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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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Schematic representation of constructs used in this  study. (A) Diagrams depicting the structure of wild-type and localization mutant β-catenin proteins. Some constructs were  tagged at the COOH terminus with either GFP (S65T mutant;  Heim et al., 1995) or a c-myc epitope (Evan et al., 1985). Shadowed boxes represent the 13 Arm repeats with a nonrepeat sequence between repeats 10 and 11. Sequences directing β-catenin  to specific intracellular compartments were added to the NH2 terminus of both wild-type and truncated forms of β-catenin. Wild-type human plakoglobin possesses an overall structure identical  to that of β-catenin and is tagged at the NH2 terminus with a c-myc  epitope (Merriam et al., 1997). (B) Linear representation of wild-type human APC protein showing conserved motifs, including  the oligomerization domain, Arm repeats, 15– and 20–amino acid  repeats, microtubule binding domain (MT binding), and discs  large binding domain (Dlg binding). The central portion of APC,  which is sufficient for β-catenin binding and downregulation  (Munemitsu et al., 1995), was tagged at the COOH terminus with  GFP (S65T mutant; Heim et al., 1995).
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Figure 1: Schematic representation of constructs used in this study. (A) Diagrams depicting the structure of wild-type and localization mutant β-catenin proteins. Some constructs were tagged at the COOH terminus with either GFP (S65T mutant; Heim et al., 1995) or a c-myc epitope (Evan et al., 1985). Shadowed boxes represent the 13 Arm repeats with a nonrepeat sequence between repeats 10 and 11. Sequences directing β-catenin to specific intracellular compartments were added to the NH2 terminus of both wild-type and truncated forms of β-catenin. Wild-type human plakoglobin possesses an overall structure identical to that of β-catenin and is tagged at the NH2 terminus with a c-myc epitope (Merriam et al., 1997). (B) Linear representation of wild-type human APC protein showing conserved motifs, including the oligomerization domain, Arm repeats, 15– and 20–amino acid repeats, microtubule binding domain (MT binding), and discs large binding domain (Dlg binding). The central portion of APC, which is sufficient for β-catenin binding and downregulation (Munemitsu et al., 1995), was tagged at the COOH terminus with GFP (S65T mutant; Heim et al., 1995).

Mentions: β-catenin is a multifunctional protein that is localized to several intracellular compartments, including the plasma membrane, cytosol, and nucleus (for review see Miller and Moon, 1996). The localization of β-catenin to each of these compartments is thought to reflect the interaction of β-catenin with various protein partners and the different functions of β-catenin in cell adhesion and signal transduction. Recent studies have suggested that the signaling function of β-catenin may be linked to accumulation of β-catenin–Lef/Tcf complexes in nuclei (Behrens et al., 1996; Molenaar et al., 1996). Although these studies suggest that the signaling function of β-catenin is carried out in the nucleus, we sought to directly test where in the cell β-catenin is required to have signaling activity. We therefore constructed a series of β-catenin localization mutants that contain additional sequences that target the resulting fusion proteins to a specific subcellular compartment (Fig. 1 A). Each fusion protein was tagged with GFP (S65T mutant; Heim et al., 1995) so that its intracellular localization could be easily determined by confocal microscopy. The wild-type (WT)–β-catenin–GFP construct was prepared as a control and was expected to mimic both the localization and function of endogenous β-catenin in various subcellular compartments. NES–β-catenin–GFP is a mutant that contains the nuclear exclusion sequence of rabbit pkI at the NH2 terminus (Wen et al., 1994, 1995). This mutant was expected to mimic the functions of cytosolic and membrane-bound β-catenin and would help test the requirement of nuclear localization for the signaling function of β-catenin. NLS–β-catenin–GFP is a mutant that contains the nuclear localization signal of the SV-40 large T-antigen (Kalderon et al., 1984; Lanford and Butel, 1984) fused to the NH2 terminus of β-catenin. This protein was expected to localize exclusively to nuclei and was produced to test the requirement for either cytosolic or membrane-localized β-catenin in signal transduction. Finally, TM–β-catenin is a mutant that contains the signal sequence and transmembrane domain of Xenopus N-cadherin fused to the NH2 terminus of β-catenin. This construct was produced to restrict β-catenin to membranes and, similar to the NES–β-catenin construct, would test whether nuclear localization is required for the signaling function of β-catenin.


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

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

Schematic representation of constructs used in this  study. (A) Diagrams depicting the structure of wild-type and localization mutant β-catenin proteins. Some constructs were  tagged at the COOH terminus with either GFP (S65T mutant;  Heim et al., 1995) or a c-myc epitope (Evan et al., 1985). Shadowed boxes represent the 13 Arm repeats with a nonrepeat sequence between repeats 10 and 11. Sequences directing β-catenin  to specific intracellular compartments were added to the NH2 terminus of both wild-type and truncated forms of β-catenin. Wild-type human plakoglobin possesses an overall structure identical  to that of β-catenin and is tagged at the NH2 terminus with a c-myc  epitope (Merriam et al., 1997). (B) Linear representation of wild-type human APC protein showing conserved motifs, including  the oligomerization domain, Arm repeats, 15– and 20–amino acid  repeats, microtubule binding domain (MT binding), and discs  large binding domain (Dlg binding). The central portion of APC,  which is sufficient for β-catenin binding and downregulation  (Munemitsu et al., 1995), was tagged at the COOH terminus with  GFP (S65T mutant; Heim et al., 1995).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic representation of constructs used in this study. (A) Diagrams depicting the structure of wild-type and localization mutant β-catenin proteins. Some constructs were tagged at the COOH terminus with either GFP (S65T mutant; Heim et al., 1995) or a c-myc epitope (Evan et al., 1985). Shadowed boxes represent the 13 Arm repeats with a nonrepeat sequence between repeats 10 and 11. Sequences directing β-catenin to specific intracellular compartments were added to the NH2 terminus of both wild-type and truncated forms of β-catenin. Wild-type human plakoglobin possesses an overall structure identical to that of β-catenin and is tagged at the NH2 terminus with a c-myc epitope (Merriam et al., 1997). (B) Linear representation of wild-type human APC protein showing conserved motifs, including the oligomerization domain, Arm repeats, 15– and 20–amino acid repeats, microtubule binding domain (MT binding), and discs large binding domain (Dlg binding). The central portion of APC, which is sufficient for β-catenin binding and downregulation (Munemitsu et al., 1995), was tagged at the COOH terminus with GFP (S65T mutant; Heim et al., 1995).
Mentions: β-catenin is a multifunctional protein that is localized to several intracellular compartments, including the plasma membrane, cytosol, and nucleus (for review see Miller and Moon, 1996). The localization of β-catenin to each of these compartments is thought to reflect the interaction of β-catenin with various protein partners and the different functions of β-catenin in cell adhesion and signal transduction. Recent studies have suggested that the signaling function of β-catenin may be linked to accumulation of β-catenin–Lef/Tcf complexes in nuclei (Behrens et al., 1996; Molenaar et al., 1996). Although these studies suggest that the signaling function of β-catenin is carried out in the nucleus, we sought to directly test where in the cell β-catenin is required to have signaling activity. We therefore constructed a series of β-catenin localization mutants that contain additional sequences that target the resulting fusion proteins to a specific subcellular compartment (Fig. 1 A). Each fusion protein was tagged with GFP (S65T mutant; Heim et al., 1995) so that its intracellular localization could be easily determined by confocal microscopy. The wild-type (WT)–β-catenin–GFP construct was prepared as a control and was expected to mimic both the localization and function of endogenous β-catenin in various subcellular compartments. NES–β-catenin–GFP is a mutant that contains the nuclear exclusion sequence of rabbit pkI at the NH2 terminus (Wen et al., 1994, 1995). This mutant was expected to mimic the functions of cytosolic and membrane-bound β-catenin and would help test the requirement of nuclear localization for the signaling function of β-catenin. NLS–β-catenin–GFP is a mutant that contains the nuclear localization signal of the SV-40 large T-antigen (Kalderon et al., 1984; Lanford and Butel, 1984) fused to the NH2 terminus of β-catenin. This protein was expected to localize exclusively to nuclei and was produced to test the requirement for either cytosolic or membrane-localized β-catenin in signal transduction. Finally, TM–β-catenin is a mutant that contains the signal sequence and transmembrane domain of Xenopus N-cadherin fused to the NH2 terminus of β-catenin. This construct was produced to restrict β-catenin to membranes and, similar to the NES–β-catenin construct, would test whether nuclear localization is required for the signaling function of β-catenin.

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