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The assembly of POSH-JNK regulates Xenopus anterior neural development
Developmental Biology 286 (2005) 256 – 269 www.elsevier.com/locate/ydbio
The assembly of POSH-JNK regulates Xenopus anterior neural development Gun-Hwa Kim, Eunjoo Park, Jin-Kwan Han* Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang, Kyungbuk 790-784, Republic of Korea Received for publication 20 April 2005, revised 14 July 2005, accepted 26 July 2005 Available online 26 August 2005
Abstract POSH (Plenty of SH3s) has distinct roles as a scaffold for specific c-Jun N-terminal kinase (JNK) signaling modules and as an E3 ubiquitin ligase. The physiological function of POSH remains unclear, however, and its possible involvement in developmental processes motivated the present study wherein the Xenopus orthologue of POSH (xPOSH) was examined for its potential role during Xenopus early embryogenesis. Loss-of-function analysis using morpholino oligonucleotides demonstrated that POSH was essential for Xenopus anterior neural development, although not Spemann organizer formation and early neurogenesis, through the formation of an active JNK signaling complex. Moreover, POSH-mediated JNK pathway was essential for apoptosis in anterior neural tissues. Finally, the present findings demonstrate that RING (Really Interesting New Gene) domain-mediated E3 ubiquitin ligase activity of POSH was not involved in POSHmediated JNK pathway in vivo. Together, these data suggest that the active JNK signaling complex formed of POSH and the JNK module is essential for the expression of anterior neural genes and apoptosis in Xenopus anterior development. D 2005 Elsevier Inc. All rights reserved. Keywords: POSH; Scaffold; JNK; Xenopus; Development; Apoptosis; Ubiquitin ligase
Introduction The c-Jun N-terminal kinase (JNK), one submember of a conserved family of mitogen-activated protein kinases (MAPK), is a serine/threonine kinase that mediates the response of cells to environmental stresses and many extracellular stimuli, including growth factors or inflammatory cytokines, for the regulation of cellular proliferation, differentiation, development, immune response, and apoptosis (Davis, 2000; Dong et al., 2002; Weston and Davis, 2002). JNK exists as three unique isoforms (JNK1, JNK2, and JNK3) and is activated by a specific protein kinase cascade module. This signaling module can be established through the sequential actions of a MAP kinase– kinase– kinase (MAPKKK), a MAP kinase –kinase (MAPKK), and a MAPK. The known MAPKKKs for activation of the JNK
* Corresponding author. Fax: +82 54 279 2199. E-mail address: [email protected] (J.-K. Han). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.07.033
pathway are MEKKs, TAK1, MLKs, and ASK1. The known MAPKKs for JNK are MKK4 and MKK7 (Weston and Davis, 2002). The diversity of signals transmitted by the JNK pathway yields a variety of biological responses, and this requires that functional signaling via the cascade be highly specified and precisely regulated. One mechanism that permits specificity and regulation of JNK signals is to employ scaffold proteins that organize multi-protein complexes via interaction between the various JNK modules (Morrison and Davis, 2003). Indeed, mathematical modeling indicates that formation of scaffold – kinase complexes can be used effectively to regulate the specificity, efficiency, and amplitude of signal propagation with MAPK modules (Levchenko et al., 2000). Studies in Saccharomyces cerevisiae have established that scaffold proteins, including Ste5p in mating response and Pbs2p in osmoregulation, can be physiologically critical components for the regulation of MAPK modules (Bardwell et al., 2001; Elion, 2001; Posas and Saito, 1997).
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The role of scaffold proteins in JNK activation in response to specific stimuli has also been identified in higher eukaryotes. h-arrestin 2 (McDonald et al., 2000), CrkII (Girardin and Yaniv, 2001), Filamin (Marti et al., 1997), IKAP (Holmberg et al., 2002), JNK interacting proteins (JIPs) (Ito et al., 1999; Kelkar et al., 2000; Lee et al., 2002; Whitmarsh et al., 1998; Yasuda et al., 1999), SKRP1/MKPX phosphatases (Chen et al., 2002; Zama et al., 2002), and POSH (Plenty of SH3s) (Xu et al., 2003) all serve as scaffold proteins for regulation of the JNK signaling pathway. While recent biochemical studies have characterized many of these scaffold proteins for their roles in organizing multi-protein complexes with certain JNK modules in the JNK pathway, little is known about their physiological functions in vivo (Morrison and Davis, 2003). POSH – comprised of a RING (Really Interesting New Gene)-finger domain, a putative active-Rac1 binding domain (RBD), and four SH3 (Src Homology 3) domains – acts as a scaffold in a multi-protein complex that links activated Rac1 and downstream modules of the JNK cascade (Tapon et al., 1998; Xu et al., 2003). POSH binds Rac1, JNK1/2, MKK4/7, and MLKs and is localized in lamellipodia by active Rac1 (Figueroa et al., 2003; Xu et al., 2003). Moreover, Akt2 negatively regulates assembly of an active JNK signaling complex by POSH (Figueroa et al., 2003). POSH was also known to be essential for HIV-1 production in infected cells (Alroy et al., 2005). However, none of these findings reveal anything about the endogenous role of POSH. In an attempt to elucidate the endogenous role of POSH in developmental processes in vivo, we examined its potential role of the Xenopus orthologue of POSH (xPOSH) during Xenopus early embryogenesis. POSH was found to be expressed mainly in the mesoderm and ectoderm that eventually become the cement gland, brain, somite, notochord, and pronephros. Analysis with antisense morpholino oligonucleotides demonstrated that POSH was essential for Xenopus anterior neural development through an active JNK signaling. Moreover, POSH-mediated JNK pathway was essential for apoptosis (programmed cell death) in head and eye regions. Finally, the present findings demonstrate that RING domain of POSH was not involved in POSHmediated JNK pathway.
Materials and methods
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Modified Ringer (MR) containing 4% Ficoll-400 (Sigma) using a Nanoliter Injector (WPI). Injected embryos were cultured in 0.33 MR until stage 8 and then transferred to 0.1 MR until they had reached the appropriate stage for the experimentation outlined below. Plasmids, RNA synthesis, and morpholino oligonucleotides For expression in Xenopus embryos, the entire coding region of xPOSH was cloned into the EcoRI and XbaI sites of the EGFP-pCS2+ vector and into the BamHI and ClaI sites of the Myc-pCS2+ vector. Capped mRNAs were synthesized from linearized plasmids using the mMessage mMachine kit (Ambion). GFP-xPOSH was linearized with ApaI, and mRNA was synthesized using the SP6 mMessage mMachine kit. Myc-xPOSH was linearized with NotI and transcribed using the same kit. The Xenopus JNK1 constructs were previously described (Kim and Han, 2005). Antisense morpholino oligonucleotides (MO) were obtained from Gene Tools. The morpholino oligonucleotide sequences were, for xPOSH MO, 5V-ATGCTTCTT TCCATGCAAAACTGTG-3V, and, for control MO, 5VCCTCTTACCTCAGTTACAATTTATA-3V. For xPOSH MO specificity, 5V-UTR and ORF xPOSH-Myc mRNA were injected with xPOSH MO or control MO into the animal region of embryos at the four-cell stage. Animal cap explants isolated at early gastrula stages were then subjected to immunoblot (IB) analysis with anti-Myc antibodies. Actin served as a specificity control. In situ hybridization and RT-PCR Whole-mount in situ hybridization was performed with digoxigenin (DIG)-labeled probes as described by Harland (1991). An antisense in situ probe against xPOSH was generated by linearizing the pGEM-T xPOSH construct with ApaI and transcribing with the SP6 RNA polymerase. RT-PCR analyses were carried out as described elsewhere (Kim and Han, in press). The forward and reverse primers for xPOSH were 5V-CAGCCGCCACCTCAATGCAA-3V and 5V-TGCCACTATGCGATGTGCCAGA-3V, respectively. Primers for Chordin, Cerberus, NCAM, XAG1, HoxB9, and Goosecoid were used as described online by De Robertis (www.hhmi.ucla.edu/derobertis/index.html). Primers for ODC, Otx2, En2, and Krox20 were also used as described online (Xenbase, www.xenbase.org/xmmr/Marker_pages/ primers.html).
Xenopus embryos and microinjection Immunoprecipitation and immunoblotting Xenopus laevis was purchased from Xenopus I (Ann Arbor, MI). Eggs were obtained from X. laevis primed with 800 units of human chorionic gonadotropin (Sigma). In vitro fertilization was performed as described previously (Newport and Kirschner, 1982), and developmental stages of the embryos were determined according to Nieuwkoop and Faber (1967). Microinjection was carried out in 0.33
HEK293FT cells (Invitrogen) were transfected with the indicated constructs for 48 h using Lipofectamine-Plus Reagent (Invitrogen) and then resuspended in immunoprecipitation (IP) buffer [50 mM HEPES/NaOH (pH 7.5), 3 mM EDTA, 3 mM CaCl2, 80 mM NaCl, 1% Triton X-100, 5 mM DTT]. Cells were disrupted and centrifuged so as to remove
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insoluble debris. The indicated antibodies were added to the supernatants and incubated at 4-C for 6 h. Protein A sepharose (Zymed) was added and the mixture was incubated at 4-C for 3 h. The immunocomplexes bound to Protein A beads were washed five times with IP buffer. Rabbit anti-myc polyclonal, mouse anti-GFP monoclonal, mouse anti-HA monoclonal (Santa Cruz Biotechnology), and mouse antiFlag monoclonal antibodies (Sigma) were used for immunoprecipitation and immunoblot analysis.
and then lysed in 1% lysis triton buffer. The samples were subsequently analyzed by 10% SDS-PAGE and transferred to a nitrocellulose (NC) membrane. Replicated membranes were blotted with anti-phospho-JNK antibodies (Cell Signaling) for activated JNK, and anti-Flag antibodies (Sigma) or anti-JNK antibodies (Cell Signaling) for total JNK. The quantity of protein in each sample was determined using BCA protein assay reagent (Pierce). TUNEL assay
JNK assays For JNK assays, HEK293FT cells and Xenopus embryos were transfected or injected with the indicated constructs
For the section in situ TUNEL staining, all embryos were fixed in MEMFA [0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO4, and 4% formaldehyde] for 1 h at room
Fig. 1. Expression pattern and antisense morpholino of Xenopus POSH (xPOSH). (A) RT-PCR analysis of temporal expression of POSH. Developmental stages are shown above each lane. Ornithine decarboxylase (ODC) was used as a loading control. (B) In situ hybridization analysis of spatial expression of POSH during early Xenopus development. (C) Scheme of the myc-tagged 5V-UTR POSH construct, the myc-tagged ORF POSH construct, and the morpholino sequence targeting the POSH 5V-UTR sequences. (D) xPOSH MO specifically inhibits the translation of its cognate mRNA. 5V-UTR (500 pg) and ORF (500 pg) xPOSH-Myc mRNA were injected with xPOSH MO (20 ng) or control MO (20 ng) into the animal region of embryos at the four-cell stage. Animal cap explants isolated at early gastrula stages were then subjected to immunoblot (IB) analysis with anti-Myc antibodies. Actin served as a specificity control.
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temperature and stored in 100% methanol at 20-C. Fixed embryos were embedded in paraffin. The blocks were sectioned with thickness of 10 – 12 Am. Sections were
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dewaxed in Xelene, detected by an in situ Cell Death Detection Kit (Roche), mounted, and analyzed on a ZEISS fluorescent microscope. Images were collected on ZEISS
Fig. 2. POSH depletion causes defective head and eye formation in Xenopus embryogenesis. (A) xPOSH MO led to a strong disruption of head and eye formation in a dose-dependent manner. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. Control (Co) MO and xPOSH MO were injected into two blastomeres of four-cell stage embryos. (B – C) Hematoxylin and eosin-stained transverse section of the head region of a tadpole embryo (a dashed line of the inner box). (B – C) xPOSH MO (20 ng) induced a reduction of eye and neural tube formation (C), while Co MO (20 ng; B) did not. (D) Coinjection of xPOSH MO (20 ng) with ORF xPOSH rescued the phenotypic defects of xPOSH MO in a dose-dependent manner. (E) Quantitative results of relative rescue in whole embryos shown in panel D.
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AxioCam MRc camera and processed using Photoshop software.
Results POSH knockdown perturbs Xenopus development To elucidate the potential role of Xenopus POSH (xPOSH: GenBank accession numberAY762093) in devel-
opmental processes in vivo, the spatiotemporal expression pattern of POSH was first analyzed during Xenopus embryogenesis. Temporally, xPOSH is expressed both maternally and zygotically throughout early development (Fig. 1A). As shown in Fig. 1B, POSH was visible in the animal hemisphere of the embryo during the cleavage and blastula stages. At the gastrula stages, POSH is expressed broadly throughout the marginal zone and animal pole tissues. At the neurula stages, it shows the restricted expression in dorsal tissue. At later stages, POSH is
Fig. 3. POSH is required for Xenopus anterior neural development, but not organizer formation and early neurogenesis. (A – B) xPOSH depletion reduced several anterior neural markers. (A) Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with xPOSH MO (20 ng) and Co MO (20 ng), allowed to develop to stage 22, and analyzed by RT-PCR for expression of the indicated markers. ODC was used as a loading control. ( ) RT denotes the no reverse transcription control sample. (B) Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO (20 ng), and analyzed by whole-mount in situ hybridization for of the regional neural markers Otx2, En2, or Krox20. Nuclear h-gal mRNA (200 pg) was coinjected to allow identification of the xPOSH MO-injected side as a lineage tracer. No; not injected, Inj; injected. (C) RT-PCR analysis showed xPOSH also did not affect the organizer markers. Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with xPOSH MO (20 ng), Co MO (20 ng), and xPOSH mRNA (2 ng). Dorsal marginal zone (DMZ) explants were isolated at stage 10.5 and analyzed by RT-PCR, using primers specific for Goosecoid (Gsc), Chordin (Chd), and Cerberus (Cer). ODC was used as a loading control. ( ) RT denotes the no reverse transcription control sample. (D) xPOSH MO did not alter expression of the organizer markers Cer or Goosecoid, and early neural marker Sox2. Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO (20 ng), cultured until stage 10.5 (left and middle) or stage 13 (right). Nuclear h-gal mRNA (200 pg) was used as a lineage tracer. No; not injected, Inj; injected.
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expressed in the cement gland, brain, somite, notochord, and pronephros. Antisense morpholino oligonucleotides (MO) are extremely useful as efficient blockers of specific mRNA translation (Heasman, 2002). To investigate the endogenous role of POSH during Xenopus early embryogenesis, a morpholino oligonucleotide was designed to be complementary to the sequence between bases 36 and 12 of the 5VUTR region of POSH mRNA (xPOSH MO; Fig. 1C). It was then possible to determine the efficiency and specificity of xPOSH MO in inhibiting translation of POSH mRNA in Xenopus embryos. xPOSH MO effectively blocked translation of mRNA containing the 5V-UTR sequences complementary to its morpholino oligonucleotide, but translation of mRNA containing only the open reading frame (ORF) of the protein was not affected. Control MO (Co MO) had no effect on translation of the POSH protein, and neither xPOSH MO nor Co MO inhibited actin translation, a specificity control (Fig. 1D). Injection of xPOSH MO into two dorsal blastomeres of embryos at the four-cell stage caused anterior truncations, microcephaly, and eye defects in a dose-dependent manner (Fig. 2A), but overexpression of ORF xPOSH did not exhibit these phenotypic defects (data not shown). Hematoxylin and an eosin-stained transverse section of the head region of a tadpole-stage embryo showed that POSH depletion led to defects in the eye and neural tube structure (Fig. 2C). The phenotypic defects caused by xPOSH MO were efficiently rescued by coinjection of xPOSH MO with ORF xPOSH mRNA in a dose-dependent manner (Figs. 2D and E), indicating that these developmental defects are specifically mediated by the effects of xPOSH MO.
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injected side (Fig. 3B), indicating that POSH is required to regulate Xenopus anterior neural development. As head and eye formation requires early action of the components established by the Spemann organizer, a dorsal signaling center in vertebrates (De Robertis and Kuroda, 2004), the disruption of anterior neural tissues in xPOSH MO-depleted embryos may be caused by impairment of the organizer. Analysis of the expression of organizer markers, Cerberus (Cer), Goosecoid (Gsc), and Chordin (Chd), was
POSH regulates Xenopus anterior neural development through JNK signaling pathway To characterize the head and eye defects in POSHdepleted embryos at the molecular level, RT-PCR analysis was carried out with several marker genes. The level of expression of the pan-neural marker NCAM (Kintner and Melton, 1987) was reduced by the xPOSH MO. XBF1, a forebrain marker (Bourguignon et al., 1998), and Otx2, a forebrain – midbrain and eye marker (Pannese et al., 1995), were also decreased in xPOSH MO-injected embryo. Moreover, expression of the mid – hindbrain boundary marker En2 (Hemmati-Brivanlou et al., 1991) was also reduced as well as the hindbrain marker Krox20 (Bradley et al., 1993). Conversely, the posterior neural marker HoxB9 (Wright et al., 1990) was not altered in xPOSH-depleted embryo (Fig. 3A). We further performed whole-mount in situ hybridization analysis using anterior neural markers. Four-cell stage embryos were injected at their single dorsal blastomere with xPOSH MO and nuclear h-gal mRNA as a lineage tracer. Consistent with RT-PCR results, expression of Otx2, En2, and Krox20 was reduced on the xPOSH MO-
Fig. 4. POSH regulates Xenopus anterior neural development through JNK signaling pathway. (A – D) Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs. (A) Coinjection of xPOSH MO (20 ng) with XeJNK1 rescued the phenotypic defects of xPOSH MO in a dose-dependent manner. In addition, coinjection of JNK1 and CO MO did not exhibit the anterior neural defects. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. (B – E) Wholemount in situ hybridization with Otx2, En2, and Krox20 as probes. The reduction of anterior neural markers caused by xPOSH MO (20 ng; C) was reversed by XeJNK1 mRNA (2 ng; D). Coinjection of JNK1 and CO MO did not alter the expression of anterior neural genes (E).
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conducted by RT-PCR. The expression of Cer, Chd, and Gsc in the xPOSH MO-injected embryos was not altered compared to that in the Co MO or ORF xPOSH-injected embryos (Fig. 3C). Likewise, whole-mount in situ hybridization analysis also demonstrated that xPOSH did not affect formation of the organizer (Fig. 3D, left and middle). We further identified whether these defects by xPOSH MO were involved in early neurogenesis which occurred via neural induction. Whole-mount in situ hybridization showed that xPOSH did not alter expression of the early neural marker Sox2 that is required for anterior and posterior neural differentiation (Sasai, 2001) (Fig. 3D, right). POSH acts as a scaffold for regulation of the JNK pathway in cultured cells (Tapon et al., 1998; Xu et al., 2003) and JNK inhibition causes the defects of neural tube and eye in Xenopus (Maurus et al., 2005). This raises a possibility that POSH function in Xenopus anterior neural development is involved in the JNK signaling. Consistent with the idea, we demonstrated that xPOSH is required for XeJNK1 activation in culture cells (Fig. 8A, lane 2) and restores the XeJNK1 phosphorylation decreased by xPOSH MO in Xenopus anterior neural tissues (Fig. 8B, lanes 2 and 3), and interacts with XeJNK1 (Fig. 8C, lane 2), indicating that xPOSH has a conserved role as a scaffold mediating JNK signaling. Next, we carried out rescue experiments to test the relationship between POSH and JNK1 in Xenopus head development. As shown in Fig. 4A, coinjection of xPOSH MO with XeJNK1 rescued in a dose-dependent manner the phenotypic defects caused by xPOSH MO. The study of molecular level using whole-mount in situ hybridization with Otx2, En2, and Krox20 as probes also demonstrated that the reduction of anterior neural markers caused by xPOSH MO was reversed by XeJNK1 mRNA (Figs. 4B –D). However, it is possible that these rescue analyses using XeJNK1 may due to JNK1’s secondary
effect which is not attributable to POSH. As shown in Figs. 4A and E, we demonstrated that both the phenotypic defects and expression of anterior neural genes in the embryos injected with XeJNK1 and CO MO were not altered compared to those in the Co MO. From the combined results, we conclude that xPOSH acts a scaffold for JNK signaling to regulate anterior neural tissue patterning in late stage, while it does not affect Spemann organizer formation and early neurogenesis in early stage. POSH-mediated JNK pathway is required for apoptosis in Xenopus anterior neural tissues A previous report has claimed that apoptosis is detected throughout the developing head region and in the eyes in Xenopus (Hensey and Gautier, 1998). Moreover, it was known that POSH is involved in apoptosis through JNK activation (Xu et al., 2003). Because the present study demonstrated that POSH is essential for the Xenopus anterior neural development, we sought to determine if the developing head and eye defects caused by xPOSH depletion is involved with apoptosis. We first examined the endogenous pattern of apoptosis in head and eye regions at a tailbud (stage 22 –23) embryo. Detection of apoptosis at single cell level was based on a section in situ DNA strand breaks-labeling (TUNEL; Terminal deoxynucleotidyl transferase mediated dUTP Nick-End Labeling) technique and then analyzed by fluorescence microscopy. TUNEL staining was strongly detected in the entire brain (Fig. 5A) and optic vesicle regions (Fig. 5B) as well as the cement gland. We next determined whether POSH is required for apoptosis observed in these tissues. A transverse (Fig. 6A) and sagittal (Figs. 6C and D) section of the head region of a tailbudstage embryo demonstrated that the apoptotic cell level of the whole brain and optic vesicle on the xPOSH MO-
Fig. 5. The endogenous pattern of apoptosis in head and eye regions at a tailbud stage embryo. Uninjected embryos were cultured until stage 22, and analyzed by the section TUNEL staining assay. (A) A sagittal section of the embryo shows that TUNEL staining was detected in the entire tissues of brain. (B) A transverse section shows that TUNEL staining was detected in the entire region of neural tube and the developing eye region. f, forebrain; ov, optic vesicle; bv, brain ventricle; os, optic stalk.
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injected side or embryo was reduced compared to that on the uninjected or control MO-injected. This apoptotic reduction was efficiently rescued by ORF xPOSH (Fig. 6E). We further determined if JNK signaling is required for the reduction of apoptosis caused by xPOSH MO. Interestingly, coexpression of xPOSH MO with XeJNK1 rescued the reduced apoptosis induced by xPOSH MO (Figs. 6B and F).
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The E3 function of RING-mediated POSH is not involved in POSH-mediated JNK pathway in vivo Ubiquitination, a highly conserved post-translational protein modification, is a multi-step process that conjugates ubiquitin to substrate proteins by sequential ATP-dependent action of three enzymes: A ubiquitin-activating enzyme
Fig. 6. POSH-mediated JNK pathway is essential for apoptosis in Xenopus head and eye regions. (A – B) A transverse section shows that the reduced apoptosis caused by xPOSH MO (10 ng; A) was reversed by XeJNK1 mRNA (1 ng; B). Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO and XeJNK1 mRNA, either alone or in combination as indicated, cultured until stage 22, and analyzed by the section TUNEL staining assay. Nuclear h-gal mRNA (200 pg) was coinjected to allow identification of the xPOSH MO-injected side as a lineage tracer. No, not injected; Inj, injected; f, forebrain; ov, optic vesicle. (C – F) A sagittal section shows that xPOSH MO (20 ng) induced a reduction of apoptotic cell level (D), while Co MO (20 ng; C) did not. Coinjection of ORF xPOSH (4 ng; E) or XeJNK1 (2 ng; F) rescued the reduced apoptosis caused by xPOSH MO. Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs.
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(E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Additionally, it was known that E3 ligases act as a key regulator of ubiquitination processes via substrate selection (Hershko and Ciechanover, 1998; Hicke and Dunn, 2003; Weissman, 2001). Previous reports have shown that the RING domain of mammalian POSH (mPOSH) has a role as an E3 ubiquitin ligase (Alroy et al., 2005; Xu et al., 2003) and its E3 function is essential for HIV-1 production (Alroy et al., 2005). Here, we demonstrated that POSHmediated JNK signaling is essential for regional patterning and apoptosis in Xenopus anterior neural development (Figs. 4 and 6). Therefore, it may be possible that E3 function of POSH is also involved in its physiological role required in Xenopus development. To investigate this possibility, we first examined whether xPOSH also had E3 ligase activity. As shown in Fig. 7B, xPOSH caused selfpolyubiquitination in vivo. Proteins containing RING-finger domains sometimes target themselves for proteasomal degradation, and this behavior was observed for xPOSH. In the absence of MG132, a proteasomal inhibitor, levels of the xPOSH protein were reduced (Fig. 7C, lane 2). In the presence of MG132, however, its degradation was inhibited (Fig. 7C, lane 3). Because the E3 activity of xPOSH was determined with ectopically induced level, we next examined if the mutation or deletion of RING domain can inhibit the E3 activity of POSH. When the RING-finger domain of xPOSH was mutated (Mut-xPOSH: Fig. 7A), the ability of POSH as E3 ubiquitin ligase was inhibited (Fig. 7B, lane 4) and, moreover, protein levels did not reduce in the lack of MG132 (Fig. 7C, lanes 4). These observations manifestly appeared in the RING domain-deleted xPOSH (DRINGxPOSH; data not shown) (Xu et al., 2003). These results indicate that Mut- and DRING-xPOSH act as a form inhibiting E3 function of xPOSH, and POSH level is at least partially regulated by the RING-dependent proteasomal pathway. To test whether E3 function of POSH is indeed required for Xenopus anterior neural development, a rescue assay was performed using xPOSH MO and its forms inhibiting E3 activity. Intriguingly, the head and eye defects induced by xPOSH MO were efficiently rescued by coexpression of Mutand DRING-xPOSH (Fig. 7D). However, overexpression of
Mut- or DRING-xPOSH, like wild type (WT) xPOSH, did not cause these phenotypic defects (data not shown). We also demonstrated that these E3-inhibiting forms can rescue the reduced apoptosis induced by xPOSH MO (Figs. 7G and H). However, N-terminus-truncated xPOSH (DN-xPOSH) lacking the RING-finger, front two SH3, and RBD domains did not rescue both the phenotypic defects and reduction of apoptosis caused by xPOSH MO (Figs. 7D and I). Because xPOSH activated JNK pathway and associated with JNK1, the data in rescue analysis had led us to determine if Mut- and DRING-xPOSH would stimulate the JNK pathway. Interestingly, both Mut- and DRING-xPOSH increased XeJNK1 phosphorylation (Fig. 8A, lanes 3 and 4), while DN-xPOSH did not trigger JNK activation (data not shown) (Tapon et al., 1998). Moreover, Mut-xPOSH rescued the reduction of JNK activity caused by xPOSH MO in Xenopus embryos (Fig. 8B, lane 4). We further sought to investigate if these xPOSH constructs interacts with XeJNK1. The immunoprecipitation analysis demonstrated that XeJNK1 physically associates with MutxPOSH and DRING-xPOSH in cells (Fig. 8C, lanes 3 and 4). However, DN-xPOSH did not interact with XeJNK1 (Fig. 8C, lane 5), indicating that POSH interacts with JNK1 via its own two front SH3 domains and RBD domain. Together, these results suggest that in Xenopus development, the function of POSH as an E3 ubiquitin ligase is not required for POSH-mediated JNK activation in vivo and its E3 function may be just required to regulate own protein level.
Discussion Recent biochemical studies have characterized many of scaffold proteins for their roles in organizing multiprotein complexes with certain JNK modules in the JNK pathway (Morrison and Davis, 2003). POSH has been known to activate JNK by acting as a scaffold for specific JNK signaling modules, but no satisfactory physiological description of its function exists to date. The present study yields novel evidence that POSHmediated JNK signaling is essential for the expression of anterior neural genes and apoptosis and the function of
Fig. 7. Mut- and DRING-xPOSH, but not DN-xPOSH, are required for Xenopus anterior neural development and apoptosis. (A) Schematic representation of POSH deletions and mutations used in the present study. (B) The RING-finger domain of xPOSH is required for self-ubiquitination in vivo. HEK293FT cells were transfected for 48 h with GFP-vector, GFP-xPOSH, and GFP-Mut xPOSH. At 24 h post-transfection, cells expressing xPOSH were treated with 2 Ag MG132. Immunoprecipitates with anti-GFP antibodies were resolved by SDS-PAGE and transferred to an NC membrane. Replicated membrane was blotted with anti-Ub antibodies. (C) xPOSH level is regulated via the proteasomal pathway in a RING-dependent fashion. xPOSH was decreased in the absence of the proteasomal inhibitor MG132, but RING-mutated xPOSH was not altered in the absence of the inhibitor. HEK293FT cell lysates were blotted with anti-GFP antibodies for xPOSH. Actin served as a loading control. (D) Coinjection of xPOSH MO (20 ng) with GFP-Mut xPOSH (2 ng) and GFP-DRING xPOSH (2 ng), but not GFP-DN xPOSH (2 ng), rescued the phenotypic defects of xPOSH MO, like WT xPOSH (4 ng). Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. (E – I) A sagittal section shows that xPOSH MO (20 ng) induced a reduction of apoptotic cell level (F), while Co MO (20 ng; E) did not. Coinjection of xPOSH MO with GFP-DRING xPOSH (2 ng; G) and GFP-Mut xPOSH (2 ng; H), but not GFP-DN xPOSH (2 ng; I), rescued the reduced apoptosis caused by xPOSH MO. Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs.
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POSH as an E3 ubiquitin ligase is not required for POSH-mediated JNK activation in Xenopus anterior neural development. Based on determination achieved via following demonstrations, our data suggest that POSH was essential for Xenopus anterior neural development through the formation of an active JNK signaling complex in a RINGindependent fashion. First, the embryonic expression pattern of xPOSH was similar to that of reported for components of the JNK signaling module that binds POSH
(Kuan et al., 1999; Lucas et al., 2002; Poitras et al., 2003; Yamanaka et al., 2002). Second, xPOSH interacted with XeJNK1 and promoted JNK activation, as previously reported for mPOSH (Xu et al., 2003). Third, POSH depletion disturbed general head formation in Xenopus, including neural tube defects with phenotypes similar to that of JNK1 and JNK2 double knockout mice (Kuan et al., 1999) and JNK inhibition in Xenopus (Maurus et al., 2005). Likewise, Xenopus MLK2 (xMLK2), potentially the target protein of POSH in the regulation of anterior neural development, knockdown also caused defects in Xenopus head formation (Poitras et al., 2003). Fourth, XeJNK1 rescued the defects caused by xPOSH depletion in anterior neural development. Finally, although the RING-mediated E3 activity of POSH is inhibited, Mut- or DRING-xPOSH normally acted as a JNK scaffold to regulate Xenopus anterior neural development. In Xenopus gastrulation, JNK signal has been known to regulate convergent extension (CE) movements in the noncanonical Wnt/planar cell polarity (Wnt/PCP) pathway (Boutros et al., 1998; Fanto et al., 2000; Habas et al., 2003; Kim and Han, 2005; Paricio et al., 1999; Strutt et al., 1997; Weber et al., 2000; Yamanaka et al., 2002). Nonetheless, the molecular mechanism by which JNK is involved in this pathway remains unknown. In the present experiments, although the protein level of xPOSH is diminished by its MO at gastrula stages (Fig. 1D), POSH depletion did not cause CE movement-defective phenotypes, such as a dorsal flexure or failure to straighten the anterioposterior (AP) axis, indicating that POSH was not required for CE movement. In line with this, xMLK2 has been known to be not expressed in the dorsal marginal zone (DMZ), where extensive CE movements occur during gastrulation, nor involved in CE
Fig. 8. Mut- and DRING-xPOSH, but not DN-xPOSH, activate and interact with XeJNK1. (A – B) xPOSH is required for JNK activation in a RINGindependent manner. (A) Immunoblots of total cell lysates prepared from HEK293FT cells were transfected for 48 h with Flag-XeJNK1 and GFPxPOSH, GFP-Mut xPOSH, or GFP-DRING xPOSH. At 24 h posttransfection, cells expressing GFP-xPOSH were treated with 2 Ag MG132. The histogram represents the ratio of JNK activity as phosphoJNK (P-JNK) level relative to Flag-XeJNK1 level in three independent experiments. The activity of the control was defined as unity. Error bars indicate the mean T SD. (B) WT and Mut-xPOSH rescue the reduction of JNK phosphorylation caused by xPOSH MO in Xenopus anterior neural tissues. Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated constructs, cultured until stage 22, and isolated the anterior neural regions. The embryo lysates were blotted with anti-phospho JNK and anti-JNK antibodies. (C) XeJNK1 physically associates with Mut-xPOSH and DRING-xPOSH in cells, but not with DN-xPOSH. HEK293FT cells were transfected for 48 h with Flag-XeJNK1, GFP-xPOSH, GFP-Mut xPOSH, GFP-DRING xPOSH, and GFP-DN xPOSH, either alone or in combination as indicated. At 24 h posttransfection, GFP-xPOSH was treated with 2 Ag MG132. Cell lysates were immunoprecipitated with anti-GFP antibodies, and immunocomplexes were blotted with anti-Flag antibodies for JNK1. The membrane was stripped and reprobed with anti-GFP antibodies for the presence of xPOSH constructs.
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movements (Poitras et al., 2003). Unlike in DMZ during gastrulation, components of the JNK-specific signaling module for POSH, including Rac1, JNK1, MKK7, and MLK2, were commonly observed in the brain region of Xenopus embryos (Lucas et al., 2002; Poitras et al., 2003; Yamanaka et al., 2002). Therefore, it is possible that the JNK pathway employs any special scaffold different than POSH to establish the regulating module that controls Xenopus CE movements. Moreover, nonetheless Sox2 expression in Xenopus early neurogenesis is regulated by apoptosis (Yeo and Gautier, 2003), POSH knockdown, which reduces apoptosis in late stage, could not alter the Sox2 expression at early neurula stage (Fig. 3D). From this finding, we propose that specificity and temporal regulation of JNK signals are tightly controlled by specific scaffold proteins that organize multi-protein complexes via interaction with the specific JNK modules in vertebrates’ development. However, it remains unknown how POSH is precisely regulated. POSH has four SH3 domains as its core conserved protein-interacting domain for binding proline-rich and identified PxxP motifs in several proteins (Ren et al., 1993). Recently, it had been known that Akt2 negatively regulates assembly of an active JNK signaling complex by POSH (Figueroa et al., 2003), supporting the possibility that POSH may be regulated by selective association of its four SH3 domains with various proteins containing proline-rich domains. Towards this end, identification and characterization of proteins that interact with POSH would be an important step in understanding the molecular mechanism that permits specificity and regulation of POSH-mediated JNK signal. Biochemical and genetic studies demonstrated that JNK has a proapoptotic role in apoptosis of neurons. In PC-12 cells, apoptosis evoked by withdrawal of trophic support such as nerve growth factor (NGF) was suppressed by inhibiting the JNK pathway (Le-Niculescu et al., 1999; Xia et al., 1995). Mice and chick deficient in both jnk1 and jnk2 had reduced apoptosis in the dorsal region of the neural folds resulting in a failure of fold closure (Kuan et al., 1999; Weil et al., 1997). These studies of cell death in the development of the central nervous system (CNS) have focused on the survival of the differentiated neurons regulated by the neurotrophic factors. It has also been known that POSH’s function as a JNK scaffold is required for the death of neurons induced by withdrawal of NGF (Xu et al., 2003). In Xenopus, a recent report had been shown that apoptosis was detected throughout the developing head region at stages prior to axonogenesis or synaptic connections (Hartenstein, 1989; Hensey and Gautier, 1998). The present investigation described for the first time that TUNEL staining in stage 22 embryos was strongly detected in the entire brain and optic vesicle regions (Fig. 5). Moreover, we demonstrated that xPOSH MO caused the reduction of apoptosis in head and eye tissues and this apoptosis defect was rescued by XeJNK1, which is consistent with data shown by in situ hybridization using anterior neural
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genes (Figs. 4B – D). However, the relationship between the expression of anterior neural genes and apoptosis regulated by POSH is unclear. Recently, it was reported that apoptosis regulates Xenopus early neurogenesis at the level of both neural and neuronal determination (Yeo and Gautier, 2003). Moreover, the apoptotic activity of msx1 was required for the Xenopus neural crest development (Tribulo et al., 2004). Thus, it is possible that apoptosis induced by POSH-mediated JNK pathway may be important for the expression of anterior neural genes at developmental stage prior to the establishment of first axons. Considering this idea, future experiments would be necessary to determine the molecular mechanism linking these two events. Recent reports suggested that Wnt pathway could mediate apoptosis via JNK signaling. The inhibition of canonical Wnt pathway by dickkopf-1 regulated c-Jun, a target of JNK signaling, for modulating apoptosis (Grotewold and Ruther, 2002; Yeo and Gautier, 2003). Xenopus frizzled 8 (Fz8) activated JNK and triggered apoptosis that is independent of its known Wnt ligands in Xenopus embryos (Lisovsky et al., 2002). In Xenopus, the head structure patterning requires inhibition of canonical Wnt signaling through several extracellular factors, such as dickkopf and cerberus (Glinka et al., 1997; Piccolo et al., 1999) and moreover, eye development is regulated by noncanonical Wnt/JNK pathway (Maurus et al., 2005; Rasmussen et al., 2001). Taken together, these results suggest that POSH-mediated JNK signaling is essential for the gene expression and apoptosis occurred in the anterior neural tissues through Wnt pathway. DN-mPOSH, which is identical with DN-xPOSH, did not induce JNK activation and apoptosis (Tapon et al., 1998), although this deletion form interacts with MLKs (Xu et al., 2003). However, it remains unknown why these characteristics appear. In the present study, we demonstrated that DNxPOSH does not interact with JNK1, nor does it activate JNK. In addition, the truncated form of POSH neither interfered with Rac-induced JNK activation (Tapon et al., 1998) nor rescued both the phenotypic defects and the apoptotic reduction caused by xPOSH depletion (Figs. 7D and I). These observations indicate that DN-POSH does not act as a dominant negative (DN) form of POSH in function of a scaffold mediating JNK signal, and the complete assembly between POSH and JNK modules is essential to meditate normal JNK signal.
Acknowledgments We would like to thank Dr. Ken W.Y. Cho, Richard M. Harland, and David G. Wilkinson for the generous gifts of probes. We are also grateful to Sun-Cheol Choi and the other members of our laboratory for reading the manuscript and providing constructive criticism. This work was supported by the Advanced Basic Research Laboratory Program (R14-2002-012-01001-0) of the KOSEF and Brain Korea 21 project.
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The assembly of POSH-JNK regulates Xenopus anterior neural development Gun-Hwa Kim, Eunjoo Park, Jin-Kwan Han* Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang, Kyungbuk 790-784, Republic of Korea Received for publication 20 April 2005, revised 14 July 2005, accepted 26 July 2005 Available online 26 August 2005
Abstract POSH (Plenty of SH3s) has distinct roles as a scaffold for specific c-Jun N-terminal kinase (JNK) signaling modules and as an E3 ubiquitin ligase. The physiological function of POSH remains unclear, however, and its possible involvement in developmental processes motivated the present study wherein the Xenopus orthologue of POSH (xPOSH) was examined for its potential role during Xenopus early embryogenesis. Loss-of-function analysis using morpholino oligonucleotides demonstrated that POSH was essential for Xenopus anterior neural development, although not Spemann organizer formation and early neurogenesis, through the formation of an active JNK signaling complex. Moreover, POSH-mediated JNK pathway was essential for apoptosis in anterior neural tissues. Finally, the present findings demonstrate that RING (Really Interesting New Gene) domain-mediated E3 ubiquitin ligase activity of POSH was not involved in POSHmediated JNK pathway in vivo. Together, these data suggest that the active JNK signaling complex formed of POSH and the JNK module is essential for the expression of anterior neural genes and apoptosis in Xenopus anterior development. D 2005 Elsevier Inc. All rights reserved. Keywords: POSH; Scaffold; JNK; Xenopus; Development; Apoptosis; Ubiquitin ligase
Introduction The c-Jun N-terminal kinase (JNK), one submember of a conserved family of mitogen-activated protein kinases (MAPK), is a serine/threonine kinase that mediates the response of cells to environmental stresses and many extracellular stimuli, including growth factors or inflammatory cytokines, for the regulation of cellular proliferation, differentiation, development, immune response, and apoptosis (Davis, 2000; Dong et al., 2002; Weston and Davis, 2002). JNK exists as three unique isoforms (JNK1, JNK2, and JNK3) and is activated by a specific protein kinase cascade module. This signaling module can be established through the sequential actions of a MAP kinase– kinase– kinase (MAPKKK), a MAP kinase –kinase (MAPKK), and a MAPK. The known MAPKKKs for activation of the JNK
* Corresponding author. Fax: +82 54 279 2199. E-mail address: [email protected] (J.-K. Han). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.07.033
pathway are MEKKs, TAK1, MLKs, and ASK1. The known MAPKKs for JNK are MKK4 and MKK7 (Weston and Davis, 2002). The diversity of signals transmitted by the JNK pathway yields a variety of biological responses, and this requires that functional signaling via the cascade be highly specified and precisely regulated. One mechanism that permits specificity and regulation of JNK signals is to employ scaffold proteins that organize multi-protein complexes via interaction between the various JNK modules (Morrison and Davis, 2003). Indeed, mathematical modeling indicates that formation of scaffold – kinase complexes can be used effectively to regulate the specificity, efficiency, and amplitude of signal propagation with MAPK modules (Levchenko et al., 2000). Studies in Saccharomyces cerevisiae have established that scaffold proteins, including Ste5p in mating response and Pbs2p in osmoregulation, can be physiologically critical components for the regulation of MAPK modules (Bardwell et al., 2001; Elion, 2001; Posas and Saito, 1997).
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The role of scaffold proteins in JNK activation in response to specific stimuli has also been identified in higher eukaryotes. h-arrestin 2 (McDonald et al., 2000), CrkII (Girardin and Yaniv, 2001), Filamin (Marti et al., 1997), IKAP (Holmberg et al., 2002), JNK interacting proteins (JIPs) (Ito et al., 1999; Kelkar et al., 2000; Lee et al., 2002; Whitmarsh et al., 1998; Yasuda et al., 1999), SKRP1/MKPX phosphatases (Chen et al., 2002; Zama et al., 2002), and POSH (Plenty of SH3s) (Xu et al., 2003) all serve as scaffold proteins for regulation of the JNK signaling pathway. While recent biochemical studies have characterized many of these scaffold proteins for their roles in organizing multi-protein complexes with certain JNK modules in the JNK pathway, little is known about their physiological functions in vivo (Morrison and Davis, 2003). POSH – comprised of a RING (Really Interesting New Gene)-finger domain, a putative active-Rac1 binding domain (RBD), and four SH3 (Src Homology 3) domains – acts as a scaffold in a multi-protein complex that links activated Rac1 and downstream modules of the JNK cascade (Tapon et al., 1998; Xu et al., 2003). POSH binds Rac1, JNK1/2, MKK4/7, and MLKs and is localized in lamellipodia by active Rac1 (Figueroa et al., 2003; Xu et al., 2003). Moreover, Akt2 negatively regulates assembly of an active JNK signaling complex by POSH (Figueroa et al., 2003). POSH was also known to be essential for HIV-1 production in infected cells (Alroy et al., 2005). However, none of these findings reveal anything about the endogenous role of POSH. In an attempt to elucidate the endogenous role of POSH in developmental processes in vivo, we examined its potential role of the Xenopus orthologue of POSH (xPOSH) during Xenopus early embryogenesis. POSH was found to be expressed mainly in the mesoderm and ectoderm that eventually become the cement gland, brain, somite, notochord, and pronephros. Analysis with antisense morpholino oligonucleotides demonstrated that POSH was essential for Xenopus anterior neural development through an active JNK signaling. Moreover, POSH-mediated JNK pathway was essential for apoptosis (programmed cell death) in head and eye regions. Finally, the present findings demonstrate that RING domain of POSH was not involved in POSHmediated JNK pathway.
Materials and methods
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Modified Ringer (MR) containing 4% Ficoll-400 (Sigma) using a Nanoliter Injector (WPI). Injected embryos were cultured in 0.33 MR until stage 8 and then transferred to 0.1 MR until they had reached the appropriate stage for the experimentation outlined below. Plasmids, RNA synthesis, and morpholino oligonucleotides For expression in Xenopus embryos, the entire coding region of xPOSH was cloned into the EcoRI and XbaI sites of the EGFP-pCS2+ vector and into the BamHI and ClaI sites of the Myc-pCS2+ vector. Capped mRNAs were synthesized from linearized plasmids using the mMessage mMachine kit (Ambion). GFP-xPOSH was linearized with ApaI, and mRNA was synthesized using the SP6 mMessage mMachine kit. Myc-xPOSH was linearized with NotI and transcribed using the same kit. The Xenopus JNK1 constructs were previously described (Kim and Han, 2005). Antisense morpholino oligonucleotides (MO) were obtained from Gene Tools. The morpholino oligonucleotide sequences were, for xPOSH MO, 5V-ATGCTTCTT TCCATGCAAAACTGTG-3V, and, for control MO, 5VCCTCTTACCTCAGTTACAATTTATA-3V. For xPOSH MO specificity, 5V-UTR and ORF xPOSH-Myc mRNA were injected with xPOSH MO or control MO into the animal region of embryos at the four-cell stage. Animal cap explants isolated at early gastrula stages were then subjected to immunoblot (IB) analysis with anti-Myc antibodies. Actin served as a specificity control. In situ hybridization and RT-PCR Whole-mount in situ hybridization was performed with digoxigenin (DIG)-labeled probes as described by Harland (1991). An antisense in situ probe against xPOSH was generated by linearizing the pGEM-T xPOSH construct with ApaI and transcribing with the SP6 RNA polymerase. RT-PCR analyses were carried out as described elsewhere (Kim and Han, in press). The forward and reverse primers for xPOSH were 5V-CAGCCGCCACCTCAATGCAA-3V and 5V-TGCCACTATGCGATGTGCCAGA-3V, respectively. Primers for Chordin, Cerberus, NCAM, XAG1, HoxB9, and Goosecoid were used as described online by De Robertis (www.hhmi.ucla.edu/derobertis/index.html). Primers for ODC, Otx2, En2, and Krox20 were also used as described online (Xenbase, www.xenbase.org/xmmr/Marker_pages/ primers.html).
Xenopus embryos and microinjection Immunoprecipitation and immunoblotting Xenopus laevis was purchased from Xenopus I (Ann Arbor, MI). Eggs were obtained from X. laevis primed with 800 units of human chorionic gonadotropin (Sigma). In vitro fertilization was performed as described previously (Newport and Kirschner, 1982), and developmental stages of the embryos were determined according to Nieuwkoop and Faber (1967). Microinjection was carried out in 0.33
HEK293FT cells (Invitrogen) were transfected with the indicated constructs for 48 h using Lipofectamine-Plus Reagent (Invitrogen) and then resuspended in immunoprecipitation (IP) buffer [50 mM HEPES/NaOH (pH 7.5), 3 mM EDTA, 3 mM CaCl2, 80 mM NaCl, 1% Triton X-100, 5 mM DTT]. Cells were disrupted and centrifuged so as to remove
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insoluble debris. The indicated antibodies were added to the supernatants and incubated at 4-C for 6 h. Protein A sepharose (Zymed) was added and the mixture was incubated at 4-C for 3 h. The immunocomplexes bound to Protein A beads were washed five times with IP buffer. Rabbit anti-myc polyclonal, mouse anti-GFP monoclonal, mouse anti-HA monoclonal (Santa Cruz Biotechnology), and mouse antiFlag monoclonal antibodies (Sigma) were used for immunoprecipitation and immunoblot analysis.
and then lysed in 1% lysis triton buffer. The samples were subsequently analyzed by 10% SDS-PAGE and transferred to a nitrocellulose (NC) membrane. Replicated membranes were blotted with anti-phospho-JNK antibodies (Cell Signaling) for activated JNK, and anti-Flag antibodies (Sigma) or anti-JNK antibodies (Cell Signaling) for total JNK. The quantity of protein in each sample was determined using BCA protein assay reagent (Pierce). TUNEL assay
JNK assays For JNK assays, HEK293FT cells and Xenopus embryos were transfected or injected with the indicated constructs
For the section in situ TUNEL staining, all embryos were fixed in MEMFA [0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO4, and 4% formaldehyde] for 1 h at room
Fig. 1. Expression pattern and antisense morpholino of Xenopus POSH (xPOSH). (A) RT-PCR analysis of temporal expression of POSH. Developmental stages are shown above each lane. Ornithine decarboxylase (ODC) was used as a loading control. (B) In situ hybridization analysis of spatial expression of POSH during early Xenopus development. (C) Scheme of the myc-tagged 5V-UTR POSH construct, the myc-tagged ORF POSH construct, and the morpholino sequence targeting the POSH 5V-UTR sequences. (D) xPOSH MO specifically inhibits the translation of its cognate mRNA. 5V-UTR (500 pg) and ORF (500 pg) xPOSH-Myc mRNA were injected with xPOSH MO (20 ng) or control MO (20 ng) into the animal region of embryos at the four-cell stage. Animal cap explants isolated at early gastrula stages were then subjected to immunoblot (IB) analysis with anti-Myc antibodies. Actin served as a specificity control.
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temperature and stored in 100% methanol at 20-C. Fixed embryos were embedded in paraffin. The blocks were sectioned with thickness of 10 – 12 Am. Sections were
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dewaxed in Xelene, detected by an in situ Cell Death Detection Kit (Roche), mounted, and analyzed on a ZEISS fluorescent microscope. Images were collected on ZEISS
Fig. 2. POSH depletion causes defective head and eye formation in Xenopus embryogenesis. (A) xPOSH MO led to a strong disruption of head and eye formation in a dose-dependent manner. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. Control (Co) MO and xPOSH MO were injected into two blastomeres of four-cell stage embryos. (B – C) Hematoxylin and eosin-stained transverse section of the head region of a tadpole embryo (a dashed line of the inner box). (B – C) xPOSH MO (20 ng) induced a reduction of eye and neural tube formation (C), while Co MO (20 ng; B) did not. (D) Coinjection of xPOSH MO (20 ng) with ORF xPOSH rescued the phenotypic defects of xPOSH MO in a dose-dependent manner. (E) Quantitative results of relative rescue in whole embryos shown in panel D.
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AxioCam MRc camera and processed using Photoshop software.
Results POSH knockdown perturbs Xenopus development To elucidate the potential role of Xenopus POSH (xPOSH: GenBank accession numberAY762093) in devel-
opmental processes in vivo, the spatiotemporal expression pattern of POSH was first analyzed during Xenopus embryogenesis. Temporally, xPOSH is expressed both maternally and zygotically throughout early development (Fig. 1A). As shown in Fig. 1B, POSH was visible in the animal hemisphere of the embryo during the cleavage and blastula stages. At the gastrula stages, POSH is expressed broadly throughout the marginal zone and animal pole tissues. At the neurula stages, it shows the restricted expression in dorsal tissue. At later stages, POSH is
Fig. 3. POSH is required for Xenopus anterior neural development, but not organizer formation and early neurogenesis. (A – B) xPOSH depletion reduced several anterior neural markers. (A) Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with xPOSH MO (20 ng) and Co MO (20 ng), allowed to develop to stage 22, and analyzed by RT-PCR for expression of the indicated markers. ODC was used as a loading control. ( ) RT denotes the no reverse transcription control sample. (B) Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO (20 ng), and analyzed by whole-mount in situ hybridization for of the regional neural markers Otx2, En2, or Krox20. Nuclear h-gal mRNA (200 pg) was coinjected to allow identification of the xPOSH MO-injected side as a lineage tracer. No; not injected, Inj; injected. (C) RT-PCR analysis showed xPOSH also did not affect the organizer markers. Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with xPOSH MO (20 ng), Co MO (20 ng), and xPOSH mRNA (2 ng). Dorsal marginal zone (DMZ) explants were isolated at stage 10.5 and analyzed by RT-PCR, using primers specific for Goosecoid (Gsc), Chordin (Chd), and Cerberus (Cer). ODC was used as a loading control. ( ) RT denotes the no reverse transcription control sample. (D) xPOSH MO did not alter expression of the organizer markers Cer or Goosecoid, and early neural marker Sox2. Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO (20 ng), cultured until stage 10.5 (left and middle) or stage 13 (right). Nuclear h-gal mRNA (200 pg) was used as a lineage tracer. No; not injected, Inj; injected.
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expressed in the cement gland, brain, somite, notochord, and pronephros. Antisense morpholino oligonucleotides (MO) are extremely useful as efficient blockers of specific mRNA translation (Heasman, 2002). To investigate the endogenous role of POSH during Xenopus early embryogenesis, a morpholino oligonucleotide was designed to be complementary to the sequence between bases 36 and 12 of the 5VUTR region of POSH mRNA (xPOSH MO; Fig. 1C). It was then possible to determine the efficiency and specificity of xPOSH MO in inhibiting translation of POSH mRNA in Xenopus embryos. xPOSH MO effectively blocked translation of mRNA containing the 5V-UTR sequences complementary to its morpholino oligonucleotide, but translation of mRNA containing only the open reading frame (ORF) of the protein was not affected. Control MO (Co MO) had no effect on translation of the POSH protein, and neither xPOSH MO nor Co MO inhibited actin translation, a specificity control (Fig. 1D). Injection of xPOSH MO into two dorsal blastomeres of embryos at the four-cell stage caused anterior truncations, microcephaly, and eye defects in a dose-dependent manner (Fig. 2A), but overexpression of ORF xPOSH did not exhibit these phenotypic defects (data not shown). Hematoxylin and an eosin-stained transverse section of the head region of a tadpole-stage embryo showed that POSH depletion led to defects in the eye and neural tube structure (Fig. 2C). The phenotypic defects caused by xPOSH MO were efficiently rescued by coinjection of xPOSH MO with ORF xPOSH mRNA in a dose-dependent manner (Figs. 2D and E), indicating that these developmental defects are specifically mediated by the effects of xPOSH MO.
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injected side (Fig. 3B), indicating that POSH is required to regulate Xenopus anterior neural development. As head and eye formation requires early action of the components established by the Spemann organizer, a dorsal signaling center in vertebrates (De Robertis and Kuroda, 2004), the disruption of anterior neural tissues in xPOSH MO-depleted embryos may be caused by impairment of the organizer. Analysis of the expression of organizer markers, Cerberus (Cer), Goosecoid (Gsc), and Chordin (Chd), was
POSH regulates Xenopus anterior neural development through JNK signaling pathway To characterize the head and eye defects in POSHdepleted embryos at the molecular level, RT-PCR analysis was carried out with several marker genes. The level of expression of the pan-neural marker NCAM (Kintner and Melton, 1987) was reduced by the xPOSH MO. XBF1, a forebrain marker (Bourguignon et al., 1998), and Otx2, a forebrain – midbrain and eye marker (Pannese et al., 1995), were also decreased in xPOSH MO-injected embryo. Moreover, expression of the mid – hindbrain boundary marker En2 (Hemmati-Brivanlou et al., 1991) was also reduced as well as the hindbrain marker Krox20 (Bradley et al., 1993). Conversely, the posterior neural marker HoxB9 (Wright et al., 1990) was not altered in xPOSH-depleted embryo (Fig. 3A). We further performed whole-mount in situ hybridization analysis using anterior neural markers. Four-cell stage embryos were injected at their single dorsal blastomere with xPOSH MO and nuclear h-gal mRNA as a lineage tracer. Consistent with RT-PCR results, expression of Otx2, En2, and Krox20 was reduced on the xPOSH MO-
Fig. 4. POSH regulates Xenopus anterior neural development through JNK signaling pathway. (A – D) Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs. (A) Coinjection of xPOSH MO (20 ng) with XeJNK1 rescued the phenotypic defects of xPOSH MO in a dose-dependent manner. In addition, coinjection of JNK1 and CO MO did not exhibit the anterior neural defects. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. (B – E) Wholemount in situ hybridization with Otx2, En2, and Krox20 as probes. The reduction of anterior neural markers caused by xPOSH MO (20 ng; C) was reversed by XeJNK1 mRNA (2 ng; D). Coinjection of JNK1 and CO MO did not alter the expression of anterior neural genes (E).
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conducted by RT-PCR. The expression of Cer, Chd, and Gsc in the xPOSH MO-injected embryos was not altered compared to that in the Co MO or ORF xPOSH-injected embryos (Fig. 3C). Likewise, whole-mount in situ hybridization analysis also demonstrated that xPOSH did not affect formation of the organizer (Fig. 3D, left and middle). We further identified whether these defects by xPOSH MO were involved in early neurogenesis which occurred via neural induction. Whole-mount in situ hybridization showed that xPOSH did not alter expression of the early neural marker Sox2 that is required for anterior and posterior neural differentiation (Sasai, 2001) (Fig. 3D, right). POSH acts as a scaffold for regulation of the JNK pathway in cultured cells (Tapon et al., 1998; Xu et al., 2003) and JNK inhibition causes the defects of neural tube and eye in Xenopus (Maurus et al., 2005). This raises a possibility that POSH function in Xenopus anterior neural development is involved in the JNK signaling. Consistent with the idea, we demonstrated that xPOSH is required for XeJNK1 activation in culture cells (Fig. 8A, lane 2) and restores the XeJNK1 phosphorylation decreased by xPOSH MO in Xenopus anterior neural tissues (Fig. 8B, lanes 2 and 3), and interacts with XeJNK1 (Fig. 8C, lane 2), indicating that xPOSH has a conserved role as a scaffold mediating JNK signaling. Next, we carried out rescue experiments to test the relationship between POSH and JNK1 in Xenopus head development. As shown in Fig. 4A, coinjection of xPOSH MO with XeJNK1 rescued in a dose-dependent manner the phenotypic defects caused by xPOSH MO. The study of molecular level using whole-mount in situ hybridization with Otx2, En2, and Krox20 as probes also demonstrated that the reduction of anterior neural markers caused by xPOSH MO was reversed by XeJNK1 mRNA (Figs. 4B –D). However, it is possible that these rescue analyses using XeJNK1 may due to JNK1’s secondary
effect which is not attributable to POSH. As shown in Figs. 4A and E, we demonstrated that both the phenotypic defects and expression of anterior neural genes in the embryos injected with XeJNK1 and CO MO were not altered compared to those in the Co MO. From the combined results, we conclude that xPOSH acts a scaffold for JNK signaling to regulate anterior neural tissue patterning in late stage, while it does not affect Spemann organizer formation and early neurogenesis in early stage. POSH-mediated JNK pathway is required for apoptosis in Xenopus anterior neural tissues A previous report has claimed that apoptosis is detected throughout the developing head region and in the eyes in Xenopus (Hensey and Gautier, 1998). Moreover, it was known that POSH is involved in apoptosis through JNK activation (Xu et al., 2003). Because the present study demonstrated that POSH is essential for the Xenopus anterior neural development, we sought to determine if the developing head and eye defects caused by xPOSH depletion is involved with apoptosis. We first examined the endogenous pattern of apoptosis in head and eye regions at a tailbud (stage 22 –23) embryo. Detection of apoptosis at single cell level was based on a section in situ DNA strand breaks-labeling (TUNEL; Terminal deoxynucleotidyl transferase mediated dUTP Nick-End Labeling) technique and then analyzed by fluorescence microscopy. TUNEL staining was strongly detected in the entire brain (Fig. 5A) and optic vesicle regions (Fig. 5B) as well as the cement gland. We next determined whether POSH is required for apoptosis observed in these tissues. A transverse (Fig. 6A) and sagittal (Figs. 6C and D) section of the head region of a tailbudstage embryo demonstrated that the apoptotic cell level of the whole brain and optic vesicle on the xPOSH MO-
Fig. 5. The endogenous pattern of apoptosis in head and eye regions at a tailbud stage embryo. Uninjected embryos were cultured until stage 22, and analyzed by the section TUNEL staining assay. (A) A sagittal section of the embryo shows that TUNEL staining was detected in the entire tissues of brain. (B) A transverse section shows that TUNEL staining was detected in the entire region of neural tube and the developing eye region. f, forebrain; ov, optic vesicle; bv, brain ventricle; os, optic stalk.
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injected side or embryo was reduced compared to that on the uninjected or control MO-injected. This apoptotic reduction was efficiently rescued by ORF xPOSH (Fig. 6E). We further determined if JNK signaling is required for the reduction of apoptosis caused by xPOSH MO. Interestingly, coexpression of xPOSH MO with XeJNK1 rescued the reduced apoptosis induced by xPOSH MO (Figs. 6B and F).
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The E3 function of RING-mediated POSH is not involved in POSH-mediated JNK pathway in vivo Ubiquitination, a highly conserved post-translational protein modification, is a multi-step process that conjugates ubiquitin to substrate proteins by sequential ATP-dependent action of three enzymes: A ubiquitin-activating enzyme
Fig. 6. POSH-mediated JNK pathway is essential for apoptosis in Xenopus head and eye regions. (A – B) A transverse section shows that the reduced apoptosis caused by xPOSH MO (10 ng; A) was reversed by XeJNK1 mRNA (1 ng; B). Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO and XeJNK1 mRNA, either alone or in combination as indicated, cultured until stage 22, and analyzed by the section TUNEL staining assay. Nuclear h-gal mRNA (200 pg) was coinjected to allow identification of the xPOSH MO-injected side as a lineage tracer. No, not injected; Inj, injected; f, forebrain; ov, optic vesicle. (C – F) A sagittal section shows that xPOSH MO (20 ng) induced a reduction of apoptotic cell level (D), while Co MO (20 ng; C) did not. Coinjection of ORF xPOSH (4 ng; E) or XeJNK1 (2 ng; F) rescued the reduced apoptosis caused by xPOSH MO. Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs.
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(E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Additionally, it was known that E3 ligases act as a key regulator of ubiquitination processes via substrate selection (Hershko and Ciechanover, 1998; Hicke and Dunn, 2003; Weissman, 2001). Previous reports have shown that the RING domain of mammalian POSH (mPOSH) has a role as an E3 ubiquitin ligase (Alroy et al., 2005; Xu et al., 2003) and its E3 function is essential for HIV-1 production (Alroy et al., 2005). Here, we demonstrated that POSHmediated JNK signaling is essential for regional patterning and apoptosis in Xenopus anterior neural development (Figs. 4 and 6). Therefore, it may be possible that E3 function of POSH is also involved in its physiological role required in Xenopus development. To investigate this possibility, we first examined whether xPOSH also had E3 ligase activity. As shown in Fig. 7B, xPOSH caused selfpolyubiquitination in vivo. Proteins containing RING-finger domains sometimes target themselves for proteasomal degradation, and this behavior was observed for xPOSH. In the absence of MG132, a proteasomal inhibitor, levels of the xPOSH protein were reduced (Fig. 7C, lane 2). In the presence of MG132, however, its degradation was inhibited (Fig. 7C, lane 3). Because the E3 activity of xPOSH was determined with ectopically induced level, we next examined if the mutation or deletion of RING domain can inhibit the E3 activity of POSH. When the RING-finger domain of xPOSH was mutated (Mut-xPOSH: Fig. 7A), the ability of POSH as E3 ubiquitin ligase was inhibited (Fig. 7B, lane 4) and, moreover, protein levels did not reduce in the lack of MG132 (Fig. 7C, lanes 4). These observations manifestly appeared in the RING domain-deleted xPOSH (DRINGxPOSH; data not shown) (Xu et al., 2003). These results indicate that Mut- and DRING-xPOSH act as a form inhibiting E3 function of xPOSH, and POSH level is at least partially regulated by the RING-dependent proteasomal pathway. To test whether E3 function of POSH is indeed required for Xenopus anterior neural development, a rescue assay was performed using xPOSH MO and its forms inhibiting E3 activity. Intriguingly, the head and eye defects induced by xPOSH MO were efficiently rescued by coexpression of Mutand DRING-xPOSH (Fig. 7D). However, overexpression of
Mut- or DRING-xPOSH, like wild type (WT) xPOSH, did not cause these phenotypic defects (data not shown). We also demonstrated that these E3-inhibiting forms can rescue the reduced apoptosis induced by xPOSH MO (Figs. 7G and H). However, N-terminus-truncated xPOSH (DN-xPOSH) lacking the RING-finger, front two SH3, and RBD domains did not rescue both the phenotypic defects and reduction of apoptosis caused by xPOSH MO (Figs. 7D and I). Because xPOSH activated JNK pathway and associated with JNK1, the data in rescue analysis had led us to determine if Mut- and DRING-xPOSH would stimulate the JNK pathway. Interestingly, both Mut- and DRING-xPOSH increased XeJNK1 phosphorylation (Fig. 8A, lanes 3 and 4), while DN-xPOSH did not trigger JNK activation (data not shown) (Tapon et al., 1998). Moreover, Mut-xPOSH rescued the reduction of JNK activity caused by xPOSH MO in Xenopus embryos (Fig. 8B, lane 4). We further sought to investigate if these xPOSH constructs interacts with XeJNK1. The immunoprecipitation analysis demonstrated that XeJNK1 physically associates with MutxPOSH and DRING-xPOSH in cells (Fig. 8C, lanes 3 and 4). However, DN-xPOSH did not interact with XeJNK1 (Fig. 8C, lane 5), indicating that POSH interacts with JNK1 via its own two front SH3 domains and RBD domain. Together, these results suggest that in Xenopus development, the function of POSH as an E3 ubiquitin ligase is not required for POSH-mediated JNK activation in vivo and its E3 function may be just required to regulate own protein level.
Discussion Recent biochemical studies have characterized many of scaffold proteins for their roles in organizing multiprotein complexes with certain JNK modules in the JNK pathway (Morrison and Davis, 2003). POSH has been known to activate JNK by acting as a scaffold for specific JNK signaling modules, but no satisfactory physiological description of its function exists to date. The present study yields novel evidence that POSHmediated JNK signaling is essential for the expression of anterior neural genes and apoptosis and the function of
Fig. 7. Mut- and DRING-xPOSH, but not DN-xPOSH, are required for Xenopus anterior neural development and apoptosis. (A) Schematic representation of POSH deletions and mutations used in the present study. (B) The RING-finger domain of xPOSH is required for self-ubiquitination in vivo. HEK293FT cells were transfected for 48 h with GFP-vector, GFP-xPOSH, and GFP-Mut xPOSH. At 24 h post-transfection, cells expressing xPOSH were treated with 2 Ag MG132. Immunoprecipitates with anti-GFP antibodies were resolved by SDS-PAGE and transferred to an NC membrane. Replicated membrane was blotted with anti-Ub antibodies. (C) xPOSH level is regulated via the proteasomal pathway in a RING-dependent fashion. xPOSH was decreased in the absence of the proteasomal inhibitor MG132, but RING-mutated xPOSH was not altered in the absence of the inhibitor. HEK293FT cell lysates were blotted with anti-GFP antibodies for xPOSH. Actin served as a loading control. (D) Coinjection of xPOSH MO (20 ng) with GFP-Mut xPOSH (2 ng) and GFP-DRING xPOSH (2 ng), but not GFP-DN xPOSH (2 ng), rescued the phenotypic defects of xPOSH MO, like WT xPOSH (4 ng). Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. (E – I) A sagittal section shows that xPOSH MO (20 ng) induced a reduction of apoptotic cell level (F), while Co MO (20 ng; E) did not. Coinjection of xPOSH MO with GFP-DRING xPOSH (2 ng; G) and GFP-Mut xPOSH (2 ng; H), but not GFP-DN xPOSH (2 ng; I), rescued the reduced apoptosis caused by xPOSH MO. Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs.
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POSH as an E3 ubiquitin ligase is not required for POSH-mediated JNK activation in Xenopus anterior neural development. Based on determination achieved via following demonstrations, our data suggest that POSH was essential for Xenopus anterior neural development through the formation of an active JNK signaling complex in a RINGindependent fashion. First, the embryonic expression pattern of xPOSH was similar to that of reported for components of the JNK signaling module that binds POSH
(Kuan et al., 1999; Lucas et al., 2002; Poitras et al., 2003; Yamanaka et al., 2002). Second, xPOSH interacted with XeJNK1 and promoted JNK activation, as previously reported for mPOSH (Xu et al., 2003). Third, POSH depletion disturbed general head formation in Xenopus, including neural tube defects with phenotypes similar to that of JNK1 and JNK2 double knockout mice (Kuan et al., 1999) and JNK inhibition in Xenopus (Maurus et al., 2005). Likewise, Xenopus MLK2 (xMLK2), potentially the target protein of POSH in the regulation of anterior neural development, knockdown also caused defects in Xenopus head formation (Poitras et al., 2003). Fourth, XeJNK1 rescued the defects caused by xPOSH depletion in anterior neural development. Finally, although the RING-mediated E3 activity of POSH is inhibited, Mut- or DRING-xPOSH normally acted as a JNK scaffold to regulate Xenopus anterior neural development. In Xenopus gastrulation, JNK signal has been known to regulate convergent extension (CE) movements in the noncanonical Wnt/planar cell polarity (Wnt/PCP) pathway (Boutros et al., 1998; Fanto et al., 2000; Habas et al., 2003; Kim and Han, 2005; Paricio et al., 1999; Strutt et al., 1997; Weber et al., 2000; Yamanaka et al., 2002). Nonetheless, the molecular mechanism by which JNK is involved in this pathway remains unknown. In the present experiments, although the protein level of xPOSH is diminished by its MO at gastrula stages (Fig. 1D), POSH depletion did not cause CE movement-defective phenotypes, such as a dorsal flexure or failure to straighten the anterioposterior (AP) axis, indicating that POSH was not required for CE movement. In line with this, xMLK2 has been known to be not expressed in the dorsal marginal zone (DMZ), where extensive CE movements occur during gastrulation, nor involved in CE
Fig. 8. Mut- and DRING-xPOSH, but not DN-xPOSH, activate and interact with XeJNK1. (A – B) xPOSH is required for JNK activation in a RINGindependent manner. (A) Immunoblots of total cell lysates prepared from HEK293FT cells were transfected for 48 h with Flag-XeJNK1 and GFPxPOSH, GFP-Mut xPOSH, or GFP-DRING xPOSH. At 24 h posttransfection, cells expressing GFP-xPOSH were treated with 2 Ag MG132. The histogram represents the ratio of JNK activity as phosphoJNK (P-JNK) level relative to Flag-XeJNK1 level in three independent experiments. The activity of the control was defined as unity. Error bars indicate the mean T SD. (B) WT and Mut-xPOSH rescue the reduction of JNK phosphorylation caused by xPOSH MO in Xenopus anterior neural tissues. Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated constructs, cultured until stage 22, and isolated the anterior neural regions. The embryo lysates were blotted with anti-phospho JNK and anti-JNK antibodies. (C) XeJNK1 physically associates with Mut-xPOSH and DRING-xPOSH in cells, but not with DN-xPOSH. HEK293FT cells were transfected for 48 h with Flag-XeJNK1, GFP-xPOSH, GFP-Mut xPOSH, GFP-DRING xPOSH, and GFP-DN xPOSH, either alone or in combination as indicated. At 24 h posttransfection, GFP-xPOSH was treated with 2 Ag MG132. Cell lysates were immunoprecipitated with anti-GFP antibodies, and immunocomplexes were blotted with anti-Flag antibodies for JNK1. The membrane was stripped and reprobed with anti-GFP antibodies for the presence of xPOSH constructs.
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movements (Poitras et al., 2003). Unlike in DMZ during gastrulation, components of the JNK-specific signaling module for POSH, including Rac1, JNK1, MKK7, and MLK2, were commonly observed in the brain region of Xenopus embryos (Lucas et al., 2002; Poitras et al., 2003; Yamanaka et al., 2002). Therefore, it is possible that the JNK pathway employs any special scaffold different than POSH to establish the regulating module that controls Xenopus CE movements. Moreover, nonetheless Sox2 expression in Xenopus early neurogenesis is regulated by apoptosis (Yeo and Gautier, 2003), POSH knockdown, which reduces apoptosis in late stage, could not alter the Sox2 expression at early neurula stage (Fig. 3D). From this finding, we propose that specificity and temporal regulation of JNK signals are tightly controlled by specific scaffold proteins that organize multi-protein complexes via interaction with the specific JNK modules in vertebrates’ development. However, it remains unknown how POSH is precisely regulated. POSH has four SH3 domains as its core conserved protein-interacting domain for binding proline-rich and identified PxxP motifs in several proteins (Ren et al., 1993). Recently, it had been known that Akt2 negatively regulates assembly of an active JNK signaling complex by POSH (Figueroa et al., 2003), supporting the possibility that POSH may be regulated by selective association of its four SH3 domains with various proteins containing proline-rich domains. Towards this end, identification and characterization of proteins that interact with POSH would be an important step in understanding the molecular mechanism that permits specificity and regulation of POSH-mediated JNK signal. Biochemical and genetic studies demonstrated that JNK has a proapoptotic role in apoptosis of neurons. In PC-12 cells, apoptosis evoked by withdrawal of trophic support such as nerve growth factor (NGF) was suppressed by inhibiting the JNK pathway (Le-Niculescu et al., 1999; Xia et al., 1995). Mice and chick deficient in both jnk1 and jnk2 had reduced apoptosis in the dorsal region of the neural folds resulting in a failure of fold closure (Kuan et al., 1999; Weil et al., 1997). These studies of cell death in the development of the central nervous system (CNS) have focused on the survival of the differentiated neurons regulated by the neurotrophic factors. It has also been known that POSH’s function as a JNK scaffold is required for the death of neurons induced by withdrawal of NGF (Xu et al., 2003). In Xenopus, a recent report had been shown that apoptosis was detected throughout the developing head region at stages prior to axonogenesis or synaptic connections (Hartenstein, 1989; Hensey and Gautier, 1998). The present investigation described for the first time that TUNEL staining in stage 22 embryos was strongly detected in the entire brain and optic vesicle regions (Fig. 5). Moreover, we demonstrated that xPOSH MO caused the reduction of apoptosis in head and eye tissues and this apoptosis defect was rescued by XeJNK1, which is consistent with data shown by in situ hybridization using anterior neural
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genes (Figs. 4B – D). However, the relationship between the expression of anterior neural genes and apoptosis regulated by POSH is unclear. Recently, it was reported that apoptosis regulates Xenopus early neurogenesis at the level of both neural and neuronal determination (Yeo and Gautier, 2003). Moreover, the apoptotic activity of msx1 was required for the Xenopus neural crest development (Tribulo et al., 2004). Thus, it is possible that apoptosis induced by POSH-mediated JNK pathway may be important for the expression of anterior neural genes at developmental stage prior to the establishment of first axons. Considering this idea, future experiments would be necessary to determine the molecular mechanism linking these two events. Recent reports suggested that Wnt pathway could mediate apoptosis via JNK signaling. The inhibition of canonical Wnt pathway by dickkopf-1 regulated c-Jun, a target of JNK signaling, for modulating apoptosis (Grotewold and Ruther, 2002; Yeo and Gautier, 2003). Xenopus frizzled 8 (Fz8) activated JNK and triggered apoptosis that is independent of its known Wnt ligands in Xenopus embryos (Lisovsky et al., 2002). In Xenopus, the head structure patterning requires inhibition of canonical Wnt signaling through several extracellular factors, such as dickkopf and cerberus (Glinka et al., 1997; Piccolo et al., 1999) and moreover, eye development is regulated by noncanonical Wnt/JNK pathway (Maurus et al., 2005; Rasmussen et al., 2001). Taken together, these results suggest that POSH-mediated JNK signaling is essential for the gene expression and apoptosis occurred in the anterior neural tissues through Wnt pathway. DN-mPOSH, which is identical with DN-xPOSH, did not induce JNK activation and apoptosis (Tapon et al., 1998), although this deletion form interacts with MLKs (Xu et al., 2003). However, it remains unknown why these characteristics appear. In the present study, we demonstrated that DNxPOSH does not interact with JNK1, nor does it activate JNK. In addition, the truncated form of POSH neither interfered with Rac-induced JNK activation (Tapon et al., 1998) nor rescued both the phenotypic defects and the apoptotic reduction caused by xPOSH depletion (Figs. 7D and I). These observations indicate that DN-POSH does not act as a dominant negative (DN) form of POSH in function of a scaffold mediating JNK signal, and the complete assembly between POSH and JNK modules is essential to meditate normal JNK signal.
Acknowledgments We would like to thank Dr. Ken W.Y. Cho, Richard M. Harland, and David G. Wilkinson for the generous gifts of probes. We are also grateful to Sun-Cheol Choi and the other members of our laboratory for reading the manuscript and providing constructive criticism. This work was supported by the Advanced Basic Research Laboratory Program (R14-2002-012-01001-0) of the KOSEF and Brain Korea 21 project.
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