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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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Localization of wild-type and mutant β-catenin–GFP  proteins in animal cap cells. The  intracellular distribution of each  mutant was determined by confocal microscopy (A–D). The WT– β-catenin–GFP protein (A) is localized to the plasma membrane,  cytosol, and nucleus in a pattern  indistinguishable from that seen  for endogenous β-catenin protein  (Yost et al., 1996). NES–β-catenin–GFP (B) is present at plasma  membrane and at high levels in  the cytoplasm. Low levels of fluorescence are detected in the nucleus, which likely reflects the fact  that the NES domain does not inhibit nuclear entry but instead  promotes the rapid export of  tagged proteins from the nucleus.  NLS–β-catenin–GFP (C) is predominantly found in the nucleus,  and very little fluorescence is observed in the cytoplasm or in association with the plasma membrane.  The TM–β-catenin–GFP mutant  (D) localizes to intracellular vesicles and organelles in apparent association with the endoplasmic  reticulum and Golgi apparatus.  TM–β-catenin–GFP fluorescence  was never detected in the nucleus.
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Figure 2: Localization of wild-type and mutant β-catenin–GFP proteins in animal cap cells. The intracellular distribution of each mutant was determined by confocal microscopy (A–D). The WT– β-catenin–GFP protein (A) is localized to the plasma membrane, cytosol, and nucleus in a pattern indistinguishable from that seen for endogenous β-catenin protein (Yost et al., 1996). NES–β-catenin–GFP (B) is present at plasma membrane and at high levels in the cytoplasm. Low levels of fluorescence are detected in the nucleus, which likely reflects the fact that the NES domain does not inhibit nuclear entry but instead promotes the rapid export of tagged proteins from the nucleus. NLS–β-catenin–GFP (C) is predominantly found in the nucleus, and very little fluorescence is observed in the cytoplasm or in association with the plasma membrane. The TM–β-catenin–GFP mutant (D) localizes to intracellular vesicles and organelles in apparent association with the endoplasmic reticulum and Golgi apparatus. TM–β-catenin–GFP fluorescence was never detected in the nucleus.

Mentions: To determine the intracellular localization of each mutant construct, Xenopus animal cap cells expressing each GFP-tagged form of β-catenin were examined by confocal microscopy. WT–β-catenin–GFP accumulates at the plasma membrane, in the cytosol, and in the nucleus (Fig. 2 A). This pattern is identical to that seen for endogenous β-catenin in animal cap cells (Yost et al., 1996), demonstrating that the GFP tag does not influence the subcellular localization of the β-catenin fusion protein. NES–β-catenin–GFP is found predominantly in the cytoplasm and at the plasma membrane, although low levels of GFP fluorescence are detected in the nucleus (Fig. 2 B). The NLS–β-catenin–GFP fusion protein is localized almost exclusively to nuclei, although faint fluorescence is detected in association with the plasma membrane (Fig. 2 C). TM–β-catenin– GFP is localized to intracellular vesicles and organelles predictive of its association with the endoplasmic reticulum and Golgi apparatus (Fig. 2 D). TM–β-catenin–GFP was never detected in the nucleus.


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

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

Localization of wild-type and mutant β-catenin–GFP  proteins in animal cap cells. The  intracellular distribution of each  mutant was determined by confocal microscopy (A–D). The WT– β-catenin–GFP protein (A) is localized to the plasma membrane,  cytosol, and nucleus in a pattern  indistinguishable from that seen  for endogenous β-catenin protein  (Yost et al., 1996). NES–β-catenin–GFP (B) is present at plasma  membrane and at high levels in  the cytoplasm. Low levels of fluorescence are detected in the nucleus, which likely reflects the fact  that the NES domain does not inhibit nuclear entry but instead  promotes the rapid export of  tagged proteins from the nucleus.  NLS–β-catenin–GFP (C) is predominantly found in the nucleus,  and very little fluorescence is observed in the cytoplasm or in association with the plasma membrane.  The TM–β-catenin–GFP mutant  (D) localizes to intracellular vesicles and organelles in apparent association with the endoplasmic  reticulum and Golgi apparatus.  TM–β-catenin–GFP fluorescence  was never detected in the nucleus.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Localization of wild-type and mutant β-catenin–GFP proteins in animal cap cells. The intracellular distribution of each mutant was determined by confocal microscopy (A–D). The WT– β-catenin–GFP protein (A) is localized to the plasma membrane, cytosol, and nucleus in a pattern indistinguishable from that seen for endogenous β-catenin protein (Yost et al., 1996). NES–β-catenin–GFP (B) is present at plasma membrane and at high levels in the cytoplasm. Low levels of fluorescence are detected in the nucleus, which likely reflects the fact that the NES domain does not inhibit nuclear entry but instead promotes the rapid export of tagged proteins from the nucleus. NLS–β-catenin–GFP (C) is predominantly found in the nucleus, and very little fluorescence is observed in the cytoplasm or in association with the plasma membrane. The TM–β-catenin–GFP mutant (D) localizes to intracellular vesicles and organelles in apparent association with the endoplasmic reticulum and Golgi apparatus. TM–β-catenin–GFP fluorescence was never detected in the nucleus.
Mentions: To determine the intracellular localization of each mutant construct, Xenopus animal cap cells expressing each GFP-tagged form of β-catenin were examined by confocal microscopy. WT–β-catenin–GFP accumulates at the plasma membrane, in the cytosol, and in the nucleus (Fig. 2 A). This pattern is identical to that seen for endogenous β-catenin in animal cap cells (Yost et al., 1996), demonstrating that the GFP tag does not influence the subcellular localization of the β-catenin fusion protein. NES–β-catenin–GFP is found predominantly in the cytoplasm and at the plasma membrane, although low levels of GFP fluorescence are detected in the nucleus (Fig. 2 B). The NLS–β-catenin–GFP fusion protein is localized almost exclusively to nuclei, although faint fluorescence is detected in association with the plasma membrane (Fig. 2 C). TM–β-catenin– GFP is localized to intracellular vesicles and organelles predictive of its association with the endoplasmic reticulum and Golgi apparatus (Fig. 2 D). TM–β-catenin–GFP was never detected in the nucleus.

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