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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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Ectopic TM–β-catenin competes with endogenous  β-catenin for binding to endogenous APC and a cadherin fraction. (A) Protein extracted from embryos injected with 1.25 ng of  TM–β-catenin 1-myc RNA was subjected to APC immunoprecipitation (APC-IP) or ConA precipitation (ConA, represents cadherin-bound fraction) followed by immunoblotting with anti-myc  antibodies. Control lysates were incubated with beads alone  (beads). These experiments show that TM–β-catenin 1-myc binds  endogenous APC and is present in ConA-bound fractions, indicating an association with cadherin. (B) To determine the effect  of overexpression of TM–β-catenin 1-myc on the levels of endogenous β-catenin associated with APC, protein extracts from control embryos or embryos injected with 1.25 ng TM–β-catenin 1-myc  were subjected to immunoprecipitation with anti-APC antibodies  followed by immunoblotting with anti–β-catenin antibodies.  These analyses show that overexpression of TM–β-catenin 1-myc  causes a decrease relative to controls in the levels of endogenous  β-catenin associated with APC. (Relative levels of endogenous  β-catenin are shown below each lane with controls set to 1.0.) (C)  Changes in the levels of endogenous β-catenin associated with a  cadherin fraction were examined by preparing ConA precipitates  from protein extracts prepared from control or TM–β-catenin  1-myc RNA–injected embryos followed by immunoblotting with  anti–β-catenin antibodies. The levels of endogenous β-catenin  present in ConA fractions decreased to 0.6 of control levels, suggesting that ectopic TM–β-catenin competes with endogenous  β-catenin for binding to cadherin. (Relative levels of endogenous  β-catenin are shown below each lane.) Molecular mass markers  indicated in A are 198, 113, and 75 kD and those indicated in B  and C are 113 and 75 kD.
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Figure 5: Ectopic TM–β-catenin competes with endogenous β-catenin for binding to endogenous APC and a cadherin fraction. (A) Protein extracted from embryos injected with 1.25 ng of TM–β-catenin 1-myc RNA was subjected to APC immunoprecipitation (APC-IP) or ConA precipitation (ConA, represents cadherin-bound fraction) followed by immunoblotting with anti-myc antibodies. Control lysates were incubated with beads alone (beads). These experiments show that TM–β-catenin 1-myc binds endogenous APC and is present in ConA-bound fractions, indicating an association with cadherin. (B) To determine the effect of overexpression of TM–β-catenin 1-myc on the levels of endogenous β-catenin associated with APC, protein extracts from control embryos or embryos injected with 1.25 ng TM–β-catenin 1-myc were subjected to immunoprecipitation with anti-APC antibodies followed by immunoblotting with anti–β-catenin antibodies. These analyses show that overexpression of TM–β-catenin 1-myc causes a decrease relative to controls in the levels of endogenous β-catenin associated with APC. (Relative levels of endogenous β-catenin are shown below each lane with controls set to 1.0.) (C) Changes in the levels of endogenous β-catenin associated with a cadherin fraction were examined by preparing ConA precipitates from protein extracts prepared from control or TM–β-catenin 1-myc RNA–injected embryos followed by immunoblotting with anti–β-catenin antibodies. The levels of endogenous β-catenin present in ConA fractions decreased to 0.6 of control levels, suggesting that ectopic TM–β-catenin competes with endogenous β-catenin for binding to cadherin. (Relative levels of endogenous β-catenin are shown below each lane.) Molecular mass markers indicated in A are 198, 113, and 75 kD and those indicated in B and C are 113 and 75 kD.

Mentions: Both APC and cadherin are potential regulators of the signaling function of β-catenin (for review see Miller and Moon, 1996) and, therefore, are candidates to play a role in the stabilization of endogenous β-catenin after overexpression of TM–β-catenin. To test whether TM–β-catenin binds endogenous APC and cadherin, protein lysates extracted from embryos injected with TM–β-catenin 1-myc RNA were subjected to immunoprecipitation with anti-APC antibodies (Fig. 5 A, APC-IP) and ConA precipitation (Fig. 5 A, ConA, represents cadherin-bound β-catenin). The association of TM–β-catenin 1-myc with APC and the ConA fraction containing cadherins was then assayed by immunoblotting with anti-myc antibodies. We found that TM–β-catenin 1-myc coimmunoprecipitated with APC (Fig. 5 A, APC-IP) and was also present in the ConA precipitates that contain cadherins (Fig. 5 A, ConA). TM–β-catenin 1-myc is not detected in lysates incubated with beads alone, demonstrating the specificity of both precipitations (Fig. 5 A, beads). Thus, ectopic TM– β-catenin interacts with endogenous APC and is present in a fraction that contains cadherins. Given this result, we asked whether the binding of TM–β-catenin to APC and its association with a fraction that contains cadherins reduces the levels of endogenous β-catenin associated with APC and the cadherin fraction. Protein extracts from embryos injected with either a control RNA or TM–β-catenin 1-myc were subjected to immunoprecipitation with anti-APC antibodies (Fig. 5 B) or ConA precipitation (Fig. 5 C), and the levels of endogenous β-catenin were subsequently determined by immunoblotting with anti–β-catenin antibodies. We observed that overexpression of TM– β-catenin 1-myc results in a decrease in the steady-state levels of endogenous β-catenin associated with APC (Fig. 5 B) and a cadherin fraction (Fig. 5 C). The levels of endogenous β-catenin that coimmunoprecipitate with APC after overexpression of TM–β-catenin 1-myc were found to decrease approximately threefold relative to control levels in two experiments and decreased approximately 1.2-fold in a third experiment. Levels of endogenous β-catenin associated with a cadherin fraction after overexpression of TM–β-catenin 1-myc were found to decrease to ∼0.6 of control levels in each of three experiments. These data suggest that the binding of ectopic β-catenin to both APC and cadherin results in a decrease in the binding of endogenous β-catenin to each protein and the accumulation of a free, signaling pool of endogenous β-catenin in the cell.


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

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

Ectopic TM–β-catenin competes with endogenous  β-catenin for binding to endogenous APC and a cadherin fraction. (A) Protein extracted from embryos injected with 1.25 ng of  TM–β-catenin 1-myc RNA was subjected to APC immunoprecipitation (APC-IP) or ConA precipitation (ConA, represents cadherin-bound fraction) followed by immunoblotting with anti-myc  antibodies. Control lysates were incubated with beads alone  (beads). These experiments show that TM–β-catenin 1-myc binds  endogenous APC and is present in ConA-bound fractions, indicating an association with cadherin. (B) To determine the effect  of overexpression of TM–β-catenin 1-myc on the levels of endogenous β-catenin associated with APC, protein extracts from control embryos or embryos injected with 1.25 ng TM–β-catenin 1-myc  were subjected to immunoprecipitation with anti-APC antibodies  followed by immunoblotting with anti–β-catenin antibodies.  These analyses show that overexpression of TM–β-catenin 1-myc  causes a decrease relative to controls in the levels of endogenous  β-catenin associated with APC. (Relative levels of endogenous  β-catenin are shown below each lane with controls set to 1.0.) (C)  Changes in the levels of endogenous β-catenin associated with a  cadherin fraction were examined by preparing ConA precipitates  from protein extracts prepared from control or TM–β-catenin  1-myc RNA–injected embryos followed by immunoblotting with  anti–β-catenin antibodies. The levels of endogenous β-catenin  present in ConA fractions decreased to 0.6 of control levels, suggesting that ectopic TM–β-catenin competes with endogenous  β-catenin for binding to cadherin. (Relative levels of endogenous  β-catenin are shown below each lane.) Molecular mass markers  indicated in A are 198, 113, and 75 kD and those indicated in B  and C are 113 and 75 kD.
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Figure 5: Ectopic TM–β-catenin competes with endogenous β-catenin for binding to endogenous APC and a cadherin fraction. (A) Protein extracted from embryos injected with 1.25 ng of TM–β-catenin 1-myc RNA was subjected to APC immunoprecipitation (APC-IP) or ConA precipitation (ConA, represents cadherin-bound fraction) followed by immunoblotting with anti-myc antibodies. Control lysates were incubated with beads alone (beads). These experiments show that TM–β-catenin 1-myc binds endogenous APC and is present in ConA-bound fractions, indicating an association with cadherin. (B) To determine the effect of overexpression of TM–β-catenin 1-myc on the levels of endogenous β-catenin associated with APC, protein extracts from control embryos or embryos injected with 1.25 ng TM–β-catenin 1-myc were subjected to immunoprecipitation with anti-APC antibodies followed by immunoblotting with anti–β-catenin antibodies. These analyses show that overexpression of TM–β-catenin 1-myc causes a decrease relative to controls in the levels of endogenous β-catenin associated with APC. (Relative levels of endogenous β-catenin are shown below each lane with controls set to 1.0.) (C) Changes in the levels of endogenous β-catenin associated with a cadherin fraction were examined by preparing ConA precipitates from protein extracts prepared from control or TM–β-catenin 1-myc RNA–injected embryos followed by immunoblotting with anti–β-catenin antibodies. The levels of endogenous β-catenin present in ConA fractions decreased to 0.6 of control levels, suggesting that ectopic TM–β-catenin competes with endogenous β-catenin for binding to cadherin. (Relative levels of endogenous β-catenin are shown below each lane.) Molecular mass markers indicated in A are 198, 113, and 75 kD and those indicated in B and C are 113 and 75 kD.
Mentions: Both APC and cadherin are potential regulators of the signaling function of β-catenin (for review see Miller and Moon, 1996) and, therefore, are candidates to play a role in the stabilization of endogenous β-catenin after overexpression of TM–β-catenin. To test whether TM–β-catenin binds endogenous APC and cadherin, protein lysates extracted from embryos injected with TM–β-catenin 1-myc RNA were subjected to immunoprecipitation with anti-APC antibodies (Fig. 5 A, APC-IP) and ConA precipitation (Fig. 5 A, ConA, represents cadherin-bound β-catenin). The association of TM–β-catenin 1-myc with APC and the ConA fraction containing cadherins was then assayed by immunoblotting with anti-myc antibodies. We found that TM–β-catenin 1-myc coimmunoprecipitated with APC (Fig. 5 A, APC-IP) and was also present in the ConA precipitates that contain cadherins (Fig. 5 A, ConA). TM–β-catenin 1-myc is not detected in lysates incubated with beads alone, demonstrating the specificity of both precipitations (Fig. 5 A, beads). Thus, ectopic TM– β-catenin interacts with endogenous APC and is present in a fraction that contains cadherins. Given this result, we asked whether the binding of TM–β-catenin to APC and its association with a fraction that contains cadherins reduces the levels of endogenous β-catenin associated with APC and the cadherin fraction. Protein extracts from embryos injected with either a control RNA or TM–β-catenin 1-myc were subjected to immunoprecipitation with anti-APC antibodies (Fig. 5 B) or ConA precipitation (Fig. 5 C), and the levels of endogenous β-catenin were subsequently determined by immunoblotting with anti–β-catenin antibodies. We observed that overexpression of TM– β-catenin 1-myc results in a decrease in the steady-state levels of endogenous β-catenin associated with APC (Fig. 5 B) and a cadherin fraction (Fig. 5 C). The levels of endogenous β-catenin that coimmunoprecipitate with APC after overexpression of TM–β-catenin 1-myc were found to decrease approximately threefold relative to control levels in two experiments and decreased approximately 1.2-fold in a third experiment. Levels of endogenous β-catenin associated with a cadherin fraction after overexpression of TM–β-catenin 1-myc were found to decrease to ∼0.6 of control levels in each of three experiments. These data suggest that the binding of ectopic β-catenin to both APC and cadherin results in a decrease in the binding of endogenous β-catenin to each protein and the accumulation of a free, signaling pool of endogenous β-catenin in the cell.

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