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First published online 12 December 2007
doi: 10.1242/dev.013540

Development 135, 367-375 (2008)
Published by The Company of Biologists 2008
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Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

Xin Zeng1,*, He Huang1,*, Keiko Tamai1,{dagger}, Xinjun Zhang1, Yuko Harada1, Chika Yokota1, Karla Almeida1, Jianbo Wang2, Brad Doble3, Jim Woodgett3, Anthony Wynshaw-Boris2, Jen-Chieh Hsieh4 and Xi He1,{ddagger}

1 The F. M. Kirby Neurobiology Center, Children's Hospital Boston, Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
2 Department of Pediatrics and Medicine, University of California, San Diego, La Jolla, CA 92093-0627, USA.
3 Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada.
4 Department of Biochemistry and Cell Biology, Center for Developmental Genetics, State University of New York, Stony Brook, Stony Brook, NY 11794, USA.


Figure 1
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  		Fig. 1. Fz function is required for Wnt-induced LRP6 phosphorylation. (A) Shisa inhibited Wnt3a-induced LRP6 phosphorylation. HEK293T cells cotransfected with LRP6 plus Flag-tagged Shisa (or a control vector) were incubated with Wnt3a conditioned medium (CM) or the control CM for 1 hour. Shisa inhibited Wnt3a-induced β-catenin accumulation in the cytosol. β-actin: a loading control. (B) L cells stably transfected with shRNAs against Fz2 and Fz7 [L (Fz-) cell] showed diminished Wnt-induced LRP6 (endogenous) phosphorylation. The L (Fz-) cells were treated for 1hour with increasing concentrations of Wnt3a CM. These L (Fz-) cells also exhibited attenuated β-catenin stabilization in response to Wnt3a compared to the control L cells. (C) Human Fz5 expression rescued Wnt3a-induced LRP6 (endogenous) phosphorylation in the L (Fz-) cells. The stable clones of L (Fz-) cell transfected with Fz5 (or the control vector) were pooled together and assayed.

 

Figure 2
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  		Fig. 2. Forced Fz-LRP6 association activates LRP6 phosphorylation. (A,B) DKK1-Fz5, but neither Fz5 nor DKK3-Fz5, activated LRP6 phosphorylation (A) and TCF/β-catenin reporter expression (B) in HEK293T cells. The expression level of DKK1-Fz5 and DKK3-Fz5 was similar, but was much lower than that of Fz5 (A) as detected by an anti-Fz5 antibody. This was probably due to the presence of the DKK moiety. (C) A diagram showing the position of the amino acid substitutions or the truncation in DKK1-Fz5 mutants. (D) DKK1-Fz5, but neither of the DKK1-Fz5 mutants, enhanced LRP6 phosphorylation. The protein level of Fz5 and DKK-Fz5 fusion proteins was detected via the Myc tag. (E) DKK1-Fz5, but neither of the two DKK1-Fz5 mutants, synergized with LRP6 to stimulate the TCF/β-catenin reporter expression in HEK293T cells.

 

Figure 3
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  		Fig. 3. Dvl is required for Wnt-induced LRP6 phosphorylation and acts via the DIX and PDZ domains. (A) Reduction of Dvl proteins diminished Wnt3a-induced Lrp6 (endogenous) phosphorylation. MEFs lacking Dvl1 and Dvl2 (Dvl1-/-;Dvl2-/-) (see Fig. S1 in the supplementary material) were infected with each of the four different lentiviral shRNAs against mouse Dvl3. After 3 days, the cells were treated with Wnt3a CM or control CM for 1 hour. Dvl3 protein level were drastically reduced by shRNA2, 3 or 4. Dvl3 protein exhibited typical Wnt-induced mobility shift (due to phosphorylation). shRNA2 and 4 were used in further experiments and yielded identical results. (B) The wild-type Dvl2 rescued LRP6 (endogenous) phosphorylation in Dvl3 knockdown Dvl1-/-;Dvl2-/- MEFs. Dvl1-/-;Dvl2-/- MEFs stably expressing Dvl2 (lanes 5 to 8) or the control vector (lanes 1 to 4) were pooled and infected with the lentiviral Dvl3 shRNA as indicated, then treated with Wnt3a or control CM for 1 hour as indicated. The exogenously expressed Dvl2 was detected by the Flag tag. (C) Dvl2{Delta}DEP, but neither Dvl2{Delta}DIX nor Dvl2{Delta}PDZ, rescued the Wnt3a-induced Lrp6 (endogenous) phosphorylation in Dvl3 knockdown Dvl1-/-;Dvl2-/- MEFs. Dvl1-/-;Dvl2-/- MEFs stably expressing the control vector (lanes 1-4), Dvl2{Delta}DIX (lanes 9-12), Dvl2{Delta}PDZ (lanes 5-8) or Dvl2{Delta}DEP (lanes 13-16) were infected with the lentiviral Dvl3 shRNA as indicated, and treated with Wnt3a CM or control CM for 1 hour as indicated. The expression of Dvl2 mutants was detected by the Flag tag.

 

Figure 4
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  		Fig. 4. Fz recruitment of axin to the plasma membrane via Dvl (Xdsh) in Xenopus embryonic explants. Synthetic mRNAs for axin (0.5 ng), Xdsh (Dvl) or Xdsh{Delta}N or Xdsh-GFP (1 ng), and human Fz5 (1 ng) were injected alone or in combinations as indicated into the Xenopus embryo. Axin and Fz proteins were detected by anti-Flag and anti-Fz5 antibodies, respectively. (A) Axin alone. (Ba-c) Xdsh-GFP plus axin. (Ca-c) Fz5 plus axin. (Da-d) Fz5 plus axin plus Xdsh-GFP. (Ea-c) Fz5 plus axin plus Xdsh. (Fa-c) Fz5 plus axin plus Xdsh{Delta}N.

 

Figure 5
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  		Fig. 5. Axin is required for Lrp6 phosphorylation via its ability to bind Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/β-catenin signaling. (A) Axin-/- and the wild-type ES cells were treated with Wnt3a or control CM. Wnt3a-induced Lrp6 (endogenous) phosphorylation was significantly reduced in Axin-/- ES cells. (B,C) Reducing Axin2 expression in Axin-/- ES cells further inhibited Lrp6 (endogenous) phosphorylation (B). Axin-/- ES cells were infected with lentiviral shRNAs against mouse Axin2. TfR, transferin receptor, loading control. The efficiency of Axin2 mRNA knockdown was assessed by RT-PCR analysis. GAPDH, a loading control (C). (D) The wild-type axin, but not the GSK3 binding mutant axin (L396Q) promoted LRP6 phosphorylation by GSK3. Axin and axin (L396Q) were cotransfected with VSVG-tagged LRP6 in the presence or absence of GSK3 in HEK293T cells. Axin and GSK3 were detected by the Flag and HA tags, respectively. (E) Gsk3{alpha} and Gsk3β share redundant function in Wnt-induced LRP6 phosphorylation. ES cells null for both Gsk3{alpha} and Gsk3β (Gsk3{alpha}-/-;β-/-), null for either Gsk3{alpha} (Gsk3{alpha}-/-) or Gsk3β (Gsk3β-/-) and the control wild-type ES cells were treated with Wnt3a or control CM for 1 hour. The endogenous Lrp6 was examined. β-actin, loading control. (F) A plasma membrane-targeted CAAX-GID blocked wnt8 (Xwnt8) signaling in Xenopus embryo explants. GID induced nr3 (Xnr3) expression; CAAX-GID, but not CAAX-GID-LP, blocked wnt8 (Xwnt8)-induced nr3 expression (each was injected at 10-50 pg mRNA/embryo). wnt8 was injected at 10 pg mRNA/embryo. -, without reverse transcriptase; Un, uninjected control embryo; EF-1{alpha}; loading control.

 

Figure 6
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  		Fig. 6. A sequential recruitment and amplification model for Wnt-induced Lrp6 phosphorylation. (A) Initiation. Wnt-induced Fz-Lrp6 complex formation promotes initial Lrp6 phosphorylation via Dvl recruitment of the axin-Gsk3 complex. (B) Amplification. Initial Lrp6 phosphorylation provides docking sites and thereby recruits additional Axin-Gsk3 complex to promote further Lrp6 phosphorylation in cis and possibly in trans if/when Lrp6 multimerizes. See Discussion for details. For clarity, β-catenin, CK1 and other proteins are omitted from the axin complex, the protein composition of which may be different with and without Wnt stimulation.

 

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© The Company of Biologists Ltd 2008