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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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GBP does not inhibit Xgsk-3 phosphorylation of a peptide substrate. Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG RNA. After 3 h, proteins were extracted and immunoprecipitated with anti-FLAG (Xgsk-3 + GBP), anti-myc (Xgsk-3 + control), or both (uninjected) antibodies. The kinase activity of immune complexes was measured by phosphorus-32 incorporation into the GSK-3–specific substrate prephosphorylated CREB peptide (p-CREB; dark bars). The nonphosphorylated CREB peptide (CREB; light bars) is not a GSK-3 substrate and was used as a control. The activity of duplicate immune complexes is shown.
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Figure 3: GBP does not inhibit Xgsk-3 phosphorylation of a peptide substrate. Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG RNA. After 3 h, proteins were extracted and immunoprecipitated with anti-FLAG (Xgsk-3 + GBP), anti-myc (Xgsk-3 + control), or both (uninjected) antibodies. The kinase activity of immune complexes was measured by phosphorus-32 incorporation into the GSK-3–specific substrate prephosphorylated CREB peptide (p-CREB; dark bars). The nonphosphorylated CREB peptide (CREB; light bars) is not a GSK-3 substrate and was used as a control. The activity of duplicate immune complexes is shown.

Mentions: The demonstration that GBP and Axin cannot bind GSK-3 simultaneously suggests that GBP might inhibit GSK-3 by removing it from the Axin complex or by preventing GSK-3 from associating with Axin. Since we previously demonstrated that GBP inhibits the in vivo phosphorylation of tau (Yost et al. 1998), a protein not thought to be involved in Wnt signaling, we wanted to examine whether GBP might also be able to inhibit GSK-3 by binding and inactivating the catalytic site. We used a modification of a published assay for GSK-3 activity that measures the ability of GSK-3 to phosphorylate the peptide substrate P-CREB, which contains a GSK-3 consensus phosphorylation site, in comparison to the negative control peptide, CREB (Wang et al. 1994). Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG (Yost et al. 1998) RNA and, after 3 h, proteins were extracted and immunoprecipitated. Anti-FLAG antibodies were used to isolate Xgsk-3 bound to GBP when both were injected; anti-myc antibodies were used to isolate Xgsk-3 when it was injected with a control RNA; and uninjected embryos were immunoprecipitated with both antibodies to measure background. The immunoprecipitates were incubated with γ-[32P]ATP in kinase buffer containing P-CREB or CREB, and the incorporated radioactivity was quantified. Western blotting showed that GBP and Xgsk-3 were both expressed and efficiently immunoprecipitated, and remained associated throughout the assay (data not shown). As shown in Fig. 3, GBP does not affect the ability of Xgsk-3 to phosphorylate this peptide substrate. This shows that GBP can inhibit GSK-3 in a way that does not inactivate its catalytic activity.


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)

GBP does not inhibit Xgsk-3 phosphorylation of a peptide substrate. Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG RNA. After 3 h, proteins were extracted and immunoprecipitated with anti-FLAG (Xgsk-3 + GBP), anti-myc (Xgsk-3 + control), or both (uninjected) antibodies. The kinase activity of immune complexes was measured by phosphorus-32 incorporation into the GSK-3–specific substrate prephosphorylated CREB peptide (p-CREB; dark bars). The nonphosphorylated CREB peptide (CREB; light bars) is not a GSK-3 substrate and was used as a control. The activity of duplicate immune complexes is shown.
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Related In: Results  -  Collection

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Figure 3: GBP does not inhibit Xgsk-3 phosphorylation of a peptide substrate. Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG RNA. After 3 h, proteins were extracted and immunoprecipitated with anti-FLAG (Xgsk-3 + GBP), anti-myc (Xgsk-3 + control), or both (uninjected) antibodies. The kinase activity of immune complexes was measured by phosphorus-32 incorporation into the GSK-3–specific substrate prephosphorylated CREB peptide (p-CREB; dark bars). The nonphosphorylated CREB peptide (CREB; light bars) is not a GSK-3 substrate and was used as a control. The activity of duplicate immune complexes is shown.
Mentions: The demonstration that GBP and Axin cannot bind GSK-3 simultaneously suggests that GBP might inhibit GSK-3 by removing it from the Axin complex or by preventing GSK-3 from associating with Axin. Since we previously demonstrated that GBP inhibits the in vivo phosphorylation of tau (Yost et al. 1998), a protein not thought to be involved in Wnt signaling, we wanted to examine whether GBP might also be able to inhibit GSK-3 by binding and inactivating the catalytic site. We used a modification of a published assay for GSK-3 activity that measures the ability of GSK-3 to phosphorylate the peptide substrate P-CREB, which contains a GSK-3 consensus phosphorylation site, in comparison to the negative control peptide, CREB (Wang et al. 1994). Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG (Yost et al. 1998) RNA and, after 3 h, proteins were extracted and immunoprecipitated. Anti-FLAG antibodies were used to isolate Xgsk-3 bound to GBP when both were injected; anti-myc antibodies were used to isolate Xgsk-3 when it was injected with a control RNA; and uninjected embryos were immunoprecipitated with both antibodies to measure background. The immunoprecipitates were incubated with γ-[32P]ATP in kinase buffer containing P-CREB or CREB, and the incorporated radioactivity was quantified. Western blotting showed that GBP and Xgsk-3 were both expressed and efficiently immunoprecipitated, and remained associated throughout the assay (data not shown). As shown in Fig. 3, GBP does not affect the ability of Xgsk-3 to phosphorylate this peptide substrate. This shows that GBP can inhibit GSK-3 in a way that does not inactivate its catalytic activity.

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