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Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements.

Kim GH, Her JH, Han JK - J. Cell Biol. (2008)

Bottom Line: Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk.Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues.Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Hyoja Dong, Pohang, Kyungbuk 790-784, Republic of Korea.

ABSTRACT
The single-pass transmembrane protein Ryk (atypical receptor related tyrosine kinase) functions as a Wnt receptor. However, Ryk's correlation with Wnt/Frizzled (Fz) signaling is poorly understood. Here, we report that Ryk regulates Xenopus laevis convergent extension (CE) movements via the beta-arrestin 2 (betaarr2)-dependent endocytic process triggered by noncanonical Wnt signaling. During X. laevis gastrulation, betaarr2-mediated endocytosis of Fz7 and dishevelled (Dvl/Dsh) actually occurs in the dorsal marginal zone tissues, which actively participate in noncanonical Wnt signaling. Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk. Ryk interacts with both Wnt11 and betaarr2, cooperates with Fz7 to mediate Wnt11-stimulated endocytosis of Dsh, and signals the noncanonical Wnt pathway in CE movements. Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues. Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.

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Ryk acts as an essential regulator of noncanonical Wnt signaling on CE movements. (A) Ryk, like Fz7, phosphorylated Dsh. Animal caps expressing myc Dsh (1 ng) alone or with the combinations of XRyk (1 ng) and Fz7 (1 ng) as indicated were subjected to Western blotting using anti-myc and anti–β-catenin antibodies. (B–E) The normal Dsh (B) and RhoA (D) distribution was translocated by Ryk to the cell membrane in animal cap cells (C and E). XRyk (1 ng) were injected with GFP Dsh (500 pg) or GFP RhoA (500 pg) into the animal regions of embryos at the four-cell stage, either alone or in combination as indicated. (F and G) Two blastomeres of four-cell stage embryos were injected at the dorsal marginal region with the indicated mRNAs or MO (1 ng myc RhoA, 1 ng Dsh, 1 ng XRyk, and 40 ng XRyk MO). The DMZ explants were dissected at stage 10.25 and cultured until stage 12. (F) Ryk activated RhoA in DMZ tissues during gastrulation, but Ryk MO inactivated them. GTP-bound RhoA in DMZ lysates was precipitated using GST-RBD and visualized by immunoblotting with an anti-myc antibody. (G) Ryk, like Dsh, induced JNK phosphorylation in CE movements. The explant lysates were blotted with anti-phospho JNK and anti-JNK antibodies. (H) Ryk physically interacted with βarr2 in vivo. HEK293FT cells were transfected with myc XRyk, GFP xβarr2, or GFP Dsh, either alone or in combination as indicated. Cell lysates were immunoprecipitated with anti-myc antibody, and the immunocomplexes were blotted with specific antibodies. Numbers to the sides of the gel blots indicate molecular mass standards in kD. (I–M) Four-cell stage embryos were injected in the animal pole with various mRNA in combination as indicated (500 pg GFP xβarr2, 1 ng Xdsh MA, 1 ng, myc XRyk, 1 ng Wnt11, 1 ng Fz7, 500 pg GFP Dsh, 10 ng xβarr2 MO, 10 ng Co MO, and 500 pg myc Rab5). Animal caps were dissected at stage 9–10 and then subjected to fluorescence analysis. (I) Normal distribution of βarr2 in animal cap cells. (J and K) Ryk-induced membrane distribution of βarr2 was relocalized by Xdsh-MA to the intracellular cluster. (L and M) βarr2 knockdown inhibited the noncanonical Wnt11/Fz7-mediated Dsh endocytosis in the presence of Ryk. Bars, 20 μm.
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fig4: Ryk acts as an essential regulator of noncanonical Wnt signaling on CE movements. (A) Ryk, like Fz7, phosphorylated Dsh. Animal caps expressing myc Dsh (1 ng) alone or with the combinations of XRyk (1 ng) and Fz7 (1 ng) as indicated were subjected to Western blotting using anti-myc and anti–β-catenin antibodies. (B–E) The normal Dsh (B) and RhoA (D) distribution was translocated by Ryk to the cell membrane in animal cap cells (C and E). XRyk (1 ng) were injected with GFP Dsh (500 pg) or GFP RhoA (500 pg) into the animal regions of embryos at the four-cell stage, either alone or in combination as indicated. (F and G) Two blastomeres of four-cell stage embryos were injected at the dorsal marginal region with the indicated mRNAs or MO (1 ng myc RhoA, 1 ng Dsh, 1 ng XRyk, and 40 ng XRyk MO). The DMZ explants were dissected at stage 10.25 and cultured until stage 12. (F) Ryk activated RhoA in DMZ tissues during gastrulation, but Ryk MO inactivated them. GTP-bound RhoA in DMZ lysates was precipitated using GST-RBD and visualized by immunoblotting with an anti-myc antibody. (G) Ryk, like Dsh, induced JNK phosphorylation in CE movements. The explant lysates were blotted with anti-phospho JNK and anti-JNK antibodies. (H) Ryk physically interacted with βarr2 in vivo. HEK293FT cells were transfected with myc XRyk, GFP xβarr2, or GFP Dsh, either alone or in combination as indicated. Cell lysates were immunoprecipitated with anti-myc antibody, and the immunocomplexes were blotted with specific antibodies. Numbers to the sides of the gel blots indicate molecular mass standards in kD. (I–M) Four-cell stage embryos were injected in the animal pole with various mRNA in combination as indicated (500 pg GFP xβarr2, 1 ng Xdsh MA, 1 ng, myc XRyk, 1 ng Wnt11, 1 ng Fz7, 500 pg GFP Dsh, 10 ng xβarr2 MO, 10 ng Co MO, and 500 pg myc Rab5). Animal caps were dissected at stage 9–10 and then subjected to fluorescence analysis. (I) Normal distribution of βarr2 in animal cap cells. (J and K) Ryk-induced membrane distribution of βarr2 was relocalized by Xdsh-MA to the intracellular cluster. (L and M) βarr2 knockdown inhibited the noncanonical Wnt11/Fz7-mediated Dsh endocytosis in the presence of Ryk. Bars, 20 μm.

Mentions: Next, we wanted to know whether Ryk signals the noncanonical Wnt pathway during CE movements. In the PCP pathway, phosphorylation of Dsh is important for its translocation to the cell membrane, and this translocation is a prerequisite for functional signaling activation (Park et al., 2005; Kim and Han, 2007). Therefore, it is possible that Ryk regulates the phosphorylation and subcellular localization of Dsh. In considering this possibility, we observed that Ryk induces the hyperphosphorylation (Fig. 4 A) and membrane accumulation of Dsh in animal cap cells (Fig. 4, B and C). In addition, we measured the activity of RhoA by using a GST-RBD fusion protein that recognizes the GTP-bound active RhoA and the phosphorylation level of JNK in CE movements. With theses assays, we found that XRyk is required for RhoA activation (Fig. 4 F) in concert with its translocation to the cell membrane of animal cap tissues (Fig. 4, D and E) and increases JNK phosphorylation in DMZ cells (Fig. 4 G). We then proceeded to determine the relationship between Ryk and βarr2. Coimmunoprecipitation and fluorescence analyses showed that Ryk is capable of binding to βarr2 (Fig. 4 H) and can redistribute βarr2 from the cytoplasm to the plasma membrane in animal cap cells (Fig. 4, I and J). Importantly, in the presence of Xdsh MA, a construct fused with the mitochondrial membrane–anchoring sequence (Park et al., 2005), the Ryk-dependent membrane accumulation of βarr2 is relocalized to intracellular clusters (Fig. 4 K), which implies that Ryk, like Fz, indirectly interacts with βarr2 via Dsh. Furthermore, we found that βarr2 knockdown inhibits the noncanonical Wnt11/Fz7-mediated Dsh endocytosis in the presence of Ryk (Fig. 4, L and M). Therefore, our observations strongly suggest that Ryk acts as a novel regulator that is essential for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements. This idea was supported by investigating the biological epistasis between Ryk and noncanonical Wnt signaling components in more detail. For this purpose, rescue experiments using a DMZ elongation assay were performed. Wnt11 overexpression inhibited the elongation of DMZ explants, and, intriguingly, this inhibition was rescued by the coexpression of XRyk MO. Moreover, the CE movements that were significantly inhibited by Ryk overexpression were restored by the functional inhibition of cytoplasmic downstream molecules of noncanonical Wnt signaling using βarr2 MO and dominant-negative forms of Dsh (Xdd1) and RhoA (RhoA-N19) (Fig. 5, A–G; and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200710188/DC1). Consistently, the CE-defective phenotypes caused by Ryk depletion were also rescued by the overexpression of Dsh, βarr2, and the constitutively active form of RhoA (RhoA-V14; Fig. S3 F), which indicates that Ryk functions upstream of the cytoplasmic molecules activated by Wnt11 in CE movements. The biochemical experiments between Dsh and Ryk showed that the cytoplasmic domain of Ryk is important to interact with Dsh (Fig. 2 F). We further tested whether this domain is required for Ryk function in CE movements. Interestingly, the XRyk ΔC rescued the CE defects caused by XRyk (Fig. 5 H) and Wnt11 (Table S1). Moreover, XRyk ΔC did not induce the hyperphosphorylation of Dsh (Fig. 5 I) or the membrane accumulation of RhoA in animal cap cells (Fig. 5, J–M). Collectively, these observations suggest that Ryk mediates the noncanonical Wnt pathway by controlling Dsh in CE movements.


Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements.

Kim GH, Her JH, Han JK - J. Cell Biol. (2008)

Ryk acts as an essential regulator of noncanonical Wnt signaling on CE movements. (A) Ryk, like Fz7, phosphorylated Dsh. Animal caps expressing myc Dsh (1 ng) alone or with the combinations of XRyk (1 ng) and Fz7 (1 ng) as indicated were subjected to Western blotting using anti-myc and anti–β-catenin antibodies. (B–E) The normal Dsh (B) and RhoA (D) distribution was translocated by Ryk to the cell membrane in animal cap cells (C and E). XRyk (1 ng) were injected with GFP Dsh (500 pg) or GFP RhoA (500 pg) into the animal regions of embryos at the four-cell stage, either alone or in combination as indicated. (F and G) Two blastomeres of four-cell stage embryos were injected at the dorsal marginal region with the indicated mRNAs or MO (1 ng myc RhoA, 1 ng Dsh, 1 ng XRyk, and 40 ng XRyk MO). The DMZ explants were dissected at stage 10.25 and cultured until stage 12. (F) Ryk activated RhoA in DMZ tissues during gastrulation, but Ryk MO inactivated them. GTP-bound RhoA in DMZ lysates was precipitated using GST-RBD and visualized by immunoblotting with an anti-myc antibody. (G) Ryk, like Dsh, induced JNK phosphorylation in CE movements. The explant lysates were blotted with anti-phospho JNK and anti-JNK antibodies. (H) Ryk physically interacted with βarr2 in vivo. HEK293FT cells were transfected with myc XRyk, GFP xβarr2, or GFP Dsh, either alone or in combination as indicated. Cell lysates were immunoprecipitated with anti-myc antibody, and the immunocomplexes were blotted with specific antibodies. Numbers to the sides of the gel blots indicate molecular mass standards in kD. (I–M) Four-cell stage embryos were injected in the animal pole with various mRNA in combination as indicated (500 pg GFP xβarr2, 1 ng Xdsh MA, 1 ng, myc XRyk, 1 ng Wnt11, 1 ng Fz7, 500 pg GFP Dsh, 10 ng xβarr2 MO, 10 ng Co MO, and 500 pg myc Rab5). Animal caps were dissected at stage 9–10 and then subjected to fluorescence analysis. (I) Normal distribution of βarr2 in animal cap cells. (J and K) Ryk-induced membrane distribution of βarr2 was relocalized by Xdsh-MA to the intracellular cluster. (L and M) βarr2 knockdown inhibited the noncanonical Wnt11/Fz7-mediated Dsh endocytosis in the presence of Ryk. Bars, 20 μm.
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fig4: Ryk acts as an essential regulator of noncanonical Wnt signaling on CE movements. (A) Ryk, like Fz7, phosphorylated Dsh. Animal caps expressing myc Dsh (1 ng) alone or with the combinations of XRyk (1 ng) and Fz7 (1 ng) as indicated were subjected to Western blotting using anti-myc and anti–β-catenin antibodies. (B–E) The normal Dsh (B) and RhoA (D) distribution was translocated by Ryk to the cell membrane in animal cap cells (C and E). XRyk (1 ng) were injected with GFP Dsh (500 pg) or GFP RhoA (500 pg) into the animal regions of embryos at the four-cell stage, either alone or in combination as indicated. (F and G) Two blastomeres of four-cell stage embryos were injected at the dorsal marginal region with the indicated mRNAs or MO (1 ng myc RhoA, 1 ng Dsh, 1 ng XRyk, and 40 ng XRyk MO). The DMZ explants were dissected at stage 10.25 and cultured until stage 12. (F) Ryk activated RhoA in DMZ tissues during gastrulation, but Ryk MO inactivated them. GTP-bound RhoA in DMZ lysates was precipitated using GST-RBD and visualized by immunoblotting with an anti-myc antibody. (G) Ryk, like Dsh, induced JNK phosphorylation in CE movements. The explant lysates were blotted with anti-phospho JNK and anti-JNK antibodies. (H) Ryk physically interacted with βarr2 in vivo. HEK293FT cells were transfected with myc XRyk, GFP xβarr2, or GFP Dsh, either alone or in combination as indicated. Cell lysates were immunoprecipitated with anti-myc antibody, and the immunocomplexes were blotted with specific antibodies. Numbers to the sides of the gel blots indicate molecular mass standards in kD. (I–M) Four-cell stage embryos were injected in the animal pole with various mRNA in combination as indicated (500 pg GFP xβarr2, 1 ng Xdsh MA, 1 ng, myc XRyk, 1 ng Wnt11, 1 ng Fz7, 500 pg GFP Dsh, 10 ng xβarr2 MO, 10 ng Co MO, and 500 pg myc Rab5). Animal caps were dissected at stage 9–10 and then subjected to fluorescence analysis. (I) Normal distribution of βarr2 in animal cap cells. (J and K) Ryk-induced membrane distribution of βarr2 was relocalized by Xdsh-MA to the intracellular cluster. (L and M) βarr2 knockdown inhibited the noncanonical Wnt11/Fz7-mediated Dsh endocytosis in the presence of Ryk. Bars, 20 μm.
Mentions: Next, we wanted to know whether Ryk signals the noncanonical Wnt pathway during CE movements. In the PCP pathway, phosphorylation of Dsh is important for its translocation to the cell membrane, and this translocation is a prerequisite for functional signaling activation (Park et al., 2005; Kim and Han, 2007). Therefore, it is possible that Ryk regulates the phosphorylation and subcellular localization of Dsh. In considering this possibility, we observed that Ryk induces the hyperphosphorylation (Fig. 4 A) and membrane accumulation of Dsh in animal cap cells (Fig. 4, B and C). In addition, we measured the activity of RhoA by using a GST-RBD fusion protein that recognizes the GTP-bound active RhoA and the phosphorylation level of JNK in CE movements. With theses assays, we found that XRyk is required for RhoA activation (Fig. 4 F) in concert with its translocation to the cell membrane of animal cap tissues (Fig. 4, D and E) and increases JNK phosphorylation in DMZ cells (Fig. 4 G). We then proceeded to determine the relationship between Ryk and βarr2. Coimmunoprecipitation and fluorescence analyses showed that Ryk is capable of binding to βarr2 (Fig. 4 H) and can redistribute βarr2 from the cytoplasm to the plasma membrane in animal cap cells (Fig. 4, I and J). Importantly, in the presence of Xdsh MA, a construct fused with the mitochondrial membrane–anchoring sequence (Park et al., 2005), the Ryk-dependent membrane accumulation of βarr2 is relocalized to intracellular clusters (Fig. 4 K), which implies that Ryk, like Fz, indirectly interacts with βarr2 via Dsh. Furthermore, we found that βarr2 knockdown inhibits the noncanonical Wnt11/Fz7-mediated Dsh endocytosis in the presence of Ryk (Fig. 4, L and M). Therefore, our observations strongly suggest that Ryk acts as a novel regulator that is essential for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements. This idea was supported by investigating the biological epistasis between Ryk and noncanonical Wnt signaling components in more detail. For this purpose, rescue experiments using a DMZ elongation assay were performed. Wnt11 overexpression inhibited the elongation of DMZ explants, and, intriguingly, this inhibition was rescued by the coexpression of XRyk MO. Moreover, the CE movements that were significantly inhibited by Ryk overexpression were restored by the functional inhibition of cytoplasmic downstream molecules of noncanonical Wnt signaling using βarr2 MO and dominant-negative forms of Dsh (Xdd1) and RhoA (RhoA-N19) (Fig. 5, A–G; and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200710188/DC1). Consistently, the CE-defective phenotypes caused by Ryk depletion were also rescued by the overexpression of Dsh, βarr2, and the constitutively active form of RhoA (RhoA-V14; Fig. S3 F), which indicates that Ryk functions upstream of the cytoplasmic molecules activated by Wnt11 in CE movements. The biochemical experiments between Dsh and Ryk showed that the cytoplasmic domain of Ryk is important to interact with Dsh (Fig. 2 F). We further tested whether this domain is required for Ryk function in CE movements. Interestingly, the XRyk ΔC rescued the CE defects caused by XRyk (Fig. 5 H) and Wnt11 (Table S1). Moreover, XRyk ΔC did not induce the hyperphosphorylation of Dsh (Fig. 5 I) or the membrane accumulation of RhoA in animal cap cells (Fig. 5, J–M). Collectively, these observations suggest that Ryk mediates the noncanonical Wnt pathway by controlling Dsh in CE movements.

Bottom Line: Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk.Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues.Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Hyoja Dong, Pohang, Kyungbuk 790-784, Republic of Korea.

ABSTRACT
The single-pass transmembrane protein Ryk (atypical receptor related tyrosine kinase) functions as a Wnt receptor. However, Ryk's correlation with Wnt/Frizzled (Fz) signaling is poorly understood. Here, we report that Ryk regulates Xenopus laevis convergent extension (CE) movements via the beta-arrestin 2 (betaarr2)-dependent endocytic process triggered by noncanonical Wnt signaling. During X. laevis gastrulation, betaarr2-mediated endocytosis of Fz7 and dishevelled (Dvl/Dsh) actually occurs in the dorsal marginal zone tissues, which actively participate in noncanonical Wnt signaling. Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk. Ryk interacts with both Wnt11 and betaarr2, cooperates with Fz7 to mediate Wnt11-stimulated endocytosis of Dsh, and signals the noncanonical Wnt pathway in CE movements. Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues. Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.

Show MeSH
Related in: MedlinePlus