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Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos.

Sheldahl LC, Slusarski DC, Pandur P, Miller JR, Kühl M, Moon RT - J. Cell Biol. (2003)

Bottom Line: Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII).Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development.These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.

ABSTRACT
Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

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XDshΔDIX activates PKC in a PTX- insensitive manner. Two-cell-stage Xenopus embryos were injected with RNAs encoding XPKCα–myc (0.5–1 ng) plus XDsh–GFP (0.5–1 ng), XDshΔDEP–GFP (0.5–1 ng), XDshΔDIX–GFP (0.5–1 ng), Rfz2 (0.5–1 ng), PTX (1–2 ng), and/or LacZ (as a control for normalizing levels of injected RNA, 0–4 ng) and cultured to stage 8, and animal caps were explanted. (a) XPKC (red) is localized primarily in the cytoplasm under control conditions. (b) Ectopic XDsh–GFP (green), which is localized to punctate structures, is relatively weak at activating translocation of XPKC (red) to the membrane. (c) XDshΔDEP–GFP (green) as well as XDshΔPDZ–GFP (not depicted) do not activate XPKC (red) membrane translocation. (c, inset) XDsh–GFP (lane 1) and XDshΔDIX–GFP (lane 2) are expressed equally, as detected by an anti-GFP Western blot. Although the protein in lane 2 migrates more rapidly on gels compared with lane 1 (not depicted), it is aligned with lane 1 to facilitate the comparison of signal intensities. (d) Expression of XDshΔDIX–GFP (green) activates translocation to the membrane of PKC (red). (e) Rfz2 (untagged) activates membrane translocation of XPKC (red). (f) Rfz-2–mediated membrane translocation of XPKC (red) is partially blocked by PTX. (g) The ability of XDshΔDIX–GFP (green) to induce membrane translocation of XPKC (red) is not blocked by PTX. (h) A schematic representing the three domains of Dsh discussed in the text.
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fig1: XDshΔDIX activates PKC in a PTX- insensitive manner. Two-cell-stage Xenopus embryos were injected with RNAs encoding XPKCα–myc (0.5–1 ng) plus XDsh–GFP (0.5–1 ng), XDshΔDEP–GFP (0.5–1 ng), XDshΔDIX–GFP (0.5–1 ng), Rfz2 (0.5–1 ng), PTX (1–2 ng), and/or LacZ (as a control for normalizing levels of injected RNA, 0–4 ng) and cultured to stage 8, and animal caps were explanted. (a) XPKC (red) is localized primarily in the cytoplasm under control conditions. (b) Ectopic XDsh–GFP (green), which is localized to punctate structures, is relatively weak at activating translocation of XPKC (red) to the membrane. (c) XDshΔDEP–GFP (green) as well as XDshΔPDZ–GFP (not depicted) do not activate XPKC (red) membrane translocation. (c, inset) XDsh–GFP (lane 1) and XDshΔDIX–GFP (lane 2) are expressed equally, as detected by an anti-GFP Western blot. Although the protein in lane 2 migrates more rapidly on gels compared with lane 1 (not depicted), it is aligned with lane 1 to facilitate the comparison of signal intensities. (d) Expression of XDshΔDIX–GFP (green) activates translocation to the membrane of PKC (red). (e) Rfz2 (untagged) activates membrane translocation of XPKC (red). (f) Rfz-2–mediated membrane translocation of XPKC (red) is partially blocked by PTX. (g) The ability of XDshΔDIX–GFP (green) to induce membrane translocation of XPKC (red) is not blocked by PTX. (h) A schematic representing the three domains of Dsh discussed in the text.

Mentions: Dsh plays a dual role in the Wnt–β-catenin and PCP pathways, and has been termed a molecular switch between the two (for review see Wharton, 2003). Dsh is comprised of different domains, including the DEP, PDZ, and DIX domains (Fig. 1 h), and analysis of Dsh deletion constructs and Drosophila genetics have been used to separate the role of Dsh in the Wnt–β-catenin pathway versus the PCP pathway (Axelrod et al., 1998; Boutros et al., 1998; Penton et al., 2002). That the PCP pathway is downstream of Wnt signaling in vertebrates was first suggested when Dsh was found to affect Wnt and Fz function during gastrulation in zebrafish (Heisenberg et al., 2000) and Xenopus (Tada and Smith, 2000; Wallingford et al., 2000). One construct, DshΔDIX, which is not competent to activate the Wnt–β-catenin pathway but can signal via the PCP pathway, has been found to rescue loss of Wnt-11 function during gastrulation in Xenopus (Tada and Smith, 2000) and zebrafish (Heisenberg et al., 2000). Finally, data from PCP signaling in flies suggest that activation of the small GTPase Rho and jun-N-terminal kinase (JNK) lies downstream of Dsh (for review see Adler and Lee, 2001). In vertebrates, activation of Rho by Wnts and Fz also involves Dsh, and the protein Daam1, which links Dsh to Rho (Habas et al., 2001).


Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos.

Sheldahl LC, Slusarski DC, Pandur P, Miller JR, Kühl M, Moon RT - J. Cell Biol. (2003)

XDshΔDIX activates PKC in a PTX- insensitive manner. Two-cell-stage Xenopus embryos were injected with RNAs encoding XPKCα–myc (0.5–1 ng) plus XDsh–GFP (0.5–1 ng), XDshΔDEP–GFP (0.5–1 ng), XDshΔDIX–GFP (0.5–1 ng), Rfz2 (0.5–1 ng), PTX (1–2 ng), and/or LacZ (as a control for normalizing levels of injected RNA, 0–4 ng) and cultured to stage 8, and animal caps were explanted. (a) XPKC (red) is localized primarily in the cytoplasm under control conditions. (b) Ectopic XDsh–GFP (green), which is localized to punctate structures, is relatively weak at activating translocation of XPKC (red) to the membrane. (c) XDshΔDEP–GFP (green) as well as XDshΔPDZ–GFP (not depicted) do not activate XPKC (red) membrane translocation. (c, inset) XDsh–GFP (lane 1) and XDshΔDIX–GFP (lane 2) are expressed equally, as detected by an anti-GFP Western blot. Although the protein in lane 2 migrates more rapidly on gels compared with lane 1 (not depicted), it is aligned with lane 1 to facilitate the comparison of signal intensities. (d) Expression of XDshΔDIX–GFP (green) activates translocation to the membrane of PKC (red). (e) Rfz2 (untagged) activates membrane translocation of XPKC (red). (f) Rfz-2–mediated membrane translocation of XPKC (red) is partially blocked by PTX. (g) The ability of XDshΔDIX–GFP (green) to induce membrane translocation of XPKC (red) is not blocked by PTX. (h) A schematic representing the three domains of Dsh discussed in the text.
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fig1: XDshΔDIX activates PKC in a PTX- insensitive manner. Two-cell-stage Xenopus embryos were injected with RNAs encoding XPKCα–myc (0.5–1 ng) plus XDsh–GFP (0.5–1 ng), XDshΔDEP–GFP (0.5–1 ng), XDshΔDIX–GFP (0.5–1 ng), Rfz2 (0.5–1 ng), PTX (1–2 ng), and/or LacZ (as a control for normalizing levels of injected RNA, 0–4 ng) and cultured to stage 8, and animal caps were explanted. (a) XPKC (red) is localized primarily in the cytoplasm under control conditions. (b) Ectopic XDsh–GFP (green), which is localized to punctate structures, is relatively weak at activating translocation of XPKC (red) to the membrane. (c) XDshΔDEP–GFP (green) as well as XDshΔPDZ–GFP (not depicted) do not activate XPKC (red) membrane translocation. (c, inset) XDsh–GFP (lane 1) and XDshΔDIX–GFP (lane 2) are expressed equally, as detected by an anti-GFP Western blot. Although the protein in lane 2 migrates more rapidly on gels compared with lane 1 (not depicted), it is aligned with lane 1 to facilitate the comparison of signal intensities. (d) Expression of XDshΔDIX–GFP (green) activates translocation to the membrane of PKC (red). (e) Rfz2 (untagged) activates membrane translocation of XPKC (red). (f) Rfz-2–mediated membrane translocation of XPKC (red) is partially blocked by PTX. (g) The ability of XDshΔDIX–GFP (green) to induce membrane translocation of XPKC (red) is not blocked by PTX. (h) A schematic representing the three domains of Dsh discussed in the text.
Mentions: Dsh plays a dual role in the Wnt–β-catenin and PCP pathways, and has been termed a molecular switch between the two (for review see Wharton, 2003). Dsh is comprised of different domains, including the DEP, PDZ, and DIX domains (Fig. 1 h), and analysis of Dsh deletion constructs and Drosophila genetics have been used to separate the role of Dsh in the Wnt–β-catenin pathway versus the PCP pathway (Axelrod et al., 1998; Boutros et al., 1998; Penton et al., 2002). That the PCP pathway is downstream of Wnt signaling in vertebrates was first suggested when Dsh was found to affect Wnt and Fz function during gastrulation in zebrafish (Heisenberg et al., 2000) and Xenopus (Tada and Smith, 2000; Wallingford et al., 2000). One construct, DshΔDIX, which is not competent to activate the Wnt–β-catenin pathway but can signal via the PCP pathway, has been found to rescue loss of Wnt-11 function during gastrulation in Xenopus (Tada and Smith, 2000) and zebrafish (Heisenberg et al., 2000). Finally, data from PCP signaling in flies suggest that activation of the small GTPase Rho and jun-N-terminal kinase (JNK) lies downstream of Dsh (for review see Adler and Lee, 2001). In vertebrates, activation of Rho by Wnts and Fz also involves Dsh, and the protein Daam1, which links Dsh to Rho (Habas et al., 2001).

Bottom Line: Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII).Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development.These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.

ABSTRACT
Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

Show MeSH
Related in: MedlinePlus