A novel GSK3-regulated APC:Axin interaction regulates Wnt signaling by driving a catalytic cycle of efficient βcatenin destruction
- University of North Carolina at Chapel Hill, United States
- National Heart, Lung, and Blood Institute, United States
Figures
Figure 1 with 2 supplements
APC2's Arm rpts provide a second means of interacting with the Axin complex.
(A) Fly APC2 and Axin. (B) Constructs used. hAPC1-1338 = the endogenous truncated hAPC1 in SW480 cells. (C and D) SW480 cells coexpressing GFP-APC2Arm rpts only and Axin-RFP, which localize adjacent …
Figure 1—figure supplement 1
Assessing levels of over-expression of Axin and APC.
(A) When overexpressed in SW480 cells, fly Axin forms puncta. (B) Plot of immunofluorescence intensities in SW480 cells transfected with GFP-APC2, or Axin-RFP, or GFP-APC2 + Axin–RFP, and stained …
Figure 1—figure supplement 2
Human APC's Arm repeat domain colocalizes with Axin.
(A) Diagrams of fly APC2 and APC2Arm rpts only. (B) SW480 cells expressing GFP-APC2Arm rpts only. APC2Arm rpts only forms cytoplasmic puncta and is unable to reduce βcat levels (insets = box in B). …
Figure 2
Axin and APC2 form structured macromolecular complexes in vivo.
SW480 cells. (A) Confocal image, GFP-APC2 and Axin-RFP. APC2 is recruited into Axin puncta. (B) Closeups, showing failure to resolve internal structure. (C–J) SIM super-resolution. (C) Axin-RFP …
Figure 3 with 1 supplement
APC2 stabilizes Axin complexes and promotes efficient βcat destruction.
(A) Stills, FRAP movie, SW480 cells transfected with GFP-APC2 (shown) and Axin-RFP. Inset = magnified APC2 signal in punctum. (B) APC2 recovers to ∼40% when in Axin puncta. Recovery curve (red); …
Figure 3—figure supplement 1
While proteasome inhibition reduces βcat destruction and causes βcat to detectably accumulate in APC2 + Axin puncta, it does not abolish the ability of APC2 to enhance Axin function in this regard.
(A and B) SW480 cells transfected with APC2 and Axin and treated with either ethanol as a control or with the proteasome inhibitor MG132. MG132 treatment elevates overall βcat levels and leads …
Figure 4
APC2’s Arm rpts and SAMPs each are required to stabilize APC2:Axin complexes.
(A) APC2 mutants. (B and C) FRAP analyses, SW480 cells. (B) APC2 needs both the Arm rpts and SAMPs to robustly associate with Axin puncta. Student's t-test. (C) Axin stabilization by APC2 is …
Figure 5 with 1 supplement
R2 and B regulate APC2 dynamics in the destruction complex, and regulate βcat removal from the destruction complex.
(A) APC2 mutants. (B–F) SW480 cells transfected with Axin-RFP and indicated GFP-APC2 constructs (B) FRAP assay. Deleting either R2 or B slows APC2's turnover in Axin puncta. (C–E) Axin-RFP, GFP-APC2 …
Figure 5—figure supplement 1
Colocalization of APC2's Arm repeat domain with Axin is controlled by R2 and B and APC2 without R2 or region B still stabilizes Axin complexes.
(A) Diagram of APC2 constructs. (B) GFP-APC2 and Axin-RFP colocalize with one another in SW480 cells and reduce βcat levels (inset = box in B). (C) Deleting APC2's SAMPs reduces colocalization with …
Figure 6 with 1 supplement
Association of Axin with APC2's Arm rpts is controlled by R2 and B.
(A) APC2 constructs. (B) Deleting either R2 or B in APC2ΔSAMPs slows APC2 recovery time and reduces recovery fraction. FRAP assay, SW480 cells transfected with GFP-tagged APC2 constructs and …
Figure 6—figure supplement 1
Binding of APC2's SAMP motif to Axin is not regulated by R2 and B.
(A) Diagram of APC2 constructs. (B) GFP-APC2ΔArm colocalizes with Axin-RFP in SW480 cells (inset = box in B). βcat is detectable in APC2ΔArm:Axin complexes. (C) Deleting R2 does not alter …
Figure 7 with 1 supplement
Axin:APC2 Arm rpts association is regulated by GSK3.
(A) R2 and B of Drosophila dAPC2, mouse mAPC1 and human hAPC1. Potential CK1(orange) and GSK3(green) phosphorylation sites. (B) APC2 mutants. (C–E) SW480 cells expressing GFP-APC2ΔSAMPs and …
Figure 7—figure supplement 1
GSK3 regulates association of APC2's Arm repeats with the Axin complex.
(A, B, D and E) GFP-APC2 and Axin-RFP expressed in SW480 cells. βcat stained via antibody. (A) APC2 and Axin strongly colocalize in cytoplasmic puncta in cells treated with EtOH as control for BIO. …
Figure 8
Mutating putative phosphorylation sites in B disrupts APC2 function.
(A) R2/B of human or fly APCs can be phosphorylated by human GSK3. In vitro kinase assay, GSK3 substrate peptide (positive control, left panels), GST-tagged humanAPC1R2/B or fly APC2R2/B fragments. …
Figure 9
Blocking potential phosphorylation at 2 conserved serines in B disrupts APC2 function in the fly.
APC2DD and APC2AA (Figure 8B) were expressed with the endogenous APC2 promoter in APC2 APC1 maternal/zygotic double mutants. (A) APC2 APC1 maternal/zygotic double mutants die as embryos (50% of …
Figure 10
Speculative Model of APC2's catalytic cycle inside the destruction complex.
(1) APC2 assembles with Axin via its Arm rpts and SAMPs. (2) APC2-bound βcat is phosphorylated by CK1 and GSK3. (3) GSK3 and CK1 phosphorylate R2/B region in APC2. (4) This induces a conformational …
Author response image 1
lnvestigating whether deleting R2 or B alters colP of þcat with Axin in SW480 cells. (A) Expression of Flag-Axin with indicated GFP-tagged APC2 constructs followed by lP of Flag-Axin and examination …
Videos
Video 1
3D reconstruction of SIM superresolution image of Axin-RFP expressed in SW480 cells (see also Figure 2D).
Volume view from Imaris 5.5 was used for reconstruction.
Video 2
3D reconstruction of SIM superresolution image of Axin-RFP (see also Figure 2E).
https://doi.org/10.7554/eLife.08022.009
Video 3
3D reconstruction of SIM superresolution image of Axin-RFP (see also Figure 2F).
https://doi.org/10.7554/eLife.08022.010
Video 4
3D reconstruction of SIM superresolution image of GFP-APC2 and Axin-RFP expressed in SW480 cells (see also Figure 2H).
Volume view from Imaris 5.5 was used for reconstruction.
Video 5
3D reconstruction of SIM superresolution image of GFP-APC2 and Axin-RFP (see also Figure 2I).
https://doi.org/10.7554/eLife.08022.012
Video 6
3D reconstruction of SIM superresolution image of GFP-APC2 and Axin-RFP (see also Figure 2J).
https://doi.org/10.7554/eLife.08022.013Tables
Table 1
Quantitation of relative expression levels of transfected versus endogenous APC and Axin
| Summary Axin overexpression | |||
|---|---|---|---|
| Experiment | I | II | III |
| Ratio GFP-FlyAxin to GFP-hAxin1 | 1.04 | 0.11 | 0.48 |
| Ratio GFP-hAxin1 to endo hAxin1 | 33.87 | 431.67 | 62.82 |
| Transfection efficiency | 30% | 42% | 36% |
| Fold overexpression level of GFP-FlyAxin to endo hAxin1 | 117.41 | 113.06 | 83.76 |
| Summary APC2 overexpresssion | |||
|---|---|---|---|
| Experiment | I | II | III |
| Ratio Flag-FlyAPC2 to Flag-hAPC1-1338 | 17.31 | 138.45 | 28.02 |
| Ratio Flag-hAPC1-1338 to endo hAPC1-1338 | 25.17 | 5.86 | 14.69 |
| Transfection efficiency | 30% | 33% | 25% |
| Fold overexpression level of Flag-FlyAPC2 to endo hAPC1-1338 | 1452.30 | 2458.54 | 1646.46 |
