Xenopus Skip Modulates Wnt/β-Catenin Signaling and Functions in Neural Crest Induction

Wang, Ying; Fu, Yu; Gao, Lei; Zhu, Guixin; Liang, Juan Boo; Gao, Chan; Huang, Biao; Fenger, Ursula; Niehrs, Christof; Chen, Ye Guang; Wu, Wei

doi:10.1074/jbc.m109.058347

Cited by 30 publications

(26 citation statements)

References 66 publications

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“…Luciferase reporter assay Luciferase reporter assays were carried out in 96-well plates, with triplicates as described previously (Wang et al, 2010a). DNA per well was: PAK1, 50 ng; Rac1 and its mutants, 50 ng; cyclin D1-luciferase reporter construct, 50 ng.…”

Section: Methodsmentioning

confidence: 99%

“…Protein stability assay, co-immunoprecipitation assay and western blot Protein stability assay, western blot and co-immunoprecipitation experiments were performed as described previously (Wang et al, 2010a). To detect the interaction between endogenous PAK1 and b-catenin, SW480 cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM dithiothreitol, supplemented with protease inhibitor cocktail, 2 mM Na 3 VO 4 and 25 mM NaF) and lysates were immunoprecipitated with anti-PAK1 antibody.…”

Section: Methodsmentioning

confidence: 99%

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A Rac1/PAK1 cascade controls β-catenin activation in colon cancer cells

et al. 2011

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P21-activated kinase 1 (PAK1) is associated with colon cancer progression and metastasis, whereas the molecular mechanism remains elusive. Here, we show that downregulation of PAK1 in colon cancer cells reduces total b-catenin level, as well as cell proliferation. Mechanistically, PAK1 directly phosphorylates b-catenin proteins at Ser675 site and this leads to more stable and transcriptional active b-catenin. Corroborating these results, PAK1 is required for full Wnt signaling, and superactivation of b-catenin is achieved by simultaneous knockdown of adenomatous polyposis coli protein and activation of PAK1. Moreover, we show that Rac1 functions upstream of PAK1 in colon cancer cells and contributes to b-catenin phosphorylation and accumulation. We conclude that a Rac1/PAK1 cascade controls b-catenin S675 phosphorylation and full activation in colon cancer cells. Supporting this conclusion, overexpression of PAK1 is observed in 70% of colon cancer samples and is correlated with massive b-catenin accumulation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Rac1/PAK1 cascade controls β-catenin activation in colon cancer cells

et al. 2011

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“…For X. laevis tmem198, the primers were 5Ј-TGAACAGTTTTATCACCCGCC-3Ј and 5Ј-TATTATGACA TCAGTATGAGAGAA-3Ј. Other primers were used as previously described (51).…”

Section: Methodsmentioning

confidence: 99%

“…The MO sequences of Xenopus laevis tmem198 (NM_001094258) were as follows: MO-1, CGGTGGGACAACAGAC GATAGATCA; MO-2, GAATCTGGTGTTAAGTAAGCATGTC. Wholemount in situ hybridization and LacZ staining were performed as described previously (51). RNA probes were hybridized at 60°C, and BM purple substrate (Roche) was applied for the chromogenic reaction.…”

Section: Methodsmentioning

confidence: 99%

Transmembrane Protein 198 Promotes LRP6 Phosphorylation and Wnt Signaling Activation

Liang

Cruciat

et al. 2011

Molecular and Cellular Biology

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Wnt/␤-catenin signaling is fundamental in embryogenesis and tissue homeostasis in metazoans. Upon Wnt stimulation, cognate coreceptors LRP5 and LRP6 ([LRP5/6] low-density lipoprotein receptor-related proteins 5 and 6) are activated via phosphorylation at key residues. Although several kinases have been implicated, the LRP5/6 activation mechanism remains unclear. Here, we report that transmembrane protein 198 (TMEM198), a previously uncharacterized seven-transmembrane protein, is able to specifically activate LRP6 in transducing Wnt signaling. TMEM198 associates with LRP6 and recruits casein kinase family proteins, via the cytoplasmic domain, to phosphorylate key residues important for LRP6 activation. In mammalian cells, TMEM198 is required for Wnt signaling and casein kinase 1-induced LRP6 phosphorylation. During Xenopus embryogenesis, maternal and zygotic tmem198 mRNAs are widely distributed in the ectoderm and mesoderm. TMEM198 is required for Wnt-mediated neural crest formation, antero-posterior patterning, and particularly engrailed-2 expression in Xenopus embryos. Thus, our results identified TMEM198 as a membrane scaffold protein that promotes LRP6 phosphorylation and Wnt signaling activation.

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“…Co-injection of Xkctd15 did not reduce the frequency or completeness of Wnt8-induced axis duplications (data not shown). A recent study showed that an activator of Wnt signaling, Skip (1-341), was inefficient in inducing axis duplications (Wang et al, 2010), suggesting that certain modulators of Wnt signaling are inefficient in early frog embryos. In zebrafish, cells responsive to b-catenin can be monitored in the transgenic TOP-dGFP reporter line (Dorsky et al, 2002).…”

Section: Kctd15 Function Is Conserved and Inhibits Nc Induction In Exmentioning

confidence: 99%

Kctd15 inhibits neural crest formation by attenuating Wnt/β-catenin signaling output

Dutta

Dawid

2010

Development

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Specification, maintenance and differentiation of neural crest (NC) cells depend on multiple signaling pathways. In Xenopus and zebrafish, low levels of Bmp and high levels of Wnt signaling cooperate in NC induction (Saint-Jeannet et al., 1997;Wilson et al., 1997;Dorsky et al., 1998;Lewis et al., 2004). As Wnt signaling regulates anterior-posterior (A-P) patterning of the neural plate (Kim et al., 2000;McGrew et al., 1997;McGrew et al., 1995), Wnt signals might induce NC through posteriorization. However, neural A-P patterning and NC induction are separable events (Wu et al., 2005). A recent study indicates that Wnt-mediated posteriorization of the neural plate border (NPB) rather than the neural plate is crucial in NC induction . Signaling pathways active in NC specification are modulated by activators and inhibitors to regulate their strength and spatial distribution (Hong and SaintJeannet, 2007;Sauka-Spengler and Bronner-Fraser, 2008;Zhao et al., 2008). Here we introduce a factor, Potassium channel tetramerization domain containing 15 (Kctd15), that has a profound influence on NC formation in zebrafish and Xenopus embryos. KCTD15 was identified in humans (Hotta et al., 2009;Willer et al., 2009) as a BTB domain-containing protein of unknown function. We show that zebrafish kctd15a and kctd15b are expressed at the NPB at the end of gastrulation. Ectopic expression of Kctd15 inhibits NC specification, whereas knockdown leads to expansion of NC markers. Simultaneous attenuation of Wnt and Kctd15 expression rescues NC specification in zebrafish embryos. We suggest that Kctd15 restricts the NC domain by interfering with the functioning or output of the Wnt/b-catenin signaling pathway. MATERIALS AND METHODS Animal maintenance and embryonic stagingZebrafish (Danio rerio) embryos from AB/TL and TOP-dGFP (Dorsky et al., 2002) lines were obtained (Westerfield, 2000), and stages are indicated as hours post fertilization (hpf) (Kimmel et al., 1995). Xenopus laevis were staged according to (Nieuwkoop and Faber, 1967). Plasmids and DNA constructsThe open reading frames (ORFs) of zebrafish kctd15a (BC083478), zebrafish kctd15b (BC078294) and X. laevis kctd15 (BC077862) were subcloned into pCS2 + . The zebrafish b-cat* construct has four point mutations; S33A, S37A, T41A and S45A. MO and mRNA injectionMorpholino oligonucleotides (MO) were targeted to 5ЈUTR regions of zebrafish kctd15a (5Ј-TCCTTCCCTCCTTGGAAGACATAGC-3Ј) and zebrafish kctd15b (5Ј-AGCTCTCCTTCCCCCTCTTGATCTT-3Ј) (Gene Tools). Embryos at 1-2 cells were injected with 1.5 ng Kctd15a plus 0.5 ng Kctd15b MO; 2 ng Wnt8.1a MO (Lewis et al., 2004); or 2-4 ng standard control MO (5Ј-CCTC TTACCCTCAGTTACAATTTATA-3Ј) per embryo. 5Ј-capped mRNAs were prepared using the mMESSAGE mMACHINE Kit (Ambion). Each zebrafish embryo was injected with 50 pg kctd15a, kctd15b, DNkctd15a, DCkctd15a, kctd10, kctd6 or kctd13 mRNA, or 5 pg b-cat* mRNA. One blastomere of 2-cell Xenopus embryos was injected with 250 pg of Xkctd15 mRNA. For rescue experiments, 1-10 pg of mRNA was injected into fish...

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Xenopus Skip Modulates Wnt/β-Catenin Signaling and Functions in Neural Crest Induction

Cited by 30 publications

References 66 publications

A Rac1/PAK1 cascade controls β-catenin activation in colon cancer cells

A Rac1/PAK1 cascade controls β-catenin activation in colon cancer cells

Transmembrane Protein 198 Promotes LRP6 Phosphorylation and Wnt Signaling Activation

Kctd15 inhibits neural crest formation by attenuating Wnt/β-catenin signaling output

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