Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of ArmΔArm Leads to the Nuclear Localization of Endogenous ArmProtein(A) Wild-type Arm protein appears in stripes that correspond to cells responding to Wg signaling.(B) Expression of ArmΔArm in an armF1a background leads to the nuclear localization of endogenous Arm.(C and D) Dark-field images reveal that expression of both ArmΔArm and ArmS10 leads to similar naked cuticle phenotypes.(E) An anti-Arm Western blot showing a faster-migrating band, which correlates with endogenous Arm's being active, and a slower-migrating band, which correlates with Arm's being inactive.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC338072&req=5

pbio-0020095-g004: Expression of ArmΔArm Leads to the Nuclear Localization of Endogenous ArmProtein(A) Wild-type Arm protein appears in stripes that correspond to cells responding to Wg signaling.(B) Expression of ArmΔArm in an armF1a background leads to the nuclear localization of endogenous Arm.(C and D) Dark-field images reveal that expression of both ArmΔArm and ArmS10 leads to similar naked cuticle phenotypes.(E) An anti-Arm Western blot showing a faster-migrating band, which correlates with endogenous Arm's being active, and a slower-migrating band, which correlates with Arm's being inactive.

Mentions: When ArmΔArm is expressed in a wild-type embryo, it strongly activates Wg signaling (Figure 4C; Chan and Struhl 2002). Chan and Struhl (2002) suggest that this is because this membrane-tethered form of Arm can signal on its own. The results presented above argue, on the other hand, that it does so by stabilizing the endogenous protein. To further test this, we asked whether expression of ArmΔArm can induce Wg signaling when endogenous Arm is replaced by signaling-deficient Arm. We turned to two novel missense mutations where the rest of the arm coding region remains intact. Because these alleles do not produce truncations through stop codons, they are immune to nonsense mRNA degradation (Wagner and Lykke-Andersen 2002). Both mutations result in amino acid substitutions close to repeat seven, a key hinge region postulated to be important in binding of TCF (Huber et al. 1997; Graham et al. 2000). Both mutants retain the phosphorylation sites required for degradation and therefore accumulate in stripes in response to Wg signal (Figure 5I and 5J). They supply apparent wild-type junctional activity and accumulate to high levels in all cells when the kinase responsible for the degradation signal (Zw3) is removed (Figure 5K and 5L). The primary phenotype of these alleles is a loss or reduction of Wnt transcriptional responses (Figure 5A and 5B). The armF1a allele produced a partial loss-of-function phenotype, and germline clone embryos show some residual naked cuticle. armLM134 produces a stronger phenotype comparable to a loss of wg function, although it may not be a signaling (see below).


A nuclear function for armadillo/beta-catenin.

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

Expression of ArmΔArm Leads to the Nuclear Localization of Endogenous ArmProtein(A) Wild-type Arm protein appears in stripes that correspond to cells responding to Wg signaling.(B) Expression of ArmΔArm in an armF1a background leads to the nuclear localization of endogenous Arm.(C and D) Dark-field images reveal that expression of both ArmΔArm and ArmS10 leads to similar naked cuticle phenotypes.(E) An anti-Arm Western blot showing a faster-migrating band, which correlates with endogenous Arm's being active, and a slower-migrating band, which correlates with Arm's being inactive.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0020095-g004: Expression of ArmΔArm Leads to the Nuclear Localization of Endogenous ArmProtein(A) Wild-type Arm protein appears in stripes that correspond to cells responding to Wg signaling.(B) Expression of ArmΔArm in an armF1a background leads to the nuclear localization of endogenous Arm.(C and D) Dark-field images reveal that expression of both ArmΔArm and ArmS10 leads to similar naked cuticle phenotypes.(E) An anti-Arm Western blot showing a faster-migrating band, which correlates with endogenous Arm's being active, and a slower-migrating band, which correlates with Arm's being inactive.
Mentions: When ArmΔArm is expressed in a wild-type embryo, it strongly activates Wg signaling (Figure 4C; Chan and Struhl 2002). Chan and Struhl (2002) suggest that this is because this membrane-tethered form of Arm can signal on its own. The results presented above argue, on the other hand, that it does so by stabilizing the endogenous protein. To further test this, we asked whether expression of ArmΔArm can induce Wg signaling when endogenous Arm is replaced by signaling-deficient Arm. We turned to two novel missense mutations where the rest of the arm coding region remains intact. Because these alleles do not produce truncations through stop codons, they are immune to nonsense mRNA degradation (Wagner and Lykke-Andersen 2002). Both mutations result in amino acid substitutions close to repeat seven, a key hinge region postulated to be important in binding of TCF (Huber et al. 1997; Graham et al. 2000). Both mutants retain the phosphorylation sites required for degradation and therefore accumulate in stripes in response to Wg signal (Figure 5I and 5J). They supply apparent wild-type junctional activity and accumulate to high levels in all cells when the kinase responsible for the degradation signal (Zw3) is removed (Figure 5K and 5L). The primary phenotype of these alleles is a loss or reduction of Wnt transcriptional responses (Figure 5A and 5B). The armF1a allele produced a partial loss-of-function phenotype, and germline clone embryos show some residual naked cuticle. armLM134 produces a stronger phenotype comparable to a loss of wg function, although it may not be a signaling (see below).

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