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Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.

Kunttas-Tatli E, Roberts DM, McCartney BM - Mol. Biol. Cell (2014)

Bottom Line: The destruction complex forms macromolecular particles we termed the destructosome.Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells.These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

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APC2 self-association is required to negatively regulate Wnt signaling in the Drosophila embryo. (A) Immunoblot of 0- to 6-h embryonic lysates demonstrates that the level of expression of GFP-APC2-FL and GFP-APC2-ΔASAD is comparable to that of endogenous APC2. (B) GFP-APC2-FL is enriched at the cell cortex with Arm in embryonic epithelia, whereas GFP-APC2-ΔASAD is primarily cytoplasmic. Scale bar, 10 μm. (C, D) Expression of GFP-APC2-FL rescued the lethality of APC2- (APC2g10) embryos and restored the wild- type cuticle phenotype, whereas the APC2-ΔASAD mutant only moderately rescued the lethality and cuticle phenotype. The numbers in D indicate the phenotypic average for each genotype (scoring criteria as in McCartney et al., 2006). Cuticle images are shown at the same scale. (E) Representative embryos showing Arm and En protein expression in wild-type (1) and APC2- (2) embryos. APC2-FL restored wild-type Arm levels and the En expression domain of APC2- (APC2g10) embryos. APC2-ΔASAD weakly suppressed Arm accumulation and restored a weak Arm stripe pattern in the epidermis. The En expression domain remains expanded in APC2- embryos expressing APC2-ΔASAD. Scale bar, 25 μm.
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Figure 6: APC2 self-association is required to negatively regulate Wnt signaling in the Drosophila embryo. (A) Immunoblot of 0- to 6-h embryonic lysates demonstrates that the level of expression of GFP-APC2-FL and GFP-APC2-ΔASAD is comparable to that of endogenous APC2. (B) GFP-APC2-FL is enriched at the cell cortex with Arm in embryonic epithelia, whereas GFP-APC2-ΔASAD is primarily cytoplasmic. Scale bar, 10 μm. (C, D) Expression of GFP-APC2-FL rescued the lethality of APC2- (APC2g10) embryos and restored the wild- type cuticle phenotype, whereas the APC2-ΔASAD mutant only moderately rescued the lethality and cuticle phenotype. The numbers in D indicate the phenotypic average for each genotype (scoring criteria as in McCartney et al., 2006). Cuticle images are shown at the same scale. (E) Representative embryos showing Arm and En protein expression in wild-type (1) and APC2- (2) embryos. APC2-FL restored wild-type Arm levels and the En expression domain of APC2- (APC2g10) embryos. APC2-ΔASAD weakly suppressed Arm accumulation and restored a weak Arm stripe pattern in the epidermis. The En expression domain remains expanded in APC2- embryos expressing APC2-ΔASAD. Scale bar, 25 μm.

Mentions: Because APC2 self-association is necessary for proper β-cat regulation in cultured cells, we asked whether APC2 self-association is also necessary for destructosome activity and the negative regulation of Wnt signaling in the more physiologically relevant context of the Drosophila embryo. We expressed GFP-tagged APC2-FL or APC2-∆ASAD in the embryo under the native APC2 promoter (McCartney et al., 2006) and found that the two tagged proteins are expressed at levels comparable to that of the endogenous wild-type protein (Figure 6A). As previously shown, APC2-FL protein expressed in APC2- (APC2g10) embryos is enriched at the cell cortex of embryonic epithelia similar to endogenous APC2 (McCartney et al., 1999; Zhou et al., 2011). However, APC2-∆ASAD exhibited limited enrichment at the cortex (Figure 6B), consistent with our observations in S2 cells (Supplemental Figure S4). We previously demonstrated that the localization of APC2 to the cell cortex requires both the N-terminal region (aa 1–490) and the most-C-terminal 30 amino acids (C30; Figure 1A; Zhou et al., 2011). Because we have now demonstrated that the function of C30 requires APC2 dimerization (McCartney and Molinar, unpublished data), it is not surprising that the ASAD is necessary for cortical localization. We previously showed that cortical localization of APC2 is not required to regulate Wnt signaling (Zhou et al., 2011); therefore, lack of APC2-∆ASAD cortical localization will not affect its destructosome activity.


Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.

Kunttas-Tatli E, Roberts DM, McCartney BM - Mol. Biol. Cell (2014)

APC2 self-association is required to negatively regulate Wnt signaling in the Drosophila embryo. (A) Immunoblot of 0- to 6-h embryonic lysates demonstrates that the level of expression of GFP-APC2-FL and GFP-APC2-ΔASAD is comparable to that of endogenous APC2. (B) GFP-APC2-FL is enriched at the cell cortex with Arm in embryonic epithelia, whereas GFP-APC2-ΔASAD is primarily cytoplasmic. Scale bar, 10 μm. (C, D) Expression of GFP-APC2-FL rescued the lethality of APC2- (APC2g10) embryos and restored the wild- type cuticle phenotype, whereas the APC2-ΔASAD mutant only moderately rescued the lethality and cuticle phenotype. The numbers in D indicate the phenotypic average for each genotype (scoring criteria as in McCartney et al., 2006). Cuticle images are shown at the same scale. (E) Representative embryos showing Arm and En protein expression in wild-type (1) and APC2- (2) embryos. APC2-FL restored wild-type Arm levels and the En expression domain of APC2- (APC2g10) embryos. APC2-ΔASAD weakly suppressed Arm accumulation and restored a weak Arm stripe pattern in the epidermis. The En expression domain remains expanded in APC2- embryos expressing APC2-ΔASAD. Scale bar, 25 μm.
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Figure 6: APC2 self-association is required to negatively regulate Wnt signaling in the Drosophila embryo. (A) Immunoblot of 0- to 6-h embryonic lysates demonstrates that the level of expression of GFP-APC2-FL and GFP-APC2-ΔASAD is comparable to that of endogenous APC2. (B) GFP-APC2-FL is enriched at the cell cortex with Arm in embryonic epithelia, whereas GFP-APC2-ΔASAD is primarily cytoplasmic. Scale bar, 10 μm. (C, D) Expression of GFP-APC2-FL rescued the lethality of APC2- (APC2g10) embryos and restored the wild- type cuticle phenotype, whereas the APC2-ΔASAD mutant only moderately rescued the lethality and cuticle phenotype. The numbers in D indicate the phenotypic average for each genotype (scoring criteria as in McCartney et al., 2006). Cuticle images are shown at the same scale. (E) Representative embryos showing Arm and En protein expression in wild-type (1) and APC2- (2) embryos. APC2-FL restored wild-type Arm levels and the En expression domain of APC2- (APC2g10) embryos. APC2-ΔASAD weakly suppressed Arm accumulation and restored a weak Arm stripe pattern in the epidermis. The En expression domain remains expanded in APC2- embryos expressing APC2-ΔASAD. Scale bar, 25 μm.
Mentions: Because APC2 self-association is necessary for proper β-cat regulation in cultured cells, we asked whether APC2 self-association is also necessary for destructosome activity and the negative regulation of Wnt signaling in the more physiologically relevant context of the Drosophila embryo. We expressed GFP-tagged APC2-FL or APC2-∆ASAD in the embryo under the native APC2 promoter (McCartney et al., 2006) and found that the two tagged proteins are expressed at levels comparable to that of the endogenous wild-type protein (Figure 6A). As previously shown, APC2-FL protein expressed in APC2- (APC2g10) embryos is enriched at the cell cortex of embryonic epithelia similar to endogenous APC2 (McCartney et al., 1999; Zhou et al., 2011). However, APC2-∆ASAD exhibited limited enrichment at the cortex (Figure 6B), consistent with our observations in S2 cells (Supplemental Figure S4). We previously demonstrated that the localization of APC2 to the cell cortex requires both the N-terminal region (aa 1–490) and the most-C-terminal 30 amino acids (C30; Figure 1A; Zhou et al., 2011). Because we have now demonstrated that the function of C30 requires APC2 dimerization (McCartney and Molinar, unpublished data), it is not surprising that the ASAD is necessary for cortical localization. We previously showed that cortical localization of APC2 is not required to regulate Wnt signaling (Zhou et al., 2011); therefore, lack of APC2-∆ASAD cortical localization will not affect its destructosome activity.

Bottom Line: The destruction complex forms macromolecular particles we termed the destructosome.Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells.These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

Show MeSH
Related in: MedlinePlus