Volume 32, Issue 5, Pages 652-661 (December 2008) APC Is Essential for Targeting Phosphorylated β-Catenin to the SCFβ-TrCP Ubiquitin Ligase  YunYun Su, Chunjiang Fu, Shinji Ishikawa, Alessandra Stella, Masayuki Kojima, Kazuhisa Shitoh, Emanuel M. Schreiber, Billy W. Day, Bo Liu  Molecular Cell  Volume 32, Issue 5, Pages 652-661 (December 2008) DOI: 10.1016/j.molcel.2008.10.023 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 APC Restores β-Catenin Ubiquitination in CRC Tumor Cells (A) Cell extracts from DLD1, HT29, and SW480 were incubated with (+) or without (−) myc-tagged wild-type APC (myc-APC) in the presence (+) or absence (−) of proteasome inhibitor ALLN. Following incubation for 4 hr at room temperature, β-catenin was immunoprecipitated (IP) by a rabbit anti-β-catenin antibody. Levels of ubiquitin-conjugated and nonconjugated β-catenin in the IP products were determined by immunoblotting (IB) with a mouse anti-β-catenin (upper panel), a rabbit anti-phospho-S33/S37/T41 β-catenin-specific antibody (second panel), and a mouse anti-ubiquitin antibody (third panel), respectively. The bottom panel shows that equal amount of myc-APC was used in relevant reactions. P-β-catenin designates phosphorylated β-catenin. (B) Phosphorylation of β-catenin was reconstituted in vitro as described in the Experimental Procedures. Components included in the reaction were designated on the top of each lane. In all reactions, β-catenin was used as a GST-fusion protein. Following the incubation, levels of Flag-tagged phospho-β-catenin (Flag-P-β-catenin) were determined by IB with a rabbit anti-phospho-S33/S37/T41 β-catenin specific antibody (upper panel). The lower panel shows that equal amount of input Flag-β-catenin was used in each reaction. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Ubiquitin Conjugation of Phosphorylated β-Catenin Requires WT APC (A) DLD1 extracts with (+) or without (−) myc-APC was immunoprecipitated by a control mouse IgG (lane 1), a mouse anti-β-TrCP (lanes 2 and 3), or a rabbit anti-β-catenin antibody (lanes 4 and 5). Levels of β-catenin (upper panel), β-TrCP (second panel), and Skp1 (bottom panel) in the IP complex were determined by IB with respective antibodies. (B) Flag-β-catenin was phosphorylated in the presence (+) or absence (−) of myc-APC in vitro. After phosphorylation, free and myc-APC-associated phospho-β-catenin were separated from the rest kinase components and incubated in DLD1, SW480, and HT29 extracts. After incubation for 4 hr at room temperature in the presence (+) or absence (−) of ALLN, levels of ubiquitin-conjugated and nonconjugated β-catenin were determined by IB with a Flag antibody. IB results from DLD1, SW480, and HT29 are shown in the first, second, and third panels, respectively. The forth and fifth panels show amount of input myc-APC and Flag-P-β-catenin. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Phosphorylated β-Catenin Not Associated with WT APC Is Rapidly Dephosphorylated (A) myc-APC associated (lanes 1–3) or free phospho-β-catenin (lanes 4 and 5) was incubated with the TNT (in vitro transcription-coupled translation) reaction mix containing a mock translated (lane 1) or 35S-labeled β-TrCP protein (lanes 2 to 5). Samples in lanes 3 and 5 were preincubated in DLD1 extracts for 10 min prior to the incubation with 35S-labeled β-TrCP. Following the incubation, proteins associated with 35S-labeled β-TrCP were immunoprecipitated with a mouse anti-β-TrCP antibody. Levels of Flag-phospho-β-catenin in the IP products were determined by IB with a rabbit anti-phospho-S33/S37/T41 β-catenin-specific antibody (upper panel). Amount of myc-APC in the same IP products was determined by IB with a myc-antibody (second panel). The third panel is an autoradiography of 35S-labeled β-TrCP from the IP complex. The forth panel is an autoradiography of input 35S-labeled β-TrCP from TNT lysates. The bottom panel indicates that equal amount of Flag-phospho-β-catenin was used in each reaction. (B) Free (left) and WT APC-associated Flag-[32P]-labeled phospho-β-catenin (right) were incubated in DLD1, SW480, and HT29 extracts for 0, 10, 20, and 30 min (min) at room temperature as indicated. After the incubation, levels of radiation signal representing Flag-[32P]-β-catenin from DLD1, SW480, and HT29 were determined by autoradiography and are shown from top to the third panels as designated. The bottom panel shows that equal amount of input Flag-[32P]-β-catenin used in each reaction. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 PP2A Is Phospho-β-Catenin-Specific Phosphatase (A) Phospho-β-catenin-specific phosphatase was purified near homogeneity from crude extracts of bovine cardiac muscle. Two micrograms purified phosphatase was resolved by 12% SDS-PAGE. Coomassie blue staining of the gel revealed two major protein subunits. (B) Mass spectral (C18-LC-nanoESI-ion trap MSn) analysis of the trypsin-digested 63 kDa subunit. Peptides with high confidence matched to the data from the NCBI database are shown in their order of elution from the C18 column. Data in the columns are as follows. Scan number, MS scan used; sequence, predicted amino acid sequence of the tryptic peptide (an asterisk after a methionine (M∗) indicates mono-oxygenation of the thioether sulfur); theoretical [M+H]+, the predicted mass-to-charge ratio for the singly charged, protonated molecular ion; peptide charge state, the actual charge state of peptide ion; crosscorrelation, values from the search; delta correlation, describes how different the first hit is from the second hit in the search results; preliminary score, the higher the value the better; rank/preliminary score, the rank of the match during the preliminary scoring; and MS/MS ions, the number of the experimental MS/MS ions matched with the theoretical MS/MS ions for the listed peptide. (C) 100 ng of above-purified protein was resolved by 12% SDS-PAGE and probed by a PP2A Aα and Cα antibodies. (D) DLD1 cytosolic extracts were treated with 10 nM okadaic acid (OA) (top panel, lane 1) or affinity beads conjugated with a control mouse IgG (lane 2) and antibodies against the respective phosphatases (designated on the top). Levels of each phosphatase in the antibody-depleted extracts were determined by IB with their respective antibodies and are shown from the second to sixth panels. Lysates confirmed to have greater than 80% of reduction in phosphatase levels were used to incubate with Flag-[32P] phospho-β-catenin for 10 min at room temperature. After incubation, levels of Flag-[32P] phospho-β-catenin were determined by direct autoradiography (upper panel). The bottom panel indicates that equal amount of Flag-[32P] phospho-β-catenin was used in each reaction. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Deficiency in PP2A Causes an Excessive Degradation of Membrane-Associated β-Catenin (A) DLD1 cells were transiently transfected with either the control GFP or the PP2A Cα-specific siRNA. After 72 hr, cells were fractioned and prepared as whole cell (W), cytosolic (C), and membrane protein lysates (M). For detection of ubiquitin conjugates, ALLN was added into the culture medium 6 hr before the harvest. Levels of β-catenin, E-cadherin, and PP2A Cα in each fraction are displayed from top to bottom panels. IB results from control siRNA transfected cells are shown on the left; those transfected with PP2A Cα-specific siRNA are shown on the right. (B) IB analysis of 293T cell lysates transfected with the above siRNA. Cell fractions and IB antibodies were essentially the same as in (A). Levels of β-catenin, E-cadherin, and PP2A Cα are displayed from top to bottom panels. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 APC Protects the β-TrCP Binding Site of Phospho-β-Catenin from PP2A-Mediated Destruction (A) 200 nM of phosphorylated β-catenin was incubated with about equal molar of myc-P-APC, HA-Axin, and β-TrCP to form respective protein complex. After 30 min of incubation, each reaction was divided into two separated groups. The control group was supplemented with 25 nM of BSA; the experimental group was mixed with 25 nM of PP2A core enzyme. Aliquots of samples were taken at 0, 10, 20, and 30 min of incubation. Levels of Flag-P-β-catenin from each of these time points were examined by IB with a rabbit anti-phospho-S33/S37/T41 β-catenin-specific antibody. IB results from the control group are shown on the left; those of the PP2A-treated group are shown on the right. Listed samples from top to bottom panels are free flag-P-β-catenin and flag-P-β-catenin preincubated with myc-P-APC, β-TrCP, and HA-Axin. (B) Schematic of APC1, APC2, APC3, APC4, and the WT APC. Protein domains and repeats sequences are designated. (C) Flag-β-catenin was phosphorylated in the presence of WT, APC4, and three mutant APC proteins as designated on the top. Following the phosphorylation, the Flag-P-β-catenin/APC protein complex was separated from other kinase components and incubated with 25 nM PP2A core enzyme for 20 min. After the incubation, levels of phospho-β-catenin from each reaction were examined by IB with a rabbit anti-phospho-S33/S37/T41 β-catenin-specific antibody (upper panel). The second and third panels indicate amount of input Flag-P-β-catenin and APC proteins detected with a flag and a myc antibody. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Phosphorylation Is Required for the Protective Function of APC (A) Flag-P-β-catenin associated with nonphosphorylated and phosphorylated APC4 (designated as P-APC4) were incubated with purified PP2A core enzyme as described in Figure 6 for 20 min. Following the incubation, levels of Flag-P-β-catenin were determined by IB with a rabbit anti-phospho-S33/S37/41 β-catenin-specific antibody (upper panel). The second and third panels show equal amount of input Flag-P-β-catenin and myc-APC4 was used in the reaction. (B) Nonphosphorylated and phosphorylated-myc-APC4/Flag-P-β-catenin (designated as APC4/P-β-cat and P-APC4/P-β-cat, respectively) protein complexes were subjected to binding competition assays by incubation with increasing amounts of GST-15 aa and GST-βBD fusion proteins (shown at molar excesses of 1-, 10-, and 100-fold), respectively. After the incubation, the reaction mix was immunoprecipitated with an anti-myc antibody. Levels of Flag-P-β-catenin that remained associated with nonphosphorylated and phosphorylated myc-APC4 in the IP products were determined by IB with a Flag antibody (upper panel). The lower panel shows amount of myc-APC4 in the IP products detected by an myc antibody. Molecular Cell 2008 32, 652-661DOI: (10.1016/j.molcel.2008.10.023) Copyright © 2008 Elsevier Inc. Terms and Conditions