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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 TM–β-catenin results in the stabilization of endogenous β-catenin. Injection of 1.25 ng of control  RNA (GFP), TM–β-catenin 1-myc RNA, or TM–β-catenin 1–9  RNA demonstrates overexpression of both forms of TM–β-catenin results in an increase in the steady-state levels of endogenous  β-catenin in both total (T) and soluble (S, non–cadherin-bound)  lysates. In this experiment, two-cell stage embryos were injected  with RNA at four sites, followed by protein extraction at stage 7.  To control for protein loading, all endogenous β-catenin bands  were normalized to α-spectrin signals from the same Western  blot. Numbers below each lane represent the relative level of endogenous β-catenin in each sample (control levels were assigned  a value of 1.0). Molecular mass markers indicated are 113 and 75 kD.
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Figure 3: Overexpression of TM–β-catenin results in the stabilization of endogenous β-catenin. Injection of 1.25 ng of control RNA (GFP), TM–β-catenin 1-myc RNA, or TM–β-catenin 1–9 RNA demonstrates overexpression of both forms of TM–β-catenin results in an increase in the steady-state levels of endogenous β-catenin in both total (T) and soluble (S, non–cadherin-bound) lysates. In this experiment, two-cell stage embryos were injected with RNA at four sites, followed by protein extraction at stage 7. To control for protein loading, all endogenous β-catenin bands were normalized to α-spectrin signals from the same Western blot. Numbers below each lane represent the relative level of endogenous β-catenin in each sample (control levels were assigned a value of 1.0). Molecular mass markers indicated are 113 and 75 kD.

Mentions: To test whether the overexpression of TM–β-catenin causes a stabilization of endogenous β-catenin in the cytosol and nucleus, we extracted protein from embryos injected with control GFP RNA or TM–β-catenin RNA and performed immunoblot analyses to determine the relative levels of endogenous β-catenin in both total (T) and soluble (S) protein fractions (Fig. 3). Soluble fractions represent lysates that have been incubated with ConA-Sepharose beads, which bind many membrane glycoproteins, including all cadherins (Fagotto et al., 1996). Thus, β-catenin present in the supernatant after incubation with ConA beads represents the soluble, non–cadherin-bound pool of β-catenin in the cell. This procedure allowed us to estimate the relative distribution of endogenous β-catenin in total homogenates (Fig. 3, T) and soluble, non–cadherin-bound pools (Fig. 3, S). Protein samples were analyzed by SDS-PAGE, and blots were probed with anti–β-catenin and anti–α-spectrin antibodies. We found that overexpression of either TM–β-catenin 1-myc or TM–β-catenin 1–9 results in an approximate twofold increase in total (T) and an approximate three- to fourfold increase in soluble (S) levels of endogenous β-catenin after normalizing β-catenin to levels of α-spectrin (numbers below each lane represent relative levels of β-catenin in TM–β-catenin–injected embryos compared to GFP-injected controls). The ability of both TM–β-catenin mutants to stabilize endogenous β-catenin was confirmed in a second, independent experiment (data not shown). Thus, overexpression of TM–β-catenin results in the elevation of both total and soluble pools of endogenous 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)

Overexpression of TM–β-catenin results in the stabilization of endogenous β-catenin. Injection of 1.25 ng of control  RNA (GFP), TM–β-catenin 1-myc RNA, or TM–β-catenin 1–9  RNA demonstrates overexpression of both forms of TM–β-catenin results in an increase in the steady-state levels of endogenous  β-catenin in both total (T) and soluble (S, non–cadherin-bound)  lysates. In this experiment, two-cell stage embryos were injected  with RNA at four sites, followed by protein extraction at stage 7.  To control for protein loading, all endogenous β-catenin bands  were normalized to α-spectrin signals from the same Western  blot. Numbers below each lane represent the relative level of endogenous β-catenin in each sample (control levels were assigned  a value of 1.0). Molecular mass markers indicated are 113 and 75 kD.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Overexpression of TM–β-catenin results in the stabilization of endogenous β-catenin. Injection of 1.25 ng of control RNA (GFP), TM–β-catenin 1-myc RNA, or TM–β-catenin 1–9 RNA demonstrates overexpression of both forms of TM–β-catenin results in an increase in the steady-state levels of endogenous β-catenin in both total (T) and soluble (S, non–cadherin-bound) lysates. In this experiment, two-cell stage embryos were injected with RNA at four sites, followed by protein extraction at stage 7. To control for protein loading, all endogenous β-catenin bands were normalized to α-spectrin signals from the same Western blot. Numbers below each lane represent the relative level of endogenous β-catenin in each sample (control levels were assigned a value of 1.0). Molecular mass markers indicated are 113 and 75 kD.
Mentions: To test whether the overexpression of TM–β-catenin causes a stabilization of endogenous β-catenin in the cytosol and nucleus, we extracted protein from embryos injected with control GFP RNA or TM–β-catenin RNA and performed immunoblot analyses to determine the relative levels of endogenous β-catenin in both total (T) and soluble (S) protein fractions (Fig. 3). Soluble fractions represent lysates that have been incubated with ConA-Sepharose beads, which bind many membrane glycoproteins, including all cadherins (Fagotto et al., 1996). Thus, β-catenin present in the supernatant after incubation with ConA beads represents the soluble, non–cadherin-bound pool of β-catenin in the cell. This procedure allowed us to estimate the relative distribution of endogenous β-catenin in total homogenates (Fig. 3, T) and soluble, non–cadherin-bound pools (Fig. 3, S). Protein samples were analyzed by SDS-PAGE, and blots were probed with anti–β-catenin and anti–α-spectrin antibodies. We found that overexpression of either TM–β-catenin 1-myc or TM–β-catenin 1–9 results in an approximate twofold increase in total (T) and an approximate three- to fourfold increase in soluble (S) levels of endogenous β-catenin after normalizing β-catenin to levels of α-spectrin (numbers below each lane represent relative levels of β-catenin in TM–β-catenin–injected embryos compared to GFP-injected controls). The ability of both TM–β-catenin mutants to stabilize endogenous β-catenin was confirmed in a second, independent experiment (data not shown). Thus, overexpression of TM–β-catenin results in the elevation of both total and soluble pools of endogenous 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