Gillis WQ et al. (2016), Gtpbp2 is a positive regulator of Wnt signaling...

XB-ART-52332

Cell Commun Signal 2016 Aug 02;141:15. doi: 10.1186/s12964-016-0138-x.

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Gtpbp2 is a positive regulator of Wnt signaling and maintains low levels of the Wnt negative regulator Axin.

Gillis WQ , Kirmizitas A , Iwasaki Y , Ki DH , Wyrick JM , Thomsen GH .

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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.

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Species referenced: Xenopus
Genes referenced: axin1 chrd ctnnb1 dvl2 frzb gsc gsk3b gtpbp2 mixer myc nodal nodal2 nodal3.1 nodal3.4 odc1 rnf146 sia1 vegt wnt8a
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	Fig. 1. Gtpbp2 is required for Wnt but not nodal target gene induction. a Four-cell Xenopus embryos injected dorsally with 25 ng of Gtpbp2 MO (middle, 93 % abnormal, n = 94), but not a control 5-base mismatch MO (left, 95 % normal, n = 54), generated tadpoles with severe axial defects that were partially rescued by co-injection of the MO and a cocktail of 0.5 ng gtpbp2a and 50 pg gtpbp2b mRNA (right panel, 56 % rescued head and anterior structures, n = 78). b Embryos injected bilaterally at the 2-cell stage with 15 ng Gtpbp2 (gtpbp2 mo) or mismatch control (co-mo) morpholino were cultured until stage 10.25 then measured for nodal3.1, siamois, and ornithine decarboxylase (odc) levels. Amounts of nodal3.1 or siamois were normalized to odc and relative levels shown as mean ± s.e.m of n = 3 with p-values from t-test. c Expression of organizer genes chordin, gsc, and frzb as well as the pan-mesodermal markers t was severely reduced in embryos injected with Gtpbp2 morpholino into two dorsal blastomeres at the four-cell stage. Expression of another T-box gene vegt was not affected. Experiments were repeated three times. d-f mRNAs encoding nodal2 (10 pg; d, e) or wnt8 (10 pg, f) were coinjected with control or Gtpbp2 morpholino into the animal pole of two cell embryos. Animal caps were cut at midblastula (stage 8), and the expression of nodal targets goosecoid (d) and mixer (e), and the Wnt8 target nodal3.1 (f), were measured using qPCR at early gastrula stage 10.5. Relative expression levels shown as mean ± s.e.m (n = 3); p-values from t-test. g Knockdown of Gtpbp2 in caps treated with wnt8 showed that Gtpbp2 is required for induction from a Wnt/β-catenin reporter. Embryos were injected with combinations of 10 pg wnt8, 4 ng GFP, 30 ng Gtpbp2 m1, Gtpbp2 m2 or control morpholinos as indicated, along with 100 pg of Super Topflash and 60 pg of TK-Renilla Luciferase plasmids. Reporter activities were normalized to renilla luciferase activity from a co-injected TK-RL construct and shown as mean ± s.e.m (n = 3) with p-values from t-test
	Fig. 2. Gtpbp2 associates with components of the β-catenin destruction complex. a HA-Gtpbp2 was coexpressed in Hek293t cells with empty vector (Myc), Myc-Axin or Myc-Gsk3b and lysates were immunoprecipitated with anti-myc antibody followed western blot assay to detect precipitated Myc-tagged proteins and co-precipitated HA-Gtpbp2. The expression HA-Gtpbp2 in cells was confirmed by western blot on cell lysates. b mRNAs encoding HA-Gtpbp2 were injected into two-cell Xenopus embryos along with mRNAs encoding either the Myc tag, Myc-Axin, Myc-Gsk3b, or Myc-Gsk3b, as indicated. Embryos were lysed at gastrula stage 11, and were immunoprecipitated with anti-myc antibody followed western blot assay to detect precipitated Myc-tagged proteins and co-precipitated HA-Gtpbp2. HA-Gtpbp2 expression in cells was evaluated by western blot on cell lysates. c mCherry-Gtpbp2 relocalizes to GFP-Dvl2 containing granules when co-expressed in Xenopus animal caps (scored at late blastula, stage 9). mCherry-Gtpbp2 distribution is diffuse in cells lacking GFP-Dsh
	Fig. 3. Gtpbp2 is required for ectopic axis induction by Wnt but not stabilized β-catenin. a-e Axis induction phenotypes. a, c Control or b, d-e Gtpbp2 morpholinos (25 ng) were injected into both ventral blastomeres at the 4 cell stage, and mRNAs encoding (a-c), Wnt8 (4 pg) or d-e ptBcat (25 pg) were injected into a single ventral vegetal blastomere at the 8–16 cell stage. The Gtpbp2 morpholino blocks induction of secondary axes from Xenopus Wnt8 but not by a phospho-resistant form of β − catenin (ptBcat). e Coinjection of Gtpbp2 morpholino along with morpholino-resistant gtpbp2 mRNA (1 ng) restored secondary axes in Wnt-injected embryos. f Counts of total injected phenotypes, including intermediate Wnt phenotypes (partial secondary axis or enlarged head). g Diagram showing epistatic relationships between Wnt pathway reagents analyzed here and in Fig. 4
	Fig. 4. Gtpbp2 epistasis experiments suggest Gtpbp2 works at the level of Axin turnover. a-d mRNAs encoding activators of the Wnt pathway, including a wnt8, b disheveled (dvl2), c kinase-dead Gsk3b (dnGsk3) or d phospho-resistant ß-catenin (ptbcat) respectively were coinjected with 40 ng control or Gtpbp2 morpholino into the animal pole of two cell Xenopus embryos. Animal caps were excised at blastula stage 8, and the levels of siamois transcript were measured using qPCR at early gastrula stage 10.5 and shown as mean ± s.e.m of n = 3. The Gtpbp2 morpholino blocks induction from wnt8, disheveled, or dominant negative dngsk3 (a-c), but not from a phospho-resistant β-catenin (ptbcat) (d)
	Fig. 5. Gtpbp2 reduces Axin protein levels in Xenopus embryos. a HA-mcherry and myc-axin mRNAs were co-injected with a morpholino (40 ng) or morpholino + gtpbp2b mRNA (2 ng), into the marginal zone of two cell embryos, and proteins were analyzed by western blot. b Quantitation of myc-Axin levels, shown as mean ± s.e.m of n = 3. c Morpholino (40 ng) or morpholino + gtpbp2 mRNA (2 ng) plus rhodamine-dextran tracer were injected in the marginal zone of two ventral blastomeres of 4 cell stage embryos. The rhodamine-dextran was used to facilitate ventral marginal zone (primarily mesoderm) dissections under a florescent dissection scope at early gastrula stage 10.5, and two of three biological replicate western blots are shown. d Quantitation of endogenous Axin shown as mean ± s.e.m of n = 3
	Fig. 6. Loss of Gtpbp2 does not affect stability of Dishevelled or Gsk3b. Xenopus embryos were co-injected in the marginal zone of two-cell blastomeres with 1 ng of mRNA encoding myc-Dvl2 or myc-Gsk3b fusion proteins and 250 ng of gfp mRNA (as loading control), along with 40 ng of control or Gtpbp2 morpholino. Embryos were lysed at stage 10.5, run on an 8 % PAGE gel, and western blots were with probed with monoclonal mouse anti-myc 9E10 antisera. Duplicate lanes correspond to independent biological replicates
	Fig. 7. Gtpbp2 is required for Wnt signaling via regulation of Axin levels. In the presence of Gtpbp2 (top), Axin protein is maintained at low levels, and all active destruction complexes can be inactivated in the presence of Wnt ligand, allowing for accumulation of cytoplasmic β-catenin. However, when Gtpbp2 levels are reduced (Loss of Function; bottom), Axin levels rise and can form additional destruction complexes with ample free Gsk3b, CkII, and APC, either allowing for additional complexes to saturate the pool of cytoplasmic β-catenin and/or allowing some complexes to escape Wnt regulation and continue to degrade β-catenin. Gtpbp2 may regulate Axin protein by direct engagement of free Axin, destruction complex-bound Axin (with proteasomal targeting), or potentially other mechanisms (e.g., mRNA translation)

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Anastas, WNT signalling pathways as therapeutic targets in cancer. 2013, Pubmed