Ambion, Austin, TX).
In situ hybridization and reverse transcription-PCR analysis In situ hybridization in Xenopus was carried out as described (Harland, 1991). The digoxygenin-labeled probe for xNET1 was synthesized using the coding region sub-cloned into pBluescript as a template. For reverse transcription (RT)-PCR analysis, RNA from Xenopus embryos was prepared with Trizol (Life Technologies, Carlsbad, CA). The cDNA was synthesized with reverse transcriptase (TRT-101; Toyobo, Osaka, Japan). The sequences of the primers for Histone H4 were as described (Iemura et al., 1998). The primers for xNET1 were: forward, 5 0 -GACAAATTGGAGTACCTC-3 0 ; reverse, 5 0 -CACCAAAGTCTCTTTTTTCTGCGG-3 0 .
Immunostaining of Xenopus embryos The procedure for whole-mount immunostaining was as described (Kurata and Ueno, 2003). The antibody used for staining somites was 12/101 (Development Studies Hybridoma Bank; Kintner and Brockes, 1984). Alkaline phosphatase-conjugated antibody was used as the secondary antibody. For immunocytochemistry of Xenopus embryonic cells, myctagged mRNA was injected into the animal pole of two-cell embryos. The animal caps were dissected from stage 9–10 embryos and fixed with MEMFA (0.1 M MOPS [pH 7.2], 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde; Harland, 1991), followed by immunostaining by a standard method using anti-myc 9E10 (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescencelabeled secondary antibodies. The localizations were determined by confocal laser-scanning microscopy, using a Carl Zeiss LSM510 microscope (Zeiss, Jena, Germany).
Glutathione-S-transferase-pull-down and RhoA activation assay The glutathione-S-transferase (GST)-pull-down assay was carried out basically as described (Ren et al., 1998; van Horck et al., 2001) with some modifications. Xenopus Cdc42 and RhoA were cloned by PCR using primers that were based on previously reported sequences (Wunnenberg-Stapleton et al., 1999; Choi and Han, 2002). Xenopus Rac was cloned as described (Kinoshita et al., 2003). These genes were fused with the GST gene in pGEX-6P-1 (Pharmacia, Piscataway, NJ). The fusion proteins were produced in E. coli BL21(DE3). The GST fusion proteins were immobilized with glutathione Sepharose 4B (Amersham Biosciences, Piscataway, NJ). Myc-xNET1-transfected HEK293 T cells were lysed at 41C in lysis buffer and spun at 15,000g for 10 min. The supernatant was mixed with the Sepharose beads and incubated for 4 hr at 41C. The beads were washed three times with the lysis buffer and analyzed by Western blotting using the anti-myc antibody. HEK293 T cells were transfected with the wild-type and mutant forms of xNET1 using Lipofectamine (Invitrogen, Carlsbad, CA). A pull-down assay using the RhoA-binding domain of Rhotekin (RBD; Ren et al., 1999) was carried out using a RhoA Activation
50 Assay kit (BK036; Cytoskeleton Inc., Denver, CO) following the manufacturer’s instructions.
xNET1 encodes a protein that is highly homologous to mammalian NET1 (Fig. 1A); it is 74.3% identical to human NET1. Mammalian NET1 contains two NLS
Immunoprecipitation and Western blotting HEK293 T cells were transiently transfected with the indicated constructs using Lipofectamine (Invitrogen). The myc-Xdsh was a kind gift from Dr. R.M. Harland (University of California, Berkeley). Cell lysates were prepared in PBS containing 0.1% Triton X-100, 50 mM NaF, 0.5 mM PMSF, and a 1/200 volume of protease inhibitor cocktail (P8340; Sigma, St. Louis, MO), and spun at 15,000g for 10 min. The indicated antibodies were added to the supernatants, and incubated at 41C overnight. Protein A/G agarose (SC-2003; Santa Cruz Biotechnology) was added, and the mixture was incubated for 1 hr in a tumbling mixer. The agarose beads were washed five times with the lysis buffer. The antibodies used for immunoprecipitation and Western blotting were anti-myc 9E10 (Santa Cruz Biotechnology) and anti-flag M2 (Sigma) antibodies.
Immunofluorescence microscopy of tissue culture cells Cells were fixed in PBS containing 4% paraformaldehyde for 10 min at 371C, washed with PBS, and then permeabilized with PBS containing 0.2% Triton X-100. Next, the cells were incubated with blocking buffer (PBS containing 10% goat serum) for 5 min followed by incubation in the first antibody solution (the blocking buffer containing the antibody) for 1 hr. The cells were washed with the wash buffer (PBS containing 0.2% gelatin) three times, and then incubated in the secondary antibody solution (the blocking buffer containing the secondary antibody) for 30 min. The cells were washed twice with the wash buffer, twice with PBS, and once with distilled water. They were then mounted on slides and observed with the laser-scanning confocal microscope. To stain actin filaments, FITC-phalloidin (Sigma) was added to the secondary antibody solution at a 1/500 dilution.
Results Identification of the xNET1 gene expressed in Xenopus embryos Almost all RhoGEFs contain conserved domains called Dbl homology (DH) and pleckstrin homology (PH) domains (Zheng, 2001; Schmidt and Hall, 2002a). These two domains form a module that is a minimal functional unit for the guanine nucleotide exchange reaction. We searched our EST database (http:// Xenopus.nibb.ac.jp) and identified six clones that encode proteins containing the DH-PH module. We focused on one clone because, when we expressed the DH-PH module of each clone in Xenopus embryos, we found that only one strongly inhibited gastrulation movements (data not shown; discussed below). Because gastrulation movements are also inhibited by constitutively active RhoA or Rac mutants (Habas et al., 2003; Tahinci and Symes, 2003), we thought this clone might effectively activate endogenous Rho GTPases. Thus, this clone, designated xNET1, was analyzed further, and the other genes will be reported elsewhere.
Fig. 1 Identification of xNET1 in the Xenopus EST library. (A) Sequence alignment of xNET1 with human NET1 (hNET1; Chan et al., 1996) and mouse NET1 (mNET1; Alberts and Treisman, 1998). The nuclear localization signals, NLS1 and 2, are indicated by brown lines. The DH and PH domains are indicated by blue and red lines, respectively. The star indicates the mutation site for mNET1(L321E) and xNET1(L267E). (B) Schematic illustration of the mutant forms of xNET1 that were constructed for this study. Deleted regions are shown in gray.
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Fig. 2 xNET1 expression in Xenopus embryos. (A) RT-PCR analysis of xNET1 expression during Xenopus development. H4 (Histone H4) was detected as a control. (B) Whole-mount in situ hybridization with antisense and sense xNET1 probes. A, anterior; P, posterior.
sequences in its N-terminal region (Schmidt and Hall, 2002b). The domain containing the NLSs is conserved between xNET1 and mammalian NET1. We investigated the expression pattern of xNET1 in Xenopus embryos. RT-PCR analysis revealed that it was expressed throughout embryogenesis (Fig. 2A). In situ hybridization showed that it was broadly expressed in the mesoderm and ectoderm during gastrulation (Fig. 2B). During the neurula and tailbud stages it was still broadly expressed, and the signal was more intense in the axial structures, such as neural tissues and somites. This expression pattern suggested that xNET1 might function during embryogenesis.
xNET1 specifically binds to RhoA Mouse NET1 has been shown to have a RhoA-specific GEF activity (Alberts and Treisman, 1998; Alberts et al., 1998). We tested to see whether xNET1 has the same activity. Myc-tagged xNET1 was expressed in HEK293 T cells, and a lysate was prepared from the transfected cells. We prepared bacterially produced
Fig. 3 xNET1 binds to and activates RhoA. (A) GST-RhoA, -Rac, and -Cdc42 were produced in bacteria and then bound to glutathione Sepharose beads. The cell lysate was prepared from HEK293 T cells transiently expressing xNET1. The lysates and beads were mixed, and the proteins that bound to the beads were analyzed by Western blotting. (B) HEK293 T cells were transfected with wild-type and mutant forms of xNET1. The cell lysates were mixed with the immobilized RhoA-binding domain (RBD), which specifically binds to GTP-bound RhoA. The bound proteins were analyzed by Western blotting with an anti-RhoA antibody. WT, wild-type xNET1; DNLS, xNET1 DNLS112; DDH, xNET1 lacking the DH domain.
RhoA, Rac, and Cdc42 fused with GST and immobilized them to glutathione Sepharose beads. We then tested whether xNET1 bound to the fusion proteins. As shown in Figure 3A, RhoA-GST pulled down xNET1, but Rac- and Cdc42-GST did not. We also tested the activation of RhoA in HEK293 T cells (Fig. 3B). The RBD was used to pull down the GTP-bound form of RhoA (Ren et al., 1999). When wild-type xNET1 was expressed in these cells, the amount of the active form of RhoA was increased. The mutant lacking the NLS had the same activity as wildtype xNET1. The DDH mutant, lacking a 20 amino acid sequence in the DH domain that is essential for the activity (Fig. 1B), did not activate RhoA. These results
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suggest that xNET1 may be a specific mediator of RhoA activation.
xNET1 is located in the nucleus Mouse NET1 is located in the nucleus (Schmidt and Hall, 2002b). To test whether xNET1 also shows the same localization, we expressed myc-tagged xNET1 in HeLa cells. Immunostaining with the anti-myc antibody revealed that xNET1 was located mainly in the nucleus (Fig. 4A). Mouse NET1 has two NLS sequences in the N terminus (Schmidt and Hall, 2002b), and these are conserved in xNET1 (Fig. 1A). When both of these signals were deleted (DNLS112; Fig. 1B), we found xNET1 was localized to the cytoplasm (Fig. 4A), indicating that these NLS regions are responsible for nuclear localization. In addition, xNET1DNLS112 promoted stress fiber formation, suggesting that cytoplasmic xNET1 activated RhoA (Ridley and Hall, 1992). We next tested the localization of xNET1 in Xenopus ectodermal cells. In addition to the DNLS112 mutant, we constructed DNLS1 and DNLS2 mutants that lacked only one of the two NLS sequences. All of these mutants were tagged with the myc epitope. The mRNAs encoding these proteins were injected near the animal pole of twocell embryos. Ectodermal explants (animal caps) were isolated at the blastula stage and immunostained with the anti-myc antibody. As shown in Figure 4B, the wildtype xNET1 was located in the nucleus in Xenopus embryonic cells. The DNLS1 and DNLS2 mutants were also located in the nucleus, but the DNLS112 mutant was located in the cell cortex and cytoplasm. This indicates that either of the two NLS sequences was sufficient to localize xNET1 to the nucleus. It has been shown that mouse NET1 is regulated by nuclear sequestration (Schmidt and Hall, 2002b). xNET1 may be regulated by a similar mechanism.
Overexpression of xNET1 inhibits gastrulation movements To examine the effect of xNET1 overexpression in Xenopus embryos, an mRNA encoding the full-length xNET1 was injected into the two dorsal blastomeres of four-cell embryos. Gastrulation movements were inhibited in these embryos, as was the extension of the anteroposterior body axis (Fig. 5A). The percentage of affected embryos increased with increasing doses of the mRNA. When xNET1 mRNA was injected into the ventral blastomeres, it did not inhibit gastrulation (data not shown). This phenotype suggests that overexpression of xNET1 inhibits the involution and extension of the dorsal mesodermal tissue. It has been shown that an active mutant of RhoA inhibits gastrulation movements (Habas et al., 2003; Tahinci and Symes, 2003). The
Fig. 4 xNET1 is localized to the nucleus. (A) Wild-type xNET1 (WT) and xNET1 lacking both NLS1 and 2 (DNLS112) tagged with the myc epitope were expressed in HeLa cells. Cells were stained with the anti-myc antibody and FITC-phalloidin. The scale bar represents 20 mm. (B) mRNAs encoding wild-type and mutant forms of xNET1 tagged with the myc epitope were expressed in the ectodermal cells of Xenopus embryos. Cells were immunostained with the anti-myc antibody. The scale bar represents 100 mm.
gastrulation-defective phenotype caused by the xNET1 mRNA injection may be due to the activation of RhoA in the embryos. Interestingly, overexpression of xNET1DNLS inhibited gastrulation movements more effectively than did the overexpression of wild-type xNET1 (Fig. 5A), suggesting that xNET1 might be active in the cytoplasm. A DH domain mutant of mouse NET1(L321E) loses its RhoGEF activity in vitro and in vivo (Alberts and Treisman, 1998). We made an xNET1(L267E) mutant
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Fig. 6 Flag-tagged xNET1 was co-expressed with myc-tagged Xenopus Dishevelled (myc-Xdsh) or myc-tagged green fluorescent protein (myc-GFP) in HEK293 T cells. The myc-tagged proteins were immunoprecipitated with the anti-myc antibody, and the precipitated proteins were analyzed by Western blotting.
xNET1 is co-immunoprecipitated with Dishevelled
Fig. 5 The phenotype of xNET1 mRNA-injected embryos. mRNAs encoding wild-type xNET1 (WT), DNLS112 (DNLS) mutant (A), or xNET1L267E(L267E) (B) were injected into two blastomeres of four-cell embryos. (C) Water was injected as a control and no phenotype was observed. (D) To examine mesoderm formation, the injected embryos were immunostained with the somite-specific antibody 12/101.
by replacing the corresponding leucine with glutamate in xNET1 (Figs. 1A, 1B). When this mutant was injected into the two dorsal blastomeres of four-cell embryos, it inhibited gastrulation movements and the extension of the anteroposterior axis (Fig. 5B). Because a dominantnegative mutant of RhoA inhibits gastrulation movements (Habas et al., 2003; Tahinci and Symes, 2003), xNET1(L267E) may prevent RhoA activation by inhibiting endogenous xNET1. Therefore, xNET1 may play an essential role in normal gastrulation movements. We examined the formation of somites by wholemount immunostaining with the 12/101 antibody (Kintner and Brockes, 1984; Fig. 5D). Somites differentiated in embryos that received injections of wild-type or mutant xNET1. This result suggests that the gastrulation-defective phenotype is likely to be caused by a defect, not in mesoderm induction, but in the movements of the mesodermal cells.
The non-canonical Wnt pathway has been shown to regulate gastrulation movements. RhoA is one of the downstream signaling components of this pathway. RhoA interacts with Dishevelled (Xdsh), and this interaction is mediated by Daam1 (Habas et al., 2001). The Wnt pathway activates RhoA, but RhoGEFs that function in this pathway have not yet been identified. Thus, we examined the relationship between xNET1 and the Wnt pathway and tested whether xNET1 interacts with Xdsh (Fig. 6). Flag-tagged xNET1 and myc-tagged Xdsh were expressed in HEK293 T cells and immunoprecipitated with the anti-myc antibody. When the immunoprecipitation was done with the anti-myc antibody, xNET1 was co-immunoprecipitated with myctagged Xdsh, but not with myc-tagged green fluorescent protein. This indicates that xNET1 and Xdsh can form a complex. The overexpression of xNET1 may affect this pathway, leading to the gastrulation-defective phenotype.
Discussion xNET1 is a RhoA-specific GEF In the present study, we have identified and characterized a RhoGEF, xNET1. The predicted amino acid sequence of xNET1 is highly homologous to that of mammalian NET1. In addition, it has very similar characteristics to mammalian NET1. Mouse NET1 is a RhoA-specific GEF (Alberts and Treisman, 1998; Alberts et al., 1998). xNET1 also seems
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to be RhoA specific. The in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. Using the RhoA pull-down assay, we showed that xNET1 activated endogenous RhoA in mammalian cells. Additionally, overexpression of xNET1 lacking the NLS promoted stress fiber formation in tissue culture cells, which also suggests that xNET1 activated RhoA (Ridley and Hall, 1992). NET1 contains two NLS sequences in the N-terminal region. This region is conserved in xNET1. xNET1 is localized mainly to the nucleus in mammalian tissue culture cells. When the putative NLS sequences were deleted, xNET1 localized to the cytoplasm in both mammalian tissue culture cells and in the ectodermal cells of Xenopus embryos. The xNET1DNLS mutant localized to the cytoplasm and promoted significant stress fiber formation in tissue culture cells, but wildtype xNET1 did not. In Xenopus embryos, overexpression of xNET1DNLS inhibited gastrulation movements more effectively than did the overexpression of wildtype xNET1. These results indicate that xNET1 may be active in the cytoplasm and inactive when sequestered in the nucleus. xNET1 may be regulated by a similar mechanism as mammalian NET1. It would be interesting to know how the localization of xNET1 is regulated.
Relationship between xNET1 and the non-canonical Wnt pathway RhoA is a component of the non-canonical Wnt signaling pathway. It interacts with Xdsh through a formin homology protein, Daam1 (Habas et al., 2001). The Wnt pathway has been shown to activate RhoA, which suggests that Xdsh may activate a specific RhoGEF. However, such a RhoA GEF has not been identified. We showed that xNET1 was co-immunoprecipitated with Xdsh. xNET1 is therefore a candidate molecule for the activation of RhoA by Xdsh. Xdsh is located in both the cytoplasm and nucleus (Cheyette et al., 2002). Xdsh that is located in the nucleus may interact with xNET1 in vivo. We tested to see whether activation of the non-canonical Wnt signaling pathway altered the localization of xNET1 in animal cap cells, but its location did not change in this experiment. Some other signaling pathways may regulate xNET1’s localization and cooperate with the Wnt pathway to regulate RhoA activity. It is also possible that other cytoplasmic RhoGEFs similar to xNET1 interact with Xdsh in the cytoplasm. Acknowledgments This work was supported by grants from the Ministry of Education, Science, and Culture of Japan to N.U. and N.K.
Roles of xNET1 in Xenopus development xNET1 is ubiquitously expressed throughout embryogenesis, suggesting that it functions from the early stages of development. In Xenopus development, both dominant-negative and constitutively active RhoA mutants inhibit gastrulation movements (Habas et al., 2003; Tahinci and Symes, 2003). Overexpression of xNET1 in Xenopus embryos severely impaired gastrulation movements. It is very likely that overexpression of xNET1 activated RhoA, leading to the inhibition of these movements. Interestingly, a mutant form of xNET1 that is expected to have no guanine exchange activity also inhibited gastrulation movements. The L321E mutation in the DH domain of mouse NET1 cannot activate RhoA in vitro or in vivo (Alberts and Treisman, 1998). The xNET1(L267E) mutant, in which the corresponding leucine was replaced with glutamate, inhibited gastrulation movements and the extension of the anteroposterior axis. Thus, xNET1(L267E) may inhibit the activation of endogenous xNET1 and RhoA by acting in a dominant-negative manner. It is unknown why overexpression of either wild-type or mutant forms led to the same phenotype. Not only is RhoA activity required, but also it must be tightly regulated during gastrulation movements. RhoA might be activated in a particular region of the mesodermal cell for cell polarity control and cell movements, and hyperactivation of RhoA might disrupt its polarity.
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