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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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Overexpression of plakoglobin causes the accumulation of endogenous β-catenin in the nucleus and the redistribution of  APC–GFP. Human plakoglobin was overexpressed in animal cap cells, and the distribution of both endogenous β-catenin (A and C)  and myc-tagged plakoglobin (B and C) was determined by confocal microscopy. Cells expressing ectopic plakoglobin possess high levels  of endogenous β-catenin in the nucleus (arrowheads in A and C) when compared to cells not expressing ectopic plakoglobin (arrows in  A and C). Overexpression of plakoglobin also results in the redistribution of APC–GFP within the cell to sites of plakoglobin accumulation. The localization of APC–GFP (D) and ectopic plakoglobin (E) in the absence of other RNAs demonstrates the different localization patterns of the two ectopic proteins. Coinjecting APC–GFP and plakoglobin, however, results in the redistribution of APC–GFP  (F) to a pattern indistinguishable from that seen for ectopic plakoglobin (G). Overlapping APC–GFP (F) and plakoglobin (G) staining  appears as yellow staining in the merged image (H). Arrows mark examples of APC–GFP and plakoglobin colocalization.
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Figure 7: Overexpression of plakoglobin causes the accumulation of endogenous β-catenin in the nucleus and the redistribution of APC–GFP. Human plakoglobin was overexpressed in animal cap cells, and the distribution of both endogenous β-catenin (A and C) and myc-tagged plakoglobin (B and C) was determined by confocal microscopy. Cells expressing ectopic plakoglobin possess high levels of endogenous β-catenin in the nucleus (arrowheads in A and C) when compared to cells not expressing ectopic plakoglobin (arrows in A and C). Overexpression of plakoglobin also results in the redistribution of APC–GFP within the cell to sites of plakoglobin accumulation. The localization of APC–GFP (D) and ectopic plakoglobin (E) in the absence of other RNAs demonstrates the different localization patterns of the two ectopic proteins. Coinjecting APC–GFP and plakoglobin, however, results in the redistribution of APC–GFP (F) to a pattern indistinguishable from that seen for ectopic plakoglobin (G). Overlapping APC–GFP (F) and plakoglobin (G) staining appears as yellow staining in the merged image (H). Arrows mark examples of APC–GFP and plakoglobin colocalization.

Mentions: Plakoglobin is closely related to β-catenin, and like β-catenin, ectopic plakoglobin accumulates in nuclei and can induce a secondary axis when overexpressed in Xenopus embryos (Karnovsky and Klymkowsky, 1995). Given that plakoglobin interacts with many of the same protein partners as β-catenin, we hypothesized that ectopic plakoglobin may compete with endogenous β-catenin for interactions with these protein partners resulting in the formation of a free, signaling pool of endogenous β-catenin in a manner similar to that seen after overexpression of the membrane-tethered β-catenin. To test this possibility, we investigated whether overexpression of plakoglobin (Fig. 1 A, h-plakoglobin) alters the distribution of endogenous β-catenin in animal cap cells. We injected RNA encoding myc-tagged plakoglobin into the animal pole of blastomeres at the four-cell stage and examined the distribution of both endogenous β-catenin (Fig. 7, A and C) and ectopic plakoglobin (Fig. 7, B and C) in animal cap cells. Ectopic myc-tagged plakoglobin accumulated in the cytoplasm of animal cap cells but was not seen in nuclei (Fig. 7, B and C). Visualization of endogenous β-catenin in the same cells (Fig. 7, A and C) revealed that elevated levels of endogenous β-catenin were present in nuclei of cells that possessed ectopic plakoglobin (Fig. 7, A and C, arrowheads), whereas no such elevation was apparent in cells that do not possess ectopic plakoglobin (Fig. 7, A and C, arrows). Therefore, overexpression of plakoglobin results in the accumulation of endogenous β-catenin in nuclei. These data are consistent with the idea that the observed signaling activity of plakoglobin may be attributable, at least in part, to the formation of a free, signaling pool of endogenous β-catenin that accumulates in the nucleus to affect target gene expression. Importantly, we observe the accumulation of endogenous β-catenin in the nucleus at levels of injected plakoglobin RNA (1–2 ng) that are less than was used by Karnovsky and Klymkowsky (1995) to establish the axis-inducing activity of plakoglobin.


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

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

Overexpression of plakoglobin causes the accumulation of endogenous β-catenin in the nucleus and the redistribution of  APC–GFP. Human plakoglobin was overexpressed in animal cap cells, and the distribution of both endogenous β-catenin (A and C)  and myc-tagged plakoglobin (B and C) was determined by confocal microscopy. Cells expressing ectopic plakoglobin possess high levels  of endogenous β-catenin in the nucleus (arrowheads in A and C) when compared to cells not expressing ectopic plakoglobin (arrows in  A and C). Overexpression of plakoglobin also results in the redistribution of APC–GFP within the cell to sites of plakoglobin accumulation. The localization of APC–GFP (D) and ectopic plakoglobin (E) in the absence of other RNAs demonstrates the different localization patterns of the two ectopic proteins. Coinjecting APC–GFP and plakoglobin, however, results in the redistribution of APC–GFP  (F) to a pattern indistinguishable from that seen for ectopic plakoglobin (G). Overlapping APC–GFP (F) and plakoglobin (G) staining  appears as yellow staining in the merged image (H). Arrows mark examples of APC–GFP and plakoglobin colocalization.
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Figure 7: Overexpression of plakoglobin causes the accumulation of endogenous β-catenin in the nucleus and the redistribution of APC–GFP. Human plakoglobin was overexpressed in animal cap cells, and the distribution of both endogenous β-catenin (A and C) and myc-tagged plakoglobin (B and C) was determined by confocal microscopy. Cells expressing ectopic plakoglobin possess high levels of endogenous β-catenin in the nucleus (arrowheads in A and C) when compared to cells not expressing ectopic plakoglobin (arrows in A and C). Overexpression of plakoglobin also results in the redistribution of APC–GFP within the cell to sites of plakoglobin accumulation. The localization of APC–GFP (D) and ectopic plakoglobin (E) in the absence of other RNAs demonstrates the different localization patterns of the two ectopic proteins. Coinjecting APC–GFP and plakoglobin, however, results in the redistribution of APC–GFP (F) to a pattern indistinguishable from that seen for ectopic plakoglobin (G). Overlapping APC–GFP (F) and plakoglobin (G) staining appears as yellow staining in the merged image (H). Arrows mark examples of APC–GFP and plakoglobin colocalization.
Mentions: Plakoglobin is closely related to β-catenin, and like β-catenin, ectopic plakoglobin accumulates in nuclei and can induce a secondary axis when overexpressed in Xenopus embryos (Karnovsky and Klymkowsky, 1995). Given that plakoglobin interacts with many of the same protein partners as β-catenin, we hypothesized that ectopic plakoglobin may compete with endogenous β-catenin for interactions with these protein partners resulting in the formation of a free, signaling pool of endogenous β-catenin in a manner similar to that seen after overexpression of the membrane-tethered β-catenin. To test this possibility, we investigated whether overexpression of plakoglobin (Fig. 1 A, h-plakoglobin) alters the distribution of endogenous β-catenin in animal cap cells. We injected RNA encoding myc-tagged plakoglobin into the animal pole of blastomeres at the four-cell stage and examined the distribution of both endogenous β-catenin (Fig. 7, A and C) and ectopic plakoglobin (Fig. 7, B and C) in animal cap cells. Ectopic myc-tagged plakoglobin accumulated in the cytoplasm of animal cap cells but was not seen in nuclei (Fig. 7, B and C). Visualization of endogenous β-catenin in the same cells (Fig. 7, A and C) revealed that elevated levels of endogenous β-catenin were present in nuclei of cells that possessed ectopic plakoglobin (Fig. 7, A and C, arrowheads), whereas no such elevation was apparent in cells that do not possess ectopic plakoglobin (Fig. 7, A and C, arrows). Therefore, overexpression of plakoglobin results in the accumulation of endogenous β-catenin in nuclei. These data are consistent with the idea that the observed signaling activity of plakoglobin may be attributable, at least in part, to the formation of a free, signaling pool of endogenous β-catenin that accumulates in the nucleus to affect target gene expression. Importantly, we observe the accumulation of endogenous β-catenin in the nucleus at levels of injected plakoglobin RNA (1–2 ng) that are less than was used by Karnovsky and Klymkowsky (1995) to establish the axis-inducing activity of plakoglobin.

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