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First published online 27 September 2005
doi: 10.1242/jcs.02589

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The adenomatous polyposis coli protein (APC) exists in two distinct soluble complexes with different functions

George A. Penman^*, Louie Leung and Inke S. Näthke

Cell and Developmental Biology, WTB, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

View larger version (35K): [in a new window]   Fig. 1. Fractionation of APC, ß-catenin and GSK3ß from different cell lines on glycerol gradients. Cell lysates from the indicated cells were fractionated on 10-40% glycerol gradients. Every other fraction was subjected to PAGE, transferred to nitrocellulose probed with antibodies against APC, ß-catenin and GSK3ß. The relative intensity of each protein was determined using a charge-coupled-device (CCD)-based enhanced chemiluminescence (ECL) detection system and was plotted for each fraction. Sedimentation of proteins with known S-values was carried out in parallel for each experiment to allow calibration for each gradient. In HeLa and MDCK cells, which express the intact ß-catenin-targeting complex, APC was present in two pools that sedimented at 10S and 23S. However, in cells that only express truncated APC or mutated ß-catenin, which cannot be phosphorylated, the relative amount of APC in the 23S complex was reduced or absent (SW480, DLD-1, HCT 116) or was shifted to a smaller size complex (HT-29).

View larger version (40K): [in a new window]   Fig. 2. Axin-1 co-sediments with the 23S APC-containing complex and is re-distributed to the 10S complex after cells were treated with GSK3ß inhibitor. Cell lysates were fractionated on 10-40% glycerol gradients as in Fig. 1. (A) Every other fraction was subjected to PAGE and probed with antibodies against APC, ß-catenin, GSK3ß, and Axin-1. (B) Intensities of the bands were measured using a CCD-based ECL detection system and then plotted as indicated. The majority of cellular Axin-1 co-sedimented with the 23S APC-containing complex. (C) Parallel samples of cells were treated with the GSK3ß inhibitor SB216763 (20 µM) for 2 hours and processed as described for A. Treatment of cells with SB216763 resulted in a relative increase in the proportion of APC and Axin-1 in smaller complexes. (D) Cell lysates from HeLa cells treated with SB for 12 hours were fractionated and probed for APC, Axin-1 and GSK3ß. Axin-1 distributes almost exclusive to the 10S complex after 12 hours of GSK3ß inhibition, similar to APC. (E) Cell lysates of cells treated with SB216763 for 2 hours were probed with antibodies against APC, ß-catenin, GSK3ß and tubulin. Only ß-catenin was increased whereas the levels of the other proteins remained constant. (F) Cell lysates from control or SB216763-treated cells were immunoprecipitated with a monoclonal antibody against APC. Immunoprecipitated material was probed with antibodies against APC and ß-catenin. Despite the elevated levels of ß-catenin in SB216763-treated cells, there was no concomitant increase in the amount of ß-catenin bound to APC.

View larger version (27K): [in a new window]   Fig. 3. Extended inhibition of GSK3ß causes a robust reduction of APC in the 23S complex. (A,B) HeLa cells were treated with the GSK3ß inhibitors, SB216763 (5 µM) (A) or LiCl (20 mM) (B) for 12 hours. Cell lysates were fractionated and the distribution of APC and ß-catenin determined as above. Inhibiting GSK3ß for 12 hours greatly reduced the amount of APC in the 23S complex and also resulted in a higher proportion of ß-catenin cofractionating with APC in the 10S complex. This effect was produced more effectively by SB216763, the more specific of the two inhibitors. These plots represent the distribution profiles of two independent experiments. (C) Immunoblots of equal amounts of total cell lysates were probed with antibodies against APC and ß-catenin. This confirmed that both GSK3ß inhibitors produced an increase in the cellular ß-catenin content, whereas the amount of APC remained constant. (D) APC was immunoprecipitated from cell lysates of SB216763 and LiCl treated HeLa cells as indicated. Probing the immunoprecipitated material with antibodies against ß-catenin revealed an increased amount of ß-catenin bound to APC when GSK3ß was inhibited. (E) Total cell lysates were also probed with an antibody against the phospho-epitope Y-216 of GSK3 or a GSK3ß-specific antibody (lower panel). Only SB216763 produced a decrease in the activating phosphorylation of GSK3 and GSK3ß at Y-216, consistent with the fact that LiCl and SB216763 inhibit GSK3 by different mechanisms.

View larger version (46K): [in a new window]   Fig. 4. Only APC in the 10S complex binds to microtubules, binding to ß-catenin and microtubules are inversely related functions of APC. (A) Fractions corresponding to the 10S and 23S complexes from control HeLa cells and HeLa cells treated with SB216763 for 12 hours as indicated were incubated with control buffers (no MTs) or taxol stabilised microtubules (+ MTs). Microtubules and associated proteins (Pel) were separated from unbound material (Sup) by sedimentation through a glycerol cushion and subjected to PAGE. The top part of each gel was transferred to nitrocellulose, followed by immunoblotting with antibodies against APC and ß-catenin. The bottom part of each gel was stained with Coomassie blue to show the distribution of tubulin. Upon addition of polymerised tubulin only APC in the 10S complex was detected bound to microtubules. ß-Catenin was not recovered bound to microtubules. (B) Total cell lysates from HeLa control cells or SB-treated HeLa cells, or DLD-1 and HCT 116 cells were incubated with control buffer (no MTs) or taxol stablised microtubules (+ MTs). Microtubules and associated proteins were recovered by centrifugation through a glycerol cushion and probed with antibodies against APC. APC co-sedimented specifically with microtubules in HeLa cells, only slightly in DLD-1 cells and extremely poorly in HCT 116 cells. (C) SW480 cells were co-transfected with GFP or GFP-tagged APC constructs and a TOP reporter plasmid carrying a TCF-specific promoter, or the FOP plasmid, a control plasmid carrying a mutated promoter, for 48 hours. The APC constructs encoded the following proteins: full length APC, APC lacking the direct microtubule binding site (residues 2168-2451) (APCdeltaMT), APC4, a fragment lacking the N-terminal 1000 amino acids, or APC4 lacking the direct microtubule binding site (APC4deltaMT), M-APC (residues 1014-2038) that have been described in detail (Zumbrunn et al., 2001), or GFP. Luciferase activity was measured in FOP- and TOP-transfected cells and the TOP:FOP ratio was calculated to control for transfection efficiency and background. Luciferase activity was significantly reduced by all APC proteins compared with GFP alone. However, APC that lacks the direct microtubule binding site was more efficient in producing this effect, regardless of whether the N-terminal domain was present or not. Similar results were observed after 24 hours of transfection. Data represent triplicate readings.