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. GST was a negative control. Left of each pair: Coomassie stained gel, right: Phosphorylation detected using P32. Asterisks (*) indicate Coomassie-stained bands that align with P32-labeled proteins. In the presence of GST alone, GSK3 autophosphorylates (lane 2). HumanAPC1R2/B is strongly phosphorylated (lane 4) while fly APC2R2/B was more weakly phosphorylated (lane 6). Representative of two experiments. (B) R2 and B of Drosophila dAPC2, mouse mAPC1 and human hAPC1. Potential CK1 (orange) and GSK3 (green) phosphorylation sites. Red Arrow in R2 = serine mutated to alanine in mutant assessed in panel E. Red arrows in B = serines mutated to aspartic acid or alanine in APC2AA or APC2DD. Magenta arrowheads = additional serines mutated in 4 serine and six serine mutations (data not shown). (C) APC2DD (2 serines in B changed to aspartic acid; arrows in B) effectively reduces βcat levels in SW480 cells (arrow vs arrowhead). Inset = box in A. (D) APC2AA (2 serines in B changed to alanine, arrows in B) is unable to target βcat for destruction (arrow vs arrowhead). (E) APC2 R2S->A (single serine in R2 changed to alanine, arrow in B) is unable to target βcat for destruction (arrow vs arrowhead). (F) Quantification, total βcat fluorescent intensity. Student's t-test. (G) FRAP, Axin-RFP + GFP-APC2 constructs. APC2AA reaches a lower recovery plateau than either wild-type APC or APC2DD.