Gtpbp2 is a positive regulator of Wnt signaling and maintains low levels of the Wnt negative regulator Axin
© The Author(s). 2016
Received: 3 March 2016
Accepted: 29 July 2016
Published: 2 August 2016
Canonical Wnt signals, transduced by stabilized β-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/β-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here.
Knockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3β but upstream of β-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b.
We conclude that Gtpbp2 is required for canonical Wnt/β-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the β-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant β-catenin signaling in cancer and other Wnt-related diseases.
KeywordsAxin GTPase Gtpbp2 Wnt signaling Xenopus embryo
Wnt signaling regulates various cell behaviors including proliferation, differentiation, polarization and migration, and is required to generate embryonic polarity as well as maintain normal tissue homeostasis [1–4]. Inappropriate activation of Wnt signaling in ventral tissues during early development can lead to the formation of a secondary axis in a variety of embryos [1, 5], and inappropriate Wnt target gene induction is thought to contribute to a broad range of neoplasms [4, 6, 7] including the majority of hereditary colorectal cancers . Moreover, besides cancers, mutations in Wnt signaling components are common in many diseases and contribute to birth defects , yet therapeutics directed at the Wnt pathway remain limited .
Central to the mechanism of canonical Wnt signaling is the stabilization of a cytoplasmic pool of β-catenin protein, which results in its subsequent nuclear translocation and induction of target genes. In the absence of Wnt ligand, cytoplasmic β-catenin is phosphorylated by glycogen synthase kinase-3 β (Gsk3b) as part of the multisubunit destruction complex that also includes the Adenomatous Polyposis Coli (Apc) and Axin proteins. Phosphorylated β-catenin is recognized by the F-box protein, β-transducin repeat-containing protein (β-TrCP), part of an E3 ubiquitin ligase that catalyzes β-catenin ubiquitination and proteasomal degradation . Upon Wnt ligand binding to Frizzled and Lrp5/6 receptors, focal accumulations of the Disheveled protein occur along with the phosphorylation of the Lrp5/6 tail by Gsk3b and Casein Kinase-1 (Ck1), which results in the subsequent recruitment and inhibition of the destruction complex, and concomitant stabilization of β-catenin [11–13].
Relative to other Wnt pathway components, Axin protein is maintained at low endogenous levels and imparts a rate-limiting step in β-catenin targeting by the destruction complex [14, 15]. Overexpression of Axin is sufficient to block β-catenin stabilization and target gene expression, and can block primary axis formation as well as ectopic secondary axis formation in Xenopus embryos triggered by overexpression of Wnt ligand or a kinase-dead form of Gsk3b (dnGsk3b) [1, 5].
Although the precise regulation of Axin turnover is not yet fully understood, several aspects of its degradation and regulation have recently been uncovered. In the presence of Wnt ligand, Axin protein is targeted for degradation while axin2 expression is itself transactivated, allowing for initial promotion and subsequent negative feedback of signaling [16, 17]. More recently, Axin protein has been recognized to undergo PolyADP-ribosylation (PARsylation) via Tankyrases 1 and 2 (Tnks1/2), which promotes subsequent Axin ubiquitination by the Ring Finger 146 (Rnf146) E3 ubiqutin ligase, prompting proteasomal degradation of Axin [18–20].
In this study we have examined the requirements for GTP binding protein 2 (Gtpbp2), a largely uncharacterized protein that is distantly related to the Eef1a (Ef1α) family of large GTPases [21, 22], in the regulation of embryonic Wnt signaling. We find that morpholino knockdown of Gtpbp2 results in axial patterning defects and reduced induction of organizer genes in Xenopus embryos. Furthermore, we demonstrate that Gtpbp2 is required for transduction of Wnt signaling and negatively correlates with Axin protein levels. Our findings reveal a new member involved in the regulation of proteins that transduce canonical Wnt signals, and illuminate Gtpbp2 as a potential drug target for diseases involving disregulated Wnt signaling.
Gtpbp2 knockdown disrupts axial patterning and reduces induction of β-catenin target genes
Gtpbp2 is required for canonical Wnt signaling
The Xenopus embryonic organizer is induced via the intersection of several signaling pathways, including Wnt/β-catenin and Nodal/Smad2/3 [3, 25]. Since Gtpbp2 can engage the BMP/Smad1 pathway, we hypothesized that Gtpbp2 might regulate nodal signals. However, knockdown of Gtpbp2 by injection of a translation-blocking morpholino (M1 of reference 24 unless noted otherwise) had no effect on Activin/Nodal direct target gene expression (goosecoid, mixer) in pluripotent Xenopus blastula ectodermal explants (animal caps) (Fig. 1d, e). The organizer genes effected in whole embryos by dorsal Gtpbp2 knockdown, above, can be directly (nodal3.1 , siamois ) or indirectly (goosecoid, chordin [28, 29]) activated by Wnt/β-catenin. Therefore, we tested whether Gtpbp2 is required for induction of the Wnt direct-response genes, nodal3.1 and siamois in animal caps. Embryos were treated with Wnt8 ligand (as injected mRNA) and either control or Gtpbp2-specific morpholinos, and animal caps were excised and scored for the induction of nodal3.1 or siamois. Wnt8 failed to induce either gene upon Gtpbp2 knockdown (Fig. 1f, and Fig. 4a discussed below). Similarly, induction of the β-catenin-responsive TOPflash reporter by wnt8 in animal caps was inhibited by knockdown of Gtpbp2, by two different morpholinos (Fig. 1g), and knockdown of Gtpbp2 reversed the stabilization of β-catenin in response to wnt8 in animal caps (Additional file 2).
Gtpbp2 binds to Axin and Gsk3b
We also examined the subcellular localization of tagged forms of Gtpbp2 in the presence or absence of coinjected dishevelled mRNA by injecting one cell of a two cell embryo with mRNA encoding mCherry-Gtpbp2, and the other cell with mRNAs encoding mCherry-Gtpbp2 and GFP-Dishevelled. We previously reported a membrane localization for mCherry tagged Gtpbp2, however injecting a lower dose of mRNA (500 pg instead of 2 ng) resulted in a more general cytoplasmic localization (Fig. 2c), consistent with the staining pattern of a Gtpbp2 antibody on human cells described in the Human Protein atlas . However, when co-expressed with GFP-tagged Disheveled, Gtpbp2 localized to cytoplasmic foci along with Dishevelled (Fig. 2c).
Gtpbp2 is required for inactivation of the β-catenin destruction complex
Gtpbp2 knockdown leads to increased Axin protein levels
To test whether Gtpbp2 could affect endogenous Axin levels in the embryo, we used an antibody raised against Xenopus Axin  to measure changes in Axin protein expression in early gastrula (stage 10.5) ventral mesoderm explants. We targeted this tissue because it has endogenously high levels of Wnt activity at this point in development . Similar to the effects we observed on ectopic myc-Axin, knockdown of both Gtpbp2 and Rnf146 caused increases in endogenous Axin levels in ventral mesoderm explants (Fig. 5c, d).
These studies are the first report of a role for Gtpbp2 in Wnt signaling and provide initial insights into the biochemical roles of this rather enigmatic GTPase. Our data show that Gtpbp2 is required for transduction of canonical Wnt signaling in Xenopus embryos and embryo explants. Inhibiting Gtpbp2 represses dorsoanterior axis formation and expression of Spemann-Mangold organizer genes, and while head loss is dramatic, the effects on organizer markers is somewhat mild, which may be due to maternal Gtpbp2 protein not effected by the morpholino. Indeed, our previous report showed a high level of maternal gtpbp2 transcripts in the egg , so cleavage stage knockdown of Gtpbp2 is likely to be partial. However, our present results clearly demonstrate that gtpbp2 morphant embryos, and zygotic wnt target genes in animal cap explants, show a near complete lack of response to ectopic induction of Wnt signaling.
Several factors have been identified which promote or suppress Axin turnover. Axin protein degradation is promoted by active Wnt signaling in response to the phosphorylation of the cytoplasmic tail of Wnt co-receptor Lrp6 [17, 31]. However, subsequent studies have demonstrated that Lrp6-mediated degradation of Axin is not required to initiate canonical Wnt signaling [12, 13], but this could be a means of promoting chronic Wnt stimulation. More recently it has been recognized that Axin protein undergoes Poly-ADP-ribosylation (PARsylation) via Tankyrases 1 and 2 (Tnks1/2) , which promotes Axin ubiquitination by the Rnf146 E3 ubiqutin ligase and consequent proteasomal degradation [19, 20]. Additionally, Axin also can be destabilized by ubiquitination by the Smurf2 ubiquitin ligase , or stabilized by Gsk3b phosphorylation , sumoylation , or by the ubiquitin-specific protease Usp34 .
Detailed investigation into how Gtpbp2 affects post-translational modifications, such as sumoylation, ubiquitination, phosphorylation, and/or PARsylation, of Axin or other proteins will likely provide more insight into the specific molecular mechanism of the Gtpbp2 protein, and resolve the apparent differences in Gtpbp2 function in Xenopus and mouse embryos.
Furthermore, because Gtpbp2 is required for normal Wnt signaling and operates downstream of proteins which engage the destruction complex, such as Apc and Lrp5 [5, 6] that are commonly mutated in Wnt-driven cancers, we suggest Gtpbp2 as a potential drug target for cancers and other diseases involving excessive Wnt signaling.
Recent work has identified crosstalk between the Wnt and BMP signaling pathways [36–39]. Smad1, a key component of the BMP signal transduction pathway, is also a target of Gsk3b. Like β-catenin, Smad1 is stabilized in response to active Wnt signaling and Gsk3b inhibition. As Gtpbp2 knockdown shows reductions in both Wnt and BMP signaling, one intriguing possibility for a common mechanism would be that the increased levels of Axin protein in Gtpbp2 morphants may also affect Smad1 levels. Although Wnt and Gsk3b regulate Smad1 levels, Axin’s role in this process is unclear. Axin has been shown to bind and regulate Smad3, however the authors of that paper mention unshown data failing to identify similar interactions between Axin and Smad1 . Therefore future studies that tease out the role of Axin and Gtpbp2 may illuminate the convergent and divergent features of Gtpbp2 in these key signaling pathways.
In summary, we have found that Gtpbp2 is a novel regulator of Wnt signaling in Xenopus embryos. We find that morpholino knockdown of Gtpbp2 results in axial patterning defects and reduced induction of organizer genes in Xenopus embryos. Furthermore, we demonstrate that Gtpbp2 is involved in the negative regulation of Axin protein levels, itself a negative regulator of Wnt signaling. Our findings reveal new insights into the tight proteolytic regulation of proteins involved in transducing canonical Wnt pathway signals, and illuminate Gtpbp2 as a potential drug target for diseases involving dysregulated Wnt signaling.
Gtpbp2 and control morpholinos were as previously described , and a morpholino targeting the translation start site of both Xenopus rnf146 homeologs was designed for this study, 5′-GCTAACCTCCCCACAACCAGCCATC -3′(GeneTools). Plasmids for gtpbp2 were previously described , and remaining plasmids were generous gifts from Ken-ichi Takemaru (Stony Brook University), Peter Klein (University of Pennsylvania) and Sergei Sokol (Mount Sinai School of Medicine), including XE10 pCS2-Xwnt-8 (wnt8) , pCS2-xnr2 (nodal2) , pCS2MT-xDsh (myc-dsh) , Xg134 pCS2MT-Gsk3 (myc-gsk3) , Xg137 pCS2-dnGsk3 (dngsk3) , XE49 pCS2MT-ptβ − catenin (ptbcat) , XE43 pCS2-GFP-Xdsh (gfp-dvl2) , pCS2-Axin MtFu1  (myc-axin). Synthetic mRNA was generated in vitro from linearized plasmids using AmpliCap™ SP6 High Yield Message Maker Kit (Cellscript). Reporter assays were conducted using the thymidine kinase promoter-Renilla luciferase (pRL-TK, Promega) and M50 Super 8x TOPFlash plasmids . Antibodies were grown from monoclonal supernatants mouse anti-Myc (9E10, 1:100), a gift from P. Klein (rabbit anti-Xenopus Axin 1066, 1:1000) , or purchased from the following: rabbit anti-HA (1:500) (RHGT-45A), ICL; mouse anti-β-tubulin (BYA-6068, 1:20,000), Accurate Chemical and Scientific Corporation; anti-GFP (B-2, 1:200), Sigma; iRDye800 conjugated affinity purified anti-mouse IgG (goat) (610-131-121, 1:5000), Rockland Immunochemicals; Alexa-fluor 680 goat anti-rabbit (A-21076, 1:5000), Molecular Probes.
Embryo microinjection and dissection
Xenopus laevis husbandry, microinjection and animal cap dissections were done as described previously . For the ventral mesoderm explants, Alexa-488 Dextran 10,000 (Molecular Probes, 5 mg/ml) was included in the injected solutions to facilitate ventral marginal zone (primarily mesoderm) dissections under a Zeiss Discovery V8 florescent dissection scope. Xenopus laevis were obtained as a non-specific wild type strain from Xenopus Express and Nasco Inc., and maintained in cycled colony.
Quantitative PCR and in situ hybridization
Embryos or explants were treated as indicated and cDNA was generated as previously described . Real-time quantitative PCR was conducted on a Roche lightcycler 480 with SYBR Green I Master (Roche), using primers sequences and conditions as described [47, 48, 49]. Target gene expression was normalized to levels of odc transcripts, and statistical analyses were conducted using R. Whole mount in situ RNA hybridization (WISH) was performed as described .
Immunoprecipitation and western blotting
Hek293t cells were co-transfected using linear PEI (1 mg/ml) with plasmids encoding HA-Gtpbp2, Myc, Myc-Gsk3b, and/or Myc-Axin. Cells were incubated overnight and lysed in NP40 lysis buffer (10 mM Tris-Cl pH 8.0, 137 mM NaCl, 10 % Glycerol, 1 % NP-40, + complete protease and phosphatase inhibitor tablets; Roche). Cell lysates were incubated with mouse anti-Myc (9E10) antibody overnight, purified using anti-IgG magnetic beads (NEB) according to the manufacturers instructions. Immunoprecipitations and whole cell lysates (1:10 of IP volumes) were blotted with mouse anti-myc (1:100) and rabbit anti-HA antibodies (1:500). For western blotting Xenopus embryos or explants were lysed in NP40 lysis buffer and extracted with an equal volume of 1,1,2-trichlorotrifluoro-ethane (Sigma), and then separated using SDS-page. Detection of western blots was conducted with IR secondary antibodies (Alexa 680 and IRdye800) scanned on a Licor Odyssey Classic Imager. Levels of ectopic myc-Axin were normalized to levels of co-injected HA-mCherry.
Two-cell stage Xenopus embryos were injected into the animal pole with mcherry-gtpbp2 mRNA (500 pg) into both cells, with one cell co-injected with gfp-dvl2 (500 pg), followed by imaging at late blastula (stage 9). Embryos were first examined live, then gently fixed in 1 % paraformaldehyde in PBS for 15 min, washed 3x in PBS, and costained with 2 mg/ml 4,6-diamino-2-phenylindole (DAPI). Embryos were then imaged on using a 10x objective on a Zeiss fluorescence microscope (Motorized Axio Imager Z1) with an ApoTome analysis unit.
Reporter assays were conducted by injecting Xenopus embryos with 100 pg Super TopFlash together with 50 pg pRL-TK reporter plasmids, and 10 pg of xwnt8 mRNA, and 30 ng of gtpbp2 M1, M2, or control morpholino as indicated. Animal caps were cut at mid-blastula stage 8 and cultured until mid-gastrula stage 11, at which point cell extracts were prepared and analyzed using Dual-Glo Luciferase Assay Reporter Assays (Promega).
CK1, Casein-kinase 1; Dsh, Dishevelled; frzb, frizzled-b; gsc, goosecoid; Gsk3b, glycogen synthase kinase-3 β; Gtpbp2, GTP-binding protein 2; β-TrCP, beta-transducin repeat-containing protein; odc, ornithine decarboxylase
We thank the Thomsen lab and local colleagues for comments on the manuscript, and S. Sokol, P. Klein, K. Takemaru for plasmids, and P. Klein for his generous gift of his polyclonal antibody to Xenopus Axin. We thank Jamina Oomen-Hajagos for help with Rnf146 morpholino design. We dedicate this work to the memory of John Miller, a graduate student who participated in early efforts on the project. Funding: This work was supported by NIH grant 5R01GM080462, and a Carol Baldwin Foundation Award (Stony Brook TRO 2014) to GHT. Support for WQG was from NIDDK 5T32DK007521 and NIGMS 1K12GM102778.
W.Q.G performed epistasis experiments, imaging of florescent-tagged proteins in Xenopus embryos, analysis of ectopic and endogenous Axin levels, and did the statistical analysis. A.K. generated the gtpbp2 constructs, designed morpholinos and characterized gross embryonic defects and reduction in endogenous nodal3.1 and siamois in gtpbp2 morphant embryos, and did the topflash reporter assays. Y.I. and D.K. conducted ancillary experiments contributing to the direction of this study. J.M.W. performed co- immunoprecipitations in HEK293T cells. W.Q.G., A.K., Y.I., and G.H.T. were involved in experimental design and analysis. W.Q.G. and A.K. contributed equally to the study. W.Q.G. wrote the manuscript, and all authors discussed results and contributed to the preparation of the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
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