Figure 7. 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 Axin-RFP. Insets = boxes in C–E (C) Control (Ethanol treated (EtOH)). Deleting the SAMPs reduces APC2:Axin colocalization (arrows). (D) 2 μM BIO enhances APC2ΔSAMPs recruitment into Axin puncta. (E) Increasing BIO to 4 μm further boosts APC2ΔSAMPs recruitment into Axin puncta. (F) Quantification of (C–E). (G) CoIP of APC2∆SAMPs with Axin in SW480 cells ± LiCl or BIO. Full length APC2 is a control. Deleting the SAMPs drastically reduces coIP but GSK inhibition partially restores this. (H) Quantification of coIP in G, >2 replicates, normalized to Axin. (I) FRAP assay, SW480 cells transfected with Axin-RFP + GFP-APC2. GSK3 inhibition decreases APC2 dynamics. (J) GSK inhibition also slows APC2ΔSAMPs dynamics. (K) Deleting R2/B in APC2ΔSAMPs abolishes effect of GSK3 inhibition on dynamics. Student's t-test. (L) GSK3 inhibition with either BIO or LiCl does not further increase the size or decrease the number of APC plus Axin puncta, nor does treatment with the proteasome inhibitor MG132. Puncta area and puncta number, from confocal images.

DOI: http://dx.doi.org/10.7554/eLife.08022.021

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. (Inset = box in A) βcat levels in APC2:Axin complexes are very low. (B) GSK3 inhibition via BIO does not alter colocalization of APC2 with Axin, but leads to βcat accumulation in APC2:Axin puncta (inset = box in B). (C) Quantification of total βcat fluorescent intensity in (A and B). (D) In control cells for LiCl treatment, βcat levels in APC2:Axin complexes are very low (inset = box in D). (E) βcat accumulates in APC2:Axin complexes when GSK3 is inhibited via LiCl (inset = box in E). (F) Quantification of total βcat fluorescent intensity in (D and E). (G) GFP-APC2ΔSAMPs is diffuse and only associates weakly with Axin (inset = box in G) in control cells for LiCl treatment. (H) GSK3 inhibition with LiCl enhances association of APC2ΔSAMPs with Axin (inset = box in H). (I) Quantification of APC2ΔSAMPs colocalization with Axin (G vs H). Student's t-test was used. (J) FRAP assay in SW480 cells transfected with Axin-RFP and GFP-APC2ΔArm. GSK3 inhibition via BIO does not alter APC2's turnover rate when association is mediated by the SAMPs. Student's t-test was used.

DOI: http://dx.doi.org/10.7554/eLife.08022.022