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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 necessary to degrade β-cat and regulate Wnt target gene expression in SW480 cells. (A) Similar to S2 cells, Axin-GFP forms cytoplasmic puncta (1) and mCh-APC2 colocalizes with Axin-GFP in SW480 cells (2). Coexpression of APC2-ΔASAD with Axin-GFP does not disrupt colocalization (3) but is also associated with defects in puncta assembly and morphology. (B) Similar to S2 cells, coexpression of Axin with APC2-FL in SW480 cells leads to fewer, larger puncta. Two-tailed z test demonstrates significant differences between the two conditions. (C, D) Expression of full-length Drosophila APC2 was sufficient to suppress the elevated levels of β-cat (2) (compare to the empty vector control [1]) in SW480 cells. The APC2-ΔASAD mutant moderately suppressed the elevated β-cat levels (3). (E) In SW480 cells, expression of APC2-FL strongly suppressed activation of Wnt targets as assessed by TOP/Flash activity compared with the empty vector control. Expression of the APC2-ΔASAD mutant suppressed target gene activation compared with the empty vector control but exhibited significantly less activity than APC2-FL. Student's t test revealed significant differences between the conditions in D and E. (F) mCh-tagged APC2-FL (2) and APC2-ΔASAD (3) were expressed at equal levels in SW480 cells used in the TOP/Flash assays. Scale bar, 10 μm.
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Figure 4: APC2 self-association is necessary to degrade β-cat and regulate Wnt target gene expression in SW480 cells. (A) Similar to S2 cells, Axin-GFP forms cytoplasmic puncta (1) and mCh-APC2 colocalizes with Axin-GFP in SW480 cells (2). Coexpression of APC2-ΔASAD with Axin-GFP does not disrupt colocalization (3) but is also associated with defects in puncta assembly and morphology. (B) Similar to S2 cells, coexpression of Axin with APC2-FL in SW480 cells leads to fewer, larger puncta. Two-tailed z test demonstrates significant differences between the two conditions. (C, D) Expression of full-length Drosophila APC2 was sufficient to suppress the elevated levels of β-cat (2) (compare to the empty vector control [1]) in SW480 cells. The APC2-ΔASAD mutant moderately suppressed the elevated β-cat levels (3). (E) In SW480 cells, expression of APC2-FL strongly suppressed activation of Wnt targets as assessed by TOP/Flash activity compared with the empty vector control. Expression of the APC2-ΔASAD mutant suppressed target gene activation compared with the empty vector control but exhibited significantly less activity than APC2-FL. Student's t test revealed significant differences between the conditions in D and E. (F) mCh-tagged APC2-FL (2) and APC2-ΔASAD (3) were expressed at equal levels in SW480 cells used in the TOP/Flash assays. Scale bar, 10 μm.

Mentions: To rule out the possibility that this is a cell type–specific effect, we examined the role of APC2 in destructosome assembly in SW480 human colon cancer cells. Similar to Drosophila S2 cells, expression of Axin-GFP in SW480 cells led to the formation of discrete cytoplasmic puncta (Figure 4A1; Fiedler et al., 2011). In the presence of Drosophila APC2-FL, Axin-GFP puncta decreased in number and increased in size (Figure 4A, 1 and 2, and B), suggesting that the role of APC proteins in destructosome assembly is conserved in human cells. Consistent with this hypothesis, expression of APC2-∆ASAD resulted in fragmented, dispersed Axin-GFP puncta (Figure 4A3), suggesting that APC2 self-association is also required for destructosome assembly in human cells.


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 necessary to degrade β-cat and regulate Wnt target gene expression in SW480 cells. (A) Similar to S2 cells, Axin-GFP forms cytoplasmic puncta (1) and mCh-APC2 colocalizes with Axin-GFP in SW480 cells (2). Coexpression of APC2-ΔASAD with Axin-GFP does not disrupt colocalization (3) but is also associated with defects in puncta assembly and morphology. (B) Similar to S2 cells, coexpression of Axin with APC2-FL in SW480 cells leads to fewer, larger puncta. Two-tailed z test demonstrates significant differences between the two conditions. (C, D) Expression of full-length Drosophila APC2 was sufficient to suppress the elevated levels of β-cat (2) (compare to the empty vector control [1]) in SW480 cells. The APC2-ΔASAD mutant moderately suppressed the elevated β-cat levels (3). (E) In SW480 cells, expression of APC2-FL strongly suppressed activation of Wnt targets as assessed by TOP/Flash activity compared with the empty vector control. Expression of the APC2-ΔASAD mutant suppressed target gene activation compared with the empty vector control but exhibited significantly less activity than APC2-FL. Student's t test revealed significant differences between the conditions in D and E. (F) mCh-tagged APC2-FL (2) and APC2-ΔASAD (3) were expressed at equal levels in SW480 cells used in the TOP/Flash assays. Scale bar, 10 μm.
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Figure 4: APC2 self-association is necessary to degrade β-cat and regulate Wnt target gene expression in SW480 cells. (A) Similar to S2 cells, Axin-GFP forms cytoplasmic puncta (1) and mCh-APC2 colocalizes with Axin-GFP in SW480 cells (2). Coexpression of APC2-ΔASAD with Axin-GFP does not disrupt colocalization (3) but is also associated with defects in puncta assembly and morphology. (B) Similar to S2 cells, coexpression of Axin with APC2-FL in SW480 cells leads to fewer, larger puncta. Two-tailed z test demonstrates significant differences between the two conditions. (C, D) Expression of full-length Drosophila APC2 was sufficient to suppress the elevated levels of β-cat (2) (compare to the empty vector control [1]) in SW480 cells. The APC2-ΔASAD mutant moderately suppressed the elevated β-cat levels (3). (E) In SW480 cells, expression of APC2-FL strongly suppressed activation of Wnt targets as assessed by TOP/Flash activity compared with the empty vector control. Expression of the APC2-ΔASAD mutant suppressed target gene activation compared with the empty vector control but exhibited significantly less activity than APC2-FL. Student's t test revealed significant differences between the conditions in D and E. (F) mCh-tagged APC2-FL (2) and APC2-ΔASAD (3) were expressed at equal levels in SW480 cells used in the TOP/Flash assays. Scale bar, 10 μm.
Mentions: To rule out the possibility that this is a cell type–specific effect, we examined the role of APC2 in destructosome assembly in SW480 human colon cancer cells. Similar to Drosophila S2 cells, expression of Axin-GFP in SW480 cells led to the formation of discrete cytoplasmic puncta (Figure 4A1; Fiedler et al., 2011). In the presence of Drosophila APC2-FL, Axin-GFP puncta decreased in number and increased in size (Figure 4A, 1 and 2, and B), suggesting that the role of APC proteins in destructosome assembly is conserved in human cells. Consistent with this hypothesis, expression of APC2-∆ASAD resulted in fragmented, dispersed Axin-GFP puncta (Figure 4A3), suggesting that APC2 self-association is also required for destructosome assembly in human cells.

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