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A nuclear function for armadillo/beta-catenin.

Tolwinski NS, Wieschaus E - PLoS Biol. (2004)

Bottom Line: The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer.We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability.Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

ABSTRACT
The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer. Wnt binding to its receptor causes the stabilization and nuclear localization of beta-catenin. Nuclear beta-catenin then functions to activate transcription in conjunction with the transcription factor TCF. A recent report has challenged this basic precept of the Wnt signaling field, arguing that the nuclear localization of beta-catenin may be unrelated to its function and that beta-catenin functions at the plasma membrane to activate this signaling pathway. Here we present evidence that the pathway in fact does depend on the nuclear localization of beta-catenin. We reexamine the functionality of various truncations of beta-catenin and find that only the most severe truncations are true signaling- mutations. Further, we define a signaling- condition and use it to show that membrane-tethered beta-catenin is insufficient to activate transcription. We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability. These alleles allow us to show that the membrane-bound form of activated beta-catenin does indeed depend on the endogenous protein. Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby. Our data clearly show that nuclear localization of beta-catenin is in fact necessary for Wnt pathway activation.

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Related in: MedlinePlus

Relief of C-Terminal Repression through the Elimination of Cby Leads to Uniform Activation of Signaling(A) A wild-type cuticle shown for comparison.(B) Expression of ArmΔArm in the armF1a background.(C) Expression of a Cby RNAi construct along with ArmΔArm in the armF1a background.(D) Expression of a Cby RNAi construct in an armF1a background.
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pbio-0020095-g006: Relief of C-Terminal Repression through the Elimination of Cby Leads to Uniform Activation of Signaling(A) A wild-type cuticle shown for comparison.(B) Expression of ArmΔArm in the armF1a background.(C) Expression of a Cby RNAi construct along with ArmΔArm in the armF1a background.(D) Expression of a Cby RNAi construct in an armF1a background.

Mentions: Although the missense mutations we have used in our studies produce (on average) weaker phenotypes, they are more effective at blocking the cell-fate transformation induced by ArmΔArm than the “medium” C-terminal truncation mutants (compare Figure 1K with Figure 5C and 5D). The comparison is somewhat indirect, owing to the necessity of expressing ArmS18 in the “medium” arm allele background in order to get intact embryos. However, we find that expression of ArmS18 in an armF1a background has no visible effect on the cuticle (data not shown). Therefore, the activity of C-terminally truncated arm alleles in response to ΔArm expression suggests that, under certain conditions, removal of the C-terminus may actually enhance the transcriptional activity of Arm. One possibility is suggested by the recent discovery of Cby (Takemaru et al. 2003), a nuclear negative regulator of the Wg pathway that binds to the C-terminus of Arm. To test whether nuclear Cby affected the transformation produced by ArmΔArm, we used RNA interference (RNAi) to reduce Cby levels in armF1a embryos with and without ArmΔArm. In the absence of ArmΔArm, i.e., in embryos where most ArmF1a protein is cytoplasmic, Cby RNAi has no effect (Figure 6D). However, when ArmΔArm is present, lowering Cby levels leads to increased naked cuticle characteristic of Wnt pathway activation (compare Figure 6B to 6C). We propose that Cby's effect on armF1a protein is dependent on ArmΔArm relocalizing Arm to the nucleus.


A nuclear function for armadillo/beta-catenin.

Tolwinski NS, Wieschaus E - PLoS Biol. (2004)

Relief of C-Terminal Repression through the Elimination of Cby Leads to Uniform Activation of Signaling(A) A wild-type cuticle shown for comparison.(B) Expression of ArmΔArm in the armF1a background.(C) Expression of a Cby RNAi construct along with ArmΔArm in the armF1a background.(D) Expression of a Cby RNAi construct in an armF1a background.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC338072&req=5

pbio-0020095-g006: Relief of C-Terminal Repression through the Elimination of Cby Leads to Uniform Activation of Signaling(A) A wild-type cuticle shown for comparison.(B) Expression of ArmΔArm in the armF1a background.(C) Expression of a Cby RNAi construct along with ArmΔArm in the armF1a background.(D) Expression of a Cby RNAi construct in an armF1a background.
Mentions: Although the missense mutations we have used in our studies produce (on average) weaker phenotypes, they are more effective at blocking the cell-fate transformation induced by ArmΔArm than the “medium” C-terminal truncation mutants (compare Figure 1K with Figure 5C and 5D). The comparison is somewhat indirect, owing to the necessity of expressing ArmS18 in the “medium” arm allele background in order to get intact embryos. However, we find that expression of ArmS18 in an armF1a background has no visible effect on the cuticle (data not shown). Therefore, the activity of C-terminally truncated arm alleles in response to ΔArm expression suggests that, under certain conditions, removal of the C-terminus may actually enhance the transcriptional activity of Arm. One possibility is suggested by the recent discovery of Cby (Takemaru et al. 2003), a nuclear negative regulator of the Wg pathway that binds to the C-terminus of Arm. To test whether nuclear Cby affected the transformation produced by ArmΔArm, we used RNA interference (RNAi) to reduce Cby levels in armF1a embryos with and without ArmΔArm. In the absence of ArmΔArm, i.e., in embryos where most ArmF1a protein is cytoplasmic, Cby RNAi has no effect (Figure 6D). However, when ArmΔArm is present, lowering Cby levels leads to increased naked cuticle characteristic of Wnt pathway activation (compare Figure 6B to 6C). We propose that Cby's effect on armF1a protein is dependent on ArmΔArm relocalizing Arm to the nucleus.

Bottom Line: The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer.We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability.Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

ABSTRACT
The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer. Wnt binding to its receptor causes the stabilization and nuclear localization of beta-catenin. Nuclear beta-catenin then functions to activate transcription in conjunction with the transcription factor TCF. A recent report has challenged this basic precept of the Wnt signaling field, arguing that the nuclear localization of beta-catenin may be unrelated to its function and that beta-catenin functions at the plasma membrane to activate this signaling pathway. Here we present evidence that the pathway in fact does depend on the nuclear localization of beta-catenin. We reexamine the functionality of various truncations of beta-catenin and find that only the most severe truncations are true signaling- mutations. Further, we define a signaling- condition and use it to show that membrane-tethered beta-catenin is insufficient to activate transcription. We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability. These alleles allow us to show that the membrane-bound form of activated beta-catenin does indeed depend on the endogenous protein. Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby. Our data clearly show that nuclear localization of beta-catenin is in fact necessary for Wnt pathway activation.

Show MeSH
Related in: MedlinePlus