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Analysis of the signaling activities of localization mutants of beta-catenin during axis specification in Xenopus.

Miller JR, Moon RT - J. Cell Biol. (1997)

Bottom Line: Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate.Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin.These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

View Article: PubMed Central - PubMed

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

ABSTRACT
In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

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Model of the Wnt signaling pathway showing competition between endogenous and ectopic β-catenin for interactions  with several protein partners, including cadherin, APC, and Lef/ Tcf. Overexpression of various β-catenin mutants that possess  the ability to interact with APC (TM–β-catenin is shown) is predicted to increase the stability of endogenous β-catenin. The accumulation of endogenous β-catenin via activation of the Wnt  pathway or expression of ectopic β-catenin both result in the increase of “free” β-catenin capable of interacting with members of  the Lef/Tcf family of transcription factors. This β-catenin transcription factor complex translocates into the nucleus, where it  regulates the expression of target genes responsible for establishing dorsal cell fate. Furthermore, phosphorylation of β-catenin by  XGSK-3 may not only regulate β-catenin stability but may also  result in competition between phosphorylated and nonphosphorylated β-catenin isoforms for binding to the cytoplasmic domain  of cadherin.
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Figure 9: Model of the Wnt signaling pathway showing competition between endogenous and ectopic β-catenin for interactions with several protein partners, including cadherin, APC, and Lef/ Tcf. Overexpression of various β-catenin mutants that possess the ability to interact with APC (TM–β-catenin is shown) is predicted to increase the stability of endogenous β-catenin. The accumulation of endogenous β-catenin via activation of the Wnt pathway or expression of ectopic β-catenin both result in the increase of “free” β-catenin capable of interacting with members of the Lef/Tcf family of transcription factors. This β-catenin transcription factor complex translocates into the nucleus, where it regulates the expression of target genes responsible for establishing dorsal cell fate. Furthermore, phosphorylation of β-catenin by XGSK-3 may not only regulate β-catenin stability but may also result in competition between phosphorylated and nonphosphorylated β-catenin isoforms for binding to the cytoplasmic domain of cadherin.

Mentions: Our analyses of the function of TM–β-catenin raise a very important caveat of overexpression analyses in Xenopus embryos. We demonstrate that overexpression of TM– β-catenin causes the elevation of a free, signaling pool of endogenous β-catenin, and it is this endogenous pool that is active in inducing an ectopic dorsal axis. Given this result, we hypothesize that the signaling activity of various β-catenin mutants (Fagotto et al., 1996) or plakoglobin mutants (Merriam et al., 1997) may be indirect, functioning by elevating levels of endogenous β-catenin in the cell (Fig. 9). In fact, a discrepancy exists in the literature regarding the signaling activity of various β-catenin mutants (Funayama et al., 1995; Fagotto et al., 1996) and similar mutants in Armadillo (Orsulic and Peifer, 1996), the Drosophila homologue of β-catenin. For example, a mutant form of β-catenin lacking both the amino and carboxyl terminus induces a secondary dorsal axis when overexpressed in Xenopus embryos (Funayama et al., 1995), yet a similar mutant form of Armadillo is unable to transduce the Wingless signal in Drosophila embryos (Peifer and Wieschaus 1990; Orsulic and Peifer 1996). Moreover, a recent study has demonstrated that the carboxy-terminal domain of β-catenin and Armadillo constitutes a transactivation domain that is required for Lef/Tcf-dependent transcriptional activation (van de Wetering et al., 1997). Although β-catenin and Armadillo possess very similar functional domains, it remains possible that this disparity in the signaling activity of various β-catenin and Armadillo mutants may be an actual difference between the functional domains required for this activity. On the other hand, this difference could be resolved by arguing that ectopically expressed β-catenin acts indirectly by competing with endogenous β-catenin for interactions with APC, a potential component of the degradative machinery. This competition would result in the stabilization of a free, signaling pool of endogenous β-catenin. In fact, examination of the published data regarding the axis-inducing activity of various β-catenin mutants shows that the ability of a given ectopically expressed β-catenin mutant to induce dorsal cell fate corresponds with its ability to bind APC (Funayama et al., 1995; Fagotto et al., 1996). This conclusion calls into question the interpretation of many β-catenin overexpression studies performed in Xenopus embryos and underscores the importance of determining the effects of overexpression on the endogenous protein and protein partners with which the overexpressed protein may interact.


Analysis of the signaling activities of localization mutants of beta-catenin during axis specification in Xenopus.

Miller JR, Moon RT - J. Cell Biol. (1997)

Model of the Wnt signaling pathway showing competition between endogenous and ectopic β-catenin for interactions  with several protein partners, including cadherin, APC, and Lef/ Tcf. Overexpression of various β-catenin mutants that possess  the ability to interact with APC (TM–β-catenin is shown) is predicted to increase the stability of endogenous β-catenin. The accumulation of endogenous β-catenin via activation of the Wnt  pathway or expression of ectopic β-catenin both result in the increase of “free” β-catenin capable of interacting with members of  the Lef/Tcf family of transcription factors. This β-catenin transcription factor complex translocates into the nucleus, where it  regulates the expression of target genes responsible for establishing dorsal cell fate. Furthermore, phosphorylation of β-catenin by  XGSK-3 may not only regulate β-catenin stability but may also  result in competition between phosphorylated and nonphosphorylated β-catenin isoforms for binding to the cytoplasmic domain  of cadherin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139814&req=5

Figure 9: Model of the Wnt signaling pathway showing competition between endogenous and ectopic β-catenin for interactions with several protein partners, including cadherin, APC, and Lef/ Tcf. Overexpression of various β-catenin mutants that possess the ability to interact with APC (TM–β-catenin is shown) is predicted to increase the stability of endogenous β-catenin. The accumulation of endogenous β-catenin via activation of the Wnt pathway or expression of ectopic β-catenin both result in the increase of “free” β-catenin capable of interacting with members of the Lef/Tcf family of transcription factors. This β-catenin transcription factor complex translocates into the nucleus, where it regulates the expression of target genes responsible for establishing dorsal cell fate. Furthermore, phosphorylation of β-catenin by XGSK-3 may not only regulate β-catenin stability but may also result in competition between phosphorylated and nonphosphorylated β-catenin isoforms for binding to the cytoplasmic domain of cadherin.
Mentions: Our analyses of the function of TM–β-catenin raise a very important caveat of overexpression analyses in Xenopus embryos. We demonstrate that overexpression of TM– β-catenin causes the elevation of a free, signaling pool of endogenous β-catenin, and it is this endogenous pool that is active in inducing an ectopic dorsal axis. Given this result, we hypothesize that the signaling activity of various β-catenin mutants (Fagotto et al., 1996) or plakoglobin mutants (Merriam et al., 1997) may be indirect, functioning by elevating levels of endogenous β-catenin in the cell (Fig. 9). In fact, a discrepancy exists in the literature regarding the signaling activity of various β-catenin mutants (Funayama et al., 1995; Fagotto et al., 1996) and similar mutants in Armadillo (Orsulic and Peifer, 1996), the Drosophila homologue of β-catenin. For example, a mutant form of β-catenin lacking both the amino and carboxyl terminus induces a secondary dorsal axis when overexpressed in Xenopus embryos (Funayama et al., 1995), yet a similar mutant form of Armadillo is unable to transduce the Wingless signal in Drosophila embryos (Peifer and Wieschaus 1990; Orsulic and Peifer 1996). Moreover, a recent study has demonstrated that the carboxy-terminal domain of β-catenin and Armadillo constitutes a transactivation domain that is required for Lef/Tcf-dependent transcriptional activation (van de Wetering et al., 1997). Although β-catenin and Armadillo possess very similar functional domains, it remains possible that this disparity in the signaling activity of various β-catenin and Armadillo mutants may be an actual difference between the functional domains required for this activity. On the other hand, this difference could be resolved by arguing that ectopically expressed β-catenin acts indirectly by competing with endogenous β-catenin for interactions with APC, a potential component of the degradative machinery. This competition would result in the stabilization of a free, signaling pool of endogenous β-catenin. In fact, examination of the published data regarding the axis-inducing activity of various β-catenin mutants shows that the ability of a given ectopically expressed β-catenin mutant to induce dorsal cell fate corresponds with its ability to bind APC (Funayama et al., 1995; Fagotto et al., 1996). This conclusion calls into question the interpretation of many β-catenin overexpression studies performed in Xenopus embryos and underscores the importance of determining the effects of overexpression on the endogenous protein and protein partners with which the overexpressed protein may interact.

Bottom Line: Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate.Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin.These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

View Article: PubMed Central - PubMed

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

ABSTRACT
In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

Show MeSH
Related in: MedlinePlus