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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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Removal of ASAD disrupts APC self-association. (A) Schematic representation of Drosophila APC2 and APC1 constructs used in the study. (B) mCherry (mCh)-tagged full-length APC2 protein (red arrow) coimmunoprecipitates untagged full-length protein (black arrow). mCh-APC2 ASAD mutants (both deletion and point mutant) and mCh-APC2-C (red arrows) fail to coimmunoprecipitate untagged APC2-FL (black arrows in 2–4). (C) Yeast two-hybrid experiments demonstrated that APC2-N can interact directly with APC2-N. Deletion of the ASAD (APC2-N-ΔASAD) or disruption of the potential coiled coil (APC2-N-ASADPro) abolishes this interaction. (D) mCh-APC1-N coimmunoprecipitates untagged APC2-FL protein but fails to coimmunoprecipitate the APC2-ΔASAD mutant (black arrow). APC2-N175K contains a mutation in the Arm repeats and retains the mCh-APC1-N interaction. (E) mCh-KAP3 coimmunoprecipitates both full-length APC2 and the APC2-ΔASAD mutant (black arrows). CL, cell lysate; DL, depleted lysate; P, pull down.
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Figure 2: Removal of ASAD disrupts APC self-association. (A) Schematic representation of Drosophila APC2 and APC1 constructs used in the study. (B) mCherry (mCh)-tagged full-length APC2 protein (red arrow) coimmunoprecipitates untagged full-length protein (black arrow). mCh-APC2 ASAD mutants (both deletion and point mutant) and mCh-APC2-C (red arrows) fail to coimmunoprecipitate untagged APC2-FL (black arrows in 2–4). (C) Yeast two-hybrid experiments demonstrated that APC2-N can interact directly with APC2-N. Deletion of the ASAD (APC2-N-ΔASAD) or disruption of the potential coiled coil (APC2-N-ASADPro) abolishes this interaction. (D) mCh-APC1-N coimmunoprecipitates untagged APC2-FL protein but fails to coimmunoprecipitate the APC2-ΔASAD mutant (black arrow). APC2-N175K contains a mutation in the Arm repeats and retains the mCh-APC1-N interaction. (E) mCh-KAP3 coimmunoprecipitates both full-length APC2 and the APC2-ΔASAD mutant (black arrows). CL, cell lysate; DL, depleted lysate; P, pull down.

Mentions: To test this hypothesis, we generated a mutant version of Drosophila APC2 lacking this region (APC2-∆ASAD; Figure 2A). In addition, we disrupted potential coiled-coil formation by changing four key hydrophobic leucine residues to proline (APC2-ASADPro; Figures 1C and 2A). To determine whether this domain is necessary to mediate APC2 self-association, we performed immunoprecipitation assays from transiently transfected Drosophila S2 cells. Previously we showed that mCherry-tagged (mCh) APC2-FL (full length) and APC2-N (aa 1–490) could coprecipitate untagged APC2-FL, unlike mCh-APC2-C (aa 491–1067; Zhou et al., 2011; Figure 2B). Neither mCh-APC2-∆ASAD nor mCh-APC2-ASADPro was able to coprecipitate untagged APC2-FL, demonstrating that the N-terminal coil is necessary for self-association (Figure 2B). Because human OD-1 mediates dimer formation through a direct protein–protein interaction (Joslyn et al., 1993), we asked whether ASAD mediates direct APC2–APC2 binding. Consistent with that model, APC2-N (Figure 2A) self-associated in a yeast two-hybrid (Y2H) assay, and this interaction was disrupted in both ASAD mutants (APC2-N-ΔASAD and APC2-N-ASADPro; Figure 2C).


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)

Removal of ASAD disrupts APC self-association. (A) Schematic representation of Drosophila APC2 and APC1 constructs used in the study. (B) mCherry (mCh)-tagged full-length APC2 protein (red arrow) coimmunoprecipitates untagged full-length protein (black arrow). mCh-APC2 ASAD mutants (both deletion and point mutant) and mCh-APC2-C (red arrows) fail to coimmunoprecipitate untagged APC2-FL (black arrows in 2–4). (C) Yeast two-hybrid experiments demonstrated that APC2-N can interact directly with APC2-N. Deletion of the ASAD (APC2-N-ΔASAD) or disruption of the potential coiled coil (APC2-N-ASADPro) abolishes this interaction. (D) mCh-APC1-N coimmunoprecipitates untagged APC2-FL protein but fails to coimmunoprecipitate the APC2-ΔASAD mutant (black arrow). APC2-N175K contains a mutation in the Arm repeats and retains the mCh-APC1-N interaction. (E) mCh-KAP3 coimmunoprecipitates both full-length APC2 and the APC2-ΔASAD mutant (black arrows). CL, cell lysate; DL, depleted lysate; P, pull down.
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Figure 2: Removal of ASAD disrupts APC self-association. (A) Schematic representation of Drosophila APC2 and APC1 constructs used in the study. (B) mCherry (mCh)-tagged full-length APC2 protein (red arrow) coimmunoprecipitates untagged full-length protein (black arrow). mCh-APC2 ASAD mutants (both deletion and point mutant) and mCh-APC2-C (red arrows) fail to coimmunoprecipitate untagged APC2-FL (black arrows in 2–4). (C) Yeast two-hybrid experiments demonstrated that APC2-N can interact directly with APC2-N. Deletion of the ASAD (APC2-N-ΔASAD) or disruption of the potential coiled coil (APC2-N-ASADPro) abolishes this interaction. (D) mCh-APC1-N coimmunoprecipitates untagged APC2-FL protein but fails to coimmunoprecipitate the APC2-ΔASAD mutant (black arrow). APC2-N175K contains a mutation in the Arm repeats and retains the mCh-APC1-N interaction. (E) mCh-KAP3 coimmunoprecipitates both full-length APC2 and the APC2-ΔASAD mutant (black arrows). CL, cell lysate; DL, depleted lysate; P, pull down.
Mentions: To test this hypothesis, we generated a mutant version of Drosophila APC2 lacking this region (APC2-∆ASAD; Figure 2A). In addition, we disrupted potential coiled-coil formation by changing four key hydrophobic leucine residues to proline (APC2-ASADPro; Figures 1C and 2A). To determine whether this domain is necessary to mediate APC2 self-association, we performed immunoprecipitation assays from transiently transfected Drosophila S2 cells. Previously we showed that mCherry-tagged (mCh) APC2-FL (full length) and APC2-N (aa 1–490) could coprecipitate untagged APC2-FL, unlike mCh-APC2-C (aa 491–1067; Zhou et al., 2011; Figure 2B). Neither mCh-APC2-∆ASAD nor mCh-APC2-ASADPro was able to coprecipitate untagged APC2-FL, demonstrating that the N-terminal coil is necessary for self-association (Figure 2B). Because human OD-1 mediates dimer formation through a direct protein–protein interaction (Joslyn et al., 1993), we asked whether ASAD mediates direct APC2–APC2 binding. Consistent with that model, APC2-N (Figure 2A) self-associated in a yeast two-hybrid (Y2H) assay, and this interaction was disrupted in both ASAD mutants (APC2-N-ΔASAD and APC2-N-ASADPro; Figure 2C).

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