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Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.

Farr GH, Ferkey DM, Yost C, Pierce SB, Weaver C, Kimelman D - J. Cell Biol. (2000)

Bottom Line: Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo.Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin.These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Center for Developmental Biology, University of Washington, Seattle, Washington 98195-7350, USA.

ABSTRACT
Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

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Dominant-negative Xgsk-3 binds Axin. Embryos were injected at the two- to eight-cell stage with 1 ng Axin-myc, 0.5 ng Xgsk-3-FLAG, and 0.5 ng dnXgsk-3-FLAG in the animal pole. Embryo extracts were precipitated with anti-FLAG antibody and detected by Western blotting (left panel). An aliquot of each sample taken before immunoprecipitation is shown in the right panel (Total Lysates). Lane numbers in the right panel refer to the same injections as shown above corresponding lane numbers in the left panel.
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Figure 4: Dominant-negative Xgsk-3 binds Axin. Embryos were injected at the two- to eight-cell stage with 1 ng Axin-myc, 0.5 ng Xgsk-3-FLAG, and 0.5 ng dnXgsk-3-FLAG in the animal pole. Embryo extracts were precipitated with anti-FLAG antibody and detected by Western blotting (left panel). An aliquot of each sample taken before immunoprecipitation is shown in the right panel (Total Lysates). Lane numbers in the right panel refer to the same injections as shown above corresponding lane numbers in the left panel.

Mentions: It has been shown in a number of studies, including our own, that mutation of a conserved lysine in the ATP binding region of GSK-3 results in a kinase-deficient mutant that acts as a dominant-negative mutant (dnXgsk-3) in Xenopus (Dominguez et al. 1995; He et al. 1995; Pierce and Kimelman 1995). The demonstration that Axin binds GSK-3 and promotes the phosphorylation of β-catenin suggests that the dnXgsk-3 might function by binding Axin and keeping it from binding endogenous Xgsk-3. However, studies in a mammalian system have shown that kinase dead GSK-3 does not bind Axin (Ikeda et al. 1998), indicating that the kinase dead dnXgsk-3 might stabilize β-catenin by an alternative mechanism. To investigate this issue, we compared Xgsk-3 and dnXgsk-3 binding to Axin in coimmunoprecipitation experiments. Axin-myc was coinjected with either Xgsk-3-FLAG or dnXgsk-3-FLAG in Xenopus embryos, and expressed proteins were immunoprecipitated with anti-FLAG antibodies. Immunocomplexes were analyzed by Western blotting with anti-myc and anti-FLAG antibodies. As expected, Axin is immunoprecipitated by Xgsk-3 (Fig. 4, lane 7). In addition, Axin is immunoprecipitated equally well by dnXgsk-3 (Fig. 4, lane 8). Thus, unlike in the mammalian system, the kinase dead mutant of Xgsk-3 binds Axin, suggesting that it stabilizes β-catenin either by displacing endogenous Xgsk-3 from the Axin complex, or by preventing association of Axin and endogenous Xgsk-3.


Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.

Farr GH, Ferkey DM, Yost C, Pierce SB, Weaver C, Kimelman D - J. Cell Biol. (2000)

Dominant-negative Xgsk-3 binds Axin. Embryos were injected at the two- to eight-cell stage with 1 ng Axin-myc, 0.5 ng Xgsk-3-FLAG, and 0.5 ng dnXgsk-3-FLAG in the animal pole. Embryo extracts were precipitated with anti-FLAG antibody and detected by Western blotting (left panel). An aliquot of each sample taken before immunoprecipitation is shown in the right panel (Total Lysates). Lane numbers in the right panel refer to the same injections as shown above corresponding lane numbers in the left panel.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169372&req=5

Figure 4: Dominant-negative Xgsk-3 binds Axin. Embryos were injected at the two- to eight-cell stage with 1 ng Axin-myc, 0.5 ng Xgsk-3-FLAG, and 0.5 ng dnXgsk-3-FLAG in the animal pole. Embryo extracts were precipitated with anti-FLAG antibody and detected by Western blotting (left panel). An aliquot of each sample taken before immunoprecipitation is shown in the right panel (Total Lysates). Lane numbers in the right panel refer to the same injections as shown above corresponding lane numbers in the left panel.
Mentions: It has been shown in a number of studies, including our own, that mutation of a conserved lysine in the ATP binding region of GSK-3 results in a kinase-deficient mutant that acts as a dominant-negative mutant (dnXgsk-3) in Xenopus (Dominguez et al. 1995; He et al. 1995; Pierce and Kimelman 1995). The demonstration that Axin binds GSK-3 and promotes the phosphorylation of β-catenin suggests that the dnXgsk-3 might function by binding Axin and keeping it from binding endogenous Xgsk-3. However, studies in a mammalian system have shown that kinase dead GSK-3 does not bind Axin (Ikeda et al. 1998), indicating that the kinase dead dnXgsk-3 might stabilize β-catenin by an alternative mechanism. To investigate this issue, we compared Xgsk-3 and dnXgsk-3 binding to Axin in coimmunoprecipitation experiments. Axin-myc was coinjected with either Xgsk-3-FLAG or dnXgsk-3-FLAG in Xenopus embryos, and expressed proteins were immunoprecipitated with anti-FLAG antibodies. Immunocomplexes were analyzed by Western blotting with anti-myc and anti-FLAG antibodies. As expected, Axin is immunoprecipitated by Xgsk-3 (Fig. 4, lane 7). In addition, Axin is immunoprecipitated equally well by dnXgsk-3 (Fig. 4, lane 8). Thus, unlike in the mammalian system, the kinase dead mutant of Xgsk-3 binds Axin, suggesting that it stabilizes β-catenin either by displacing endogenous Xgsk-3 from the Axin complex, or by preventing association of Axin and endogenous Xgsk-3.

Bottom Line: Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo.Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin.These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Center for Developmental Biology, University of Washington, Seattle, Washington 98195-7350, USA.

ABSTRACT
Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

Show MeSH
Related in: MedlinePlus