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Biochimica et Biophysica Acta 1773 (2007) 120–130 www.elsevier.com/locate/bbamcr

Characterization of a 60S complex of the adenomatous polyposis coli tumor suppressor ⁎ Sreekala Mahadevaiyer, Chong Xu, Barry M. Gumbiner

Department of Cell Biology, University of Virginia Health Sciences Center, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA Received 1 February 2006; received in revised form 27 September 2006; accepted 13 October 2006 Available online 20 October 2006

Abstract

The tumor suppressor protein adenomatous polyposis coli (APC) is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and roles in cytoskeletal regulation and cell polarity. The soluble pool of APC protein in colon epithelial tumor cells exists in two distinct complexes fractionating at ∼20S and ∼60S in size. The 20S complex contains components of the β-catenin destruction complex and probably functions in the Wnt pathway. In this study, we characterized the molecular nature of the 60S APC- containing complex by examining known potential binding partners of APC. 60S APC did not contain EB1 or diaphanous, that have been reported to interact with APC and are implicated in plus end stabilization. Nor did the two other microtubule associated proteins, MAP4 or KAP3, which is thought to link APC to motor proteins, associate with the 60S complex. Minor fractions of α-, γ-tubulin and IQGAP1, a Rac1 and CDC42 effector that interacts with APC, specifically associated with APC in the 60S fraction. We propose that 60S APC is a discrete high molecular weight complex with a novel function in cytoskeletal regulation in epithelial cells apart from its well established role in targeting catenin destruction or its proposed role in microtubule plus end stabilization. © 2006 Elsevier B.V. All rights reserved.

Keywords: APC; fractionation; tubulin; microtubule; IQGAP1; EB1; diaphanous; epithelial cell

1. Introduction resides in its ability to bind axin and downregulate β-catenin and this function is lost in many colorectal cells carrying The function of adenomatous polyposis coli (APC) as a APC mutation [14]. Studies on , Xenopus and “gatekeeper” in colon is well established Caenorhabditis elegans have also revealed that APC has a role [1–4]. The APC gene product is a 312 kDa protein which has in the normal functioning of the Wnt/β-catenin signaling been proposed to play a role in a wide variety of cellular pathway [15–18]. processes including the , APC has also been shown to interact with and regulation, microtubule assembly, cell fate determination, cell regulate their dynamics in several different ways [19,20]. adhesion and migration, chromosomal stability and Endogenous full length APC localizes to some but not all [5,6]. The best characterized biological function of APC protein growing microtubule plus ends in subconfluent epithelial cells lies in its ability to downregulate β-catenin, a key player in cell [21,40], but when over expressed APC protein distributes adhesion and in signal transduction in Wnt signaling pathway along the microtubules and moves along a subset of [7–10]. APC binds to the axin [11,12], which microtubules towards the plus end [19,20,22]. Movement of binds GSK3β and in turn mediates phosphorylation of β- APC along the microtubules can be attributed to its ability to catenin [13] thereby targeting β-catenin for degradation by interact with kinesin superfamily of plus end directed motor proteosomes [14]. The tumor suppressor function of APC proteins KIF3a–KIF3b, through its association with KAP3 protein [23]. APC binds to microtubules either directly through its basic amino acid rich region near the C-terminus ⁎ Corresponding author. Tel.: +1 434 243 9290; fax: +1 434 982 3912. or indirectly through EB1, a microtubule end binding protein E-mail address: [email protected] (B.M. Gumbiner). [24]. The direct microtubule binding region of APC appears to

0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2006.10.006 S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130 121 be important for APC's involvement in microtubule stabiliza- tion [19,21,25] and microtubules that are associated with APC were found to be resistant to drug induced depolymerization both in vivo and in vitro. However the C-terminal APC fragment lacking the direct microtubule binding domain can promote microtubule assembly in presence of EB1 in vitro [24]. Another study has also shown that APC-EB1 complex at the microtubule plus ends interacts with formin protein diaphanous (mDia) and functions downstream of lysopho- sphatidic acid (LPA)-Rho-mDia signaling pathway [26]. This complex may contribute to microtubule stabilization either by directly capping microtubule plus ends or indirectly by contri- buting to other factors that may cap the plus ends in migrating cells [26]. It has also been suggested that association of APC with plus end of microtubule is essential for cell polarization and migration in astrocytes [27]. Another proposed role of APC resides in its ability to bind to IQGAP1, a protein involved in cytoskeletal organization. APC Fig. 1. Schematic representation of the protocol for fractionation of the 60S APC interacts with IQGAP1 and form a tripartite complex with complex. Nuclei and unlysed cells were removed from the cell homogenate by activated Rac1 and Cdc42 and this complex has been proposed low speed centrifugation and the post nuclear fraction (PNF) was subjected to to link the with microtubule dynamics during high speed centrifugation to separate particulate/membrane fraction (P100) from cytosol (S100). In some experiments the S100 fraction was centrifuged at very cell polarization and directional migration [28]. high speed in order to pellet the 60S pool of proteins (60S pellet). Both the S100 Although APC has been shown to play roles in many and the 60S pellet sample were further fractionated by velocity sedimentation for cellular processes and interact with a variety of proteins, very size analysis. The 60S pellet was also analyzed by cation exchange column and little attention has been paid to understand biochemical nature by immunoprecipitation experiments. of APC complexes present in the cell. Analyses of the interaction of components of destruction complex in Wnt PMSF, 0.017 mg/ml; leupeptin, 0.002 mg/ml; aprotinin, 0.004 mg/ml; signaling pathway [8,12,29,30] suggest that it functions in antipain, 0.01 mg/ml; benzamidine, 0.05 mg/ml; ST inhibitor, 0.01 mg/ml; cytosol. APC has also been shown to localize at the plasma iodoacetamide, 0.1 mg/ml. Cells were homogenized in a dounce homogenizer membrane, perhaps colocalizing with apical plasma mem- and the nuclei and the cell debri were removed by centrifugation at 5000 rpm brane [31], cortical actin or adherens junction [32]. Analysis for 30 min. 1 M KCl was added to post nuclear fractions (PNF) to bring up the salt concentration to isotonic level (140 mM). PNFs were subjected to of the subcellular distribution of APC and the destruction high speed centrifugation at 100,000×g for 1 h to remove particulate and complex in polarized human colon epithelial tumor cells membrane fractions. Supernatant containing the soluble proteins (S100) was carried out in our laboratory revealed that the cytosolic pool collected for further experiments. To pellet the 60S fraction (60S pellet) from of APC fractionated into two high molecular weight the soluble pool of proteins, the S100 fraction was spun at 55,000 rpm for complexes of ∼20S and ∼60S in size [31]. Most of the 48 min in a Beckman Coulter TLS55 rotor. The supernatant from the centrifugation was then used as the “20S” sample for following immunopre- cellular axin cofractionated with the 20S fraction and none of cipitation experiments. the components of destruction complex fractionated with the 60S fraction of APC raising the question of its function in 2.2. Velocity sedimentation analysis cells. In order to understand the function of the 60S APC complex, we decided to determine whether it contains any Aliquots of either the S100 or the 60S pellet containing 1.5–2 mg total known APC binding partners. We employed subcellular and protein was loaded on top of 4.5 ml of a continuous 10–40% sucrose gradient protein fractionation as well as immunoprecipitation analyses that was layered over 0.5 ml of a 66% sucrose cushion. The gradients were spun at 55,000 rpm in a Beckman Coulter SW55 rotor for 4.5 h. Fractions were to identify the proteins associating with APC in the 60S collected and the proteins were precipitated with 10% trichloro acetic acid complex. (TCA) for analysis by western blotting. Catalase (4–5S pool as monomer derived from 11.5S tetramer pool) and thyroglobulin (19S) were spun in parallel 2. Materials and methods gradients to determine the sedimentation coefficients below 20S. To estimate S values >19S, a software program for the calculation of velocity gradients was 2.1. Cell fractionation used based on a method developed by McEwen [33].

The cell line HCT116 was obtained from American 2.3. Equilibrium density gradient centrifugation Tissue Culture Center and maintained in McCoy's 5a media containing 10% FBS. Cells were grown in the media until they were completely confluent. All To analyze whether the 60S pool of APC has any potential membrane cell fractionations, as outlined in Fig. 1, were carried out at 4 °C. Confluent association, the 60S APC pellet was resuspended in a 55% sucrose solution (in monolayer cells were washed briefly with phosphate buffered saline (PBS) buffer A: 100 mM NaCl, 20 mM Tris–HCl, pH 7.8, 5 mM EDTA) and loaded and harvested by scraping in PBS and pelleting at 500 g for 5 min. Pelleted onto 0.5 ml of a 66% sucrose cushion. 5 ml of a continuous 20–50% sucrose cells were resuspended in hypotonic lysis buffer containing 30 mM HEPES, gradient was poured on top (in buffer A). The samples were spun in a Beckman pH 7.8 and 10 mM KCl and incubated for 30 min in ice. All the buffers Coulter SW 55Ti rotor at 45,000 rpm for 16 h at 4 °C. Fractions were analyzed contained the following protease inhibitors at the indicated concentrations: by western blotting for APC. 122 S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130

2.4. Immunoblotting and incubated at 4 °C for 2 h with either 5 μg anti-APC Ab (C20 rabbit polyclonal IgG; Santa Cruz Biotechnology Inc.) or APC Ab preincubated with For the detection of APC, samples were separated on 3% agarose gels under 100 M excess blocking peptide (Santa Cruz Biotechnology Inc) as control denaturing conditions and transferred by capillary transfer to nitrocellulose followed by 1 h incubation with protein A beads. Beads were pelleted and membrane overnight as described [34]. Western blotting was performed as washed five times with Tris buffer, 50 mM, pH 7.4 with 140 Mm NaCl; 0.1% described [15]. Proteins were separated on 10% SDS polyacrylamide gel for the NP-40; 0.1% Deoxycholate; 0.01% SDS or Tris buffer 20 Mm pH 7.5, 140 mM detection of , EB1 and actin and on 7.5% gel for the detection of E- KCl and 0.1% Triton X100. , diaphanous, MAP4, KAP3 and IQGAP1, and then transferred to nitrocellulose membrane by semidry transfer. The following primary antibodies 2.6. Cation exchange chromatography were used: APC, anti-APC COOH terminus (Ab-2, mouse IgG1; Research Products), α-tubulin, monoclonal anti-α-tubulin (DM1A), γ-tubulin, The 60S pellet was resuspended in isotonic buffer and was applied to a monoclonal anti-γ-tubulin (GTU88), moncoclonal anti actin (AC-40) and cation exchange Hitrap SP column (GE Healthcare-Amersham Biosciences). MAP4 (Sigma -Aldrich Inc), monoclonal anti-EB1, diaphanous, (p140mDia) Proteins were eluted with 30 mL linear gradient of NaCl in HEPES buffer (10– and KAP3A (BD Transduction Laboratories). IQGAP1 MAb was kindly 900 mM), pH 7.2 and analyzed by western blotting for distribution of APC, α provided by Dr. George Bloom (University of Virginia, Charlottesville, VA). and γ-tubulins and IQGAP1.

2.5. Immunoprecipitation 3. Results The S100 and 20S fractions were directly subject to immunoprecipitation. The P100 pellet from the cell fractionation was resuspended in 1% TX-100 An analysis of distinct cytoplasmic pools of APC from contained in 20 mM Tris HCl pH 7.8 and 140 mM KCl, and centrifuged for HCT116 human epithelial colon cancer cells, which express 10 min at 14,000 rpm to spin down the insoluble debris. The 60S pellet was resuspended in HEPES (30 mM) buffer, pH 7.8 containing 140 mM KCl. The wild type APC [35], was carried out as illustrated in Fig. 1. samples were precleared with rabbit IgG (Jackson Laboratories) and Protein A Velocity sedimentation analysis of the cytosolic S100 fraction (GE Healthcare-Amersham Biosciences), then divided into two equal samples showed that 30–40% of the APC sedimented around 60S. The

Fig. 2. 60S APC complex is not associated with membranes or β-catenin. (a) The P100, S100 and the 60S pellet were analyzed for APC and E-cadherin distribution in the left panel. The 60S pellet fraction was subjected to equilibrium density gradient centrifugation and the fractions analyzed for APC and E-cadherin, a membrane marker as shown on the right panel. (b) APC and β-catenin from the 60S pellet were immunoprecipitated with respective antibodies and the immunocomplexes were analyzed for both proteins. Control experiments were carried out in parallel where the 60S pellet sample was incubated with non-immune IgG. (c) Immunoprecipitation was also carried out on the S100 fraction that contained both 20S and 60S pools of APC. S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130 123

60S pelleted APC eluted as a broad single peak on a sephacryl 500 gel filtration column where bluedextran (∼2000 kDa) was eluted (data not shown), which is comparable to the calculated molecular weight of particles sedimenting around 60S. We could not detect any E-cadherin in either the S100 fraction or the 60S pellet, indicating that these fractions were free of membranes (Fig. 2a). Moreover the 60S APC complex failed to float on equilibrium density gradients (Fig. 2a). This ruled out any association of the 60S APC with membrane fragments. Analysis of soluble APC pools from two different epithelial cancer cell lines revealed that axin sedimented around 20S indicating that this pool represents components of the β-catenin destruction complex; with none in the 60S fraction of APC [31]. Nonetheless APC has multiple β-catenin binding sites, and a fraction of β-catenin could potentially be associated with the 60S APC fraction in these cells. However we could not detect any coimmunoprecipitation of β-catenin with APC from the 60S pellet (Fig. 2b) in contrast to the total cytosolic pool of APC or the 20S fraction where β-catenin did coimmunoprecipitate with APC (Fig. 2c and Fig. 9). The absence of β-catenin in the 60S fraction further confirms our earlier report that the 60S APC complex is not likely to represent the β-catenin destruction complex as part of the Wnt signaling pathway. We propose that Fig. 3. Microtubule associated proteins do not copurify with 60S APC. The S100 fraction was fractionated by velocity sedimentation. Nine fractions were the 60S APC complex is a distinct cytosolic protein complex collected and analyzed by western blotting for APC, diaphanous, EB1, MAP4 with molecular weight of 2000–3000 kDa and does not and KAP3. APC from the soluble pool fractionated into two distinct complexes associate with membranes or the β-catenin destruction of 20S and 60S. None of the microtubule binding proteins distributed in complex. fractions where the 60S APC complex was detected. Proteins with known sedimentation values were run in parallel to determine the sedimentation coefficients. 3.1. 60S APC is not associated with proteins involved in microtubule plus end stabilization or microtubule transport protein KAP3 containing complex that has been reported to move along the microtubule toward the plus end. We decided to ask whether the 60S APC fraction represents the complex of APC associated with the microtubule plus end 3.2. Potential interaction of α-andγ-tubulins with the 60S binding proteins with possible function in microtubule APC complex stabilization and cytoskeletal regulation [26]. The fractionation patterns of both EB1 and diaphanous, which have previously APC can interact with microtubules directly through its basic been shown to interact with APC, during velocity sedimentation amino acid rich domain present near its carboxy terminal [6], of the S100 fraction was very distinct from the APC distribution and we therefore asked whether it interacts with tubulin in the (Fig. 3). EB1 sedimented as a distinct pool around ∼5S while 60S complex. α-Tubulin could be detected in all fractions diaphanous fractionated around 5–10S. We could not detect across the velocity gradient with the majority of the tubulin either of the proteins in the fractions sedimenting around 60S. protein fractionating in the lighter fractions around 5S–20S, and Small amounts of both the proteins pelleted during differential small amounts trailing into the region around 60S pool of centrifugation to enrich the 60S pellet, but neither protein proteins (Fig. 4a). The sedimentation pattern of both APC and cofractionated when the 60S pellet was refractionated by α-tubulin in velocity sizing gradients of the S100 fraction did velocity sedimentation nor coimmunoprecipitated with APC not change even after the cells were preincubated with (data not shown). Therefore the 60S APC complex identified in microtubule depolymerizing agent nocodazole (33 μM) (data our study does not appear to be a part of the microtubule plus not shown), indicating that the tubulin containing complexes or end stabilization complex containing EB1 and diaphanous. We polymers fractionating around 60S were resistant to drug also tested whether 60S APC is a part of the transport complex induced depolymerization. We also asked whether 60S APC that could potentially move along the microtubules using the interacts with other microtubule associated proteins, like kinesin family of plus-end directed motor proteins through its MAP4, the most abundant non-tubulin component of micro- interaction via KAP3 [23]. The soluble pool of KAP3 tubule that has been shown to stabilize microtubules both in sedimented around 10S; with none detectable in the heavy vivo and in vitro [36]. However the majority of the MAP4 fraction where 60S APC was present (Fig. 3). KAP3 also failed protein did not copurify with 60S APC, and instead fractionated to immunoprecipitate with APC from 60S pellet (data not around 5–10S (Fig. 3). Even though trace amounts of this shown). Therefore we do not think that the 60S APC is the APC protein were detectable in fractions sedimenting around 20– 124 S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130

Fig. 4. Minor fractions of α-tubulin and γ-tubulin cofractionate with 60S APC. (a) The S100 fraction was fractionated by velocity sedimentation as described in Fig. 3 and the fractions were analyzed on western blots with antibodies against α- and γ-tubulin. (b) The 60S pellet was resuspended and refractionated by velocity sedimentation and the fractions analyzed as described above. 90% of APC in the 60S pellet refractionated at 60S. (c) The 60S pellet was immunoprecipitated with anti- APC Ab or APC Ab preincubated with 100 M excess blocking peptide. Immunoprecipitates were analyzed by western blot for APC, α-tubulin and γ-tubulin. A small fraction of both the tubulins from this pool specifically co-immunoprecipitated with APC, with majority of the tubulins left in the supernatant.

40S, MAP4 failed to coimmunoprecipitate with APC from the complex, proteins from the 60S pellet were fractionated by 60S pellet (Supplemental Fig. 1). cation exchange chromatography (Fig. 5). Most of the APC SinceAPChasalsobeenreportedtolocalizeto protein from 60S pellet eluted as a tight peak at salt centrosomes [37], we decided to analyze the distribution of concentration of 330–430 mM (fractions 9–11). α-Tubulin γ-tubulin, an integral component of the centrosomes, with a was distributed as a broad peak at salt concentrations ranging proposed role in microtubule minus end nucleation [38]. γ- from 250 mM to 550 mM (fractions 7–14) with peak fractions Tubulin was also distributed broadly across the gradient and overlapping with the APC peak fractions, while γ-tubulin enriched around 25–40S (Fig. 4a). This pool likely represents exhibited a very broad elution profile at salt concentrations from the soluble γ-tubulin ring complex known to sediment 250 mM to 750 mM (fractions 7–17) which overlapped with the around 40S [39]. The distribution profile of γ-tubulin also APC and α-tubulin profile. Therefore, although the α and γ- overlapped with 60S APC fraction across the gradient, tubulins in the 60S fraction did not entirely copurify with APC, although it was not enriched in the peak fractions of 60S it is possible that the peak fractions represent a complex with APC complex. APC. In order to determine whether the small amount of either α- To directly test whether either form of tubulin physically or γ- tubulin sedimenting in the 60S fractions are associated associate with APC in the 60S fraction, the 60S pellet was with the 60S APC complex, we refractionated the 60S pellet on subjected to coimmunoprecipitation analysis. Minor fractions the sucrose velocity gradients as described earlier (Fig. 4b). As of the 60S pelleted α- and γ- tubulins coimmunoprecipitated expected 90% of the APC sedimented around 60S, confirming with APC from this pool (Fig. 4c). Even though majority of that pelleted APC from the soluble pool in our high speed the tubulin proteins from the 60S pellet did not coimmuno- centrifugation step largely comprised of the 60S APC. The precipitate with APC, the small fractions associating with majority of the α and γ- tubulin enriched in the 60S pellet APCappearedtobespecificbecausetubulindidnot cosedimented with APC. To assess whether either form of coimmunoprecipitate in controls preincubated with 100 M tubulin present in the 60S pellet copurifies with APC as a single excess of specific antibody blocking peptide. Furthermore we S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130 125

3.3. IQGAP1 is a potential component of the 60S APC complex

IQGAP1, an effector of Rac1 and CDC42, has been shown to interact with APC [28]. Activated Rac1/CDC42 form a ternary complex with APC and IQGAP1 in migrating Vero cells. It has been hypothesized that this complex could potentially link actin cytoskeletal network and microtubule dynamics during cell polarization and directional migration. IQGAP1 from the S100 fraction distributed broadly across the sucrose gradient, with most of the protein sedimenting around 5–20S (Fig. 6a). However, we could detect small amounts of IQGAP1 in fractions where 60S APC complex sedimented. When the 60S pellet was refractionated by velocity sedimentation, a fraction of IQGAP1 enriched in the same fraction where most of the APC sedimented (Fig. 6b). IQGAP1 from the 60S pellet eluted from the ion exchange column at salt concentrations of 400– 500 mM. The peak was separated from the peak of APC (Fig. 5), which suggests that most of the IQGAP1 in 60S pellet is not directly associated with APC. However a small fraction of the 60S pelleted IQGAP1 was also coimmunoprecipitated with APC from this pellet compared to control preincubated with 100 M excess antibody specific blocking peptide (Fig. 6c). Actin from S100 fractions sedimented around 5–10S and was not detectable in the 60S fraction. Our data suggest that a fraction of IQGAP1 specifically associates with APC in the 60S complex, even though most of the IQGAP1 did not copurify with APC in our analysis.

3.4. Interaction of APC with putative binding partners in different cell fractions.

Because we did not observe any of the reported interactions Fig. 5. Cation exchange chromatographic analysis of 60S APC and its potential of EB1, KAP3, and mDia with APC in the 60S complex, we binding partners. The 60S pellet was resuspended in isotonic buffer and applied examined whether these interactions might occur in different to a cation exchange Hitrap SP column. Proteins were eluted with 30 mL linear subcellular fractions. Of particular interest is the P100 fraction salt gradient. Fractions were analyzed by western blotting for distribution of APC, tubulins and IQGAP1. containing membranes and membrane-cytoskeletal elements, since some of the microtubule-associated proteins are proposed to interact with APC at or near the cell cortex [23,26]. EB1, did not observe any association of other known binding KAP3, mDIA, and MAP4 were detectable in the P100 (P) partners of APC in our immunoprecipitation analysis, fraction (Figs. 7 and 8), but at extremely low levels compared to indicating that there were no general nonspecific interactions their amounts in the cytosolic fraction (S100, S). Note that detected by coimmunoprecipitation. Also because APC is not virtually all the E-cadherin fractionated in the P100 fraction, associated with most of the tubulin species separated by demonstrating complete pelleting of plasma membranes. Such copurification and coimmunoprecipitation, it is clear that APC low amounts of microtubule-associated proteins in the P100 does not nonselectively bind to all tubulin polymers in the fraction could be due either to minor contamination with cells. We could not determine the stoichiometry of APC and cytosolic proteins or to very small amounts present in the tubulin interaction since the detection method and the membrane-cortex. Nonetheless, the proportions of these sensitivity of the antibodies used widely differed. However proteins present in the P100 fraction relative to the S100 is considering that the endogenous APC levels are extremely very much less than the proportion of APC in the P100 (∼ half), low in the cells compared to α-tubulin, which is an abundant indicating that significant amounts are not stably associated molecule, and that the endogenous APC has been shown to with membrane-cortical associated APC. We did not detect any colocalize only with a subset of microtubules in the cells [6], EB1, KAP3, mDIA, or MAP4 in immunoprecipitates of APC we think that the small amount of tubulin associating with the solubilized from the P100 fraction (Fig. 7, IP), but this is not 60S APC is quite significant. This raises the possibility that surprising given how little of these proteins were present in the this APC complex has some novel function in cytoskeletal P100 to begin with. regulation in epithelial cells apart from its proposed role in Greater amounts of IQGAP1, α-tubulin, and γ-tubulin were microtubule plus end stabilization. present in the P100 fraction, presumably due either to their 126 S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130

Fig. 6. Association of a minor fraction of IQGAP1 with 60S APC. (a) The S100 fraction was fractionated by velocity sedimentation. Nine fractions were collected and analyzed by western blotting for APC, IQGAP1 and actin. (b) The 60S pellet was resuspended and refractionated by velocity sedimentation and fractions analyzed as described above. * Σ Non specific protein band (c) Immunoprecipitation of the 60S pellet protein with anti-APC Ab or APC Ab preincubated with 100 M excess blocking peptide. Immunoprecipitates were analyzed by western blotting for APC, actin and IQGAP1. membrane association or to their presence in large cyto- pitated with APC in the 20S fraction (Fig. 9), in contrast to skeletal elements, or both. We attempted to determine 60S APC (Fig. 2b). whether these proteins in the P100 co-immunoprecipitate with APC. As reported previously [31], it is difficult to 4. Discussion solubilize APC from the P100 membrane fraction (compare lanes I and S2 In Fig. 7). We did not detect IQGAP1, α- Confluent HCT116 cells have two distinct soluble pools of tubulin, or γ-tubulin in immunoprecipitates of APC solubi- APC of size 20S and 60S [31]. The 20S APC fraction is lized from the P100 fraction. Of course, this does not associated with axin, but we could not detect any of the exclude the possibility that any of the tubulin or micro- components of β-catenin destruction complex in the 60S APC tubule-associated proteins interact with APC in the insoluble fraction. However APC can potentially bind to β-catenin fraction, but they clearly do not remain associated after through its multiple binding sites. The absence of β-catenin in detergent solubilization. the APC coimmunoprecipitate from the 60S complex We also examined whether the tubulins and microtubule- indicates that it either has a very low affinity for the form associated proteins associate with APC in the total cytosolic of APC in this fraction, or else its binding sites are sheltered (S100) fraction, which includes both the 60S and 20S APC by other protein interactions. β-catenin in these cells is complexes (Fig. 8). As observed for the 60S complex, small capable of interacting with APC since it coimmunoprecipi- amounts of IQGAP1, α-tubulin, and γ-tubulin specifically co- tated with APC in the total cytosolic fraction S100 or the 20S immunoprecipitate with APC (IP) and their immunoprecipita- fraction (Figs. 8 and 9). APC in the 60S fraction was not tion is inhibited by a specific antibody blocking peptide associated with any membranes either. Therefore we propose (B.P.). In contrast to what is observed for the 60S complex, β- that the 60S fraction of APC identified in confluent HCT116 catenin specifically co-immunoprecipitates with APC in the cells is a discrete soluble high molecular weight protein S100 fraction, presumably due to their interactions in the 20S complex that is distinct from the complex regulating β- APC complex [31]. We did not detect any mDia, KAP3, catenin destruction. MAP4, or EB1 in APC immunoprecipitates from the S100 APC is also known to have a role in microtubule function fraction (Fig. 8; IP). [6]. Because α-tubulin specifically coimmunoprecipitates with Since we observed an interaction of APC with IQGAP1, APC in the 60S fraction, the 60S APC complex may have a role α-tubulin, and γ-tubulin in both the 60S fraction and the total in microtubule function or dynamics. We do not believe that the cytosolic fraction S100, we decided to ask whether these small fraction of the α-tubulin in our immunoprecipitation interactions exist in the 20S APC fraction. Small amounts of analysis was due to some non specific interaction occurring IQGAP1, α-tubulin, and γ-tubulin specifically coimmuno- after cell lysis, because we did not see nonspecific association of precipitated with APC in the 20S fraction (Fig. 9). As APC with other known binding partners like EB1, KAP3, expected, significant amounts of β-catenin coimmunopreci- β-catenin etc. The fact that the APC fractionates differently S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130 127

The association of α-tubulin with the 60S APC complex raised the possibility that it represents a microtubule plus end associating complex. APC has been proposed to play a role in microtubule plus end stabilization in migrating cells [26] through its association with EB1 and mDia. It has also been reported that APC can utilize kinesin family of motor proteins KIF3A–KIF3B via its interaction with KAP3 to move along the microtubule toward the growing plus end [23]. However none of these microtubule plus end binding partners of APC, EB1, diaphanous (mDia) or the kinesin associated protein KAP3 could be found associated with the 60S APC complex. The absence of these interactions suggests that the 60S APC is unlikely to be the APC complex involved in microtubule plus end stabilization [26], and the absence of KAP3 suggests that 60S APC is not simply derived from the complex being transported along the microtubules toward the plus end tips. The reported interactions of APC with the microtubule binding proteins such as EB1, KAP3 and mDia were not observed in either the total cytosolic fraction S100 or the solubilized membrane/dense fraction P100 in highly confluent HCT116 cells. These findings for EB1 are consistent with previous publications, in which co-immunoprecipitation with APC has not been observed despite other evidence for interactions [24,44]. Similarly, the published co-immunopreci- pitation of mDia was barely detectable, despite the other evidence for interactions [26]. KAP3 has been reported to co- immunoprecipitate with APC in MDCK cells [23]. The reason we did not observe the KAP3 co-immunoprecipitation with APC in HCT116 cells is not clear. However, a possible explanation is that the interactions of some of these proteins with APC may be very transient and only occur during cell polarization in migrating cells [23,26,44], whereas our studies were all performed with confluent epithelial monolayers, a state of needed for the formation of the 60S complex in epithelial cells [31]. It is important to note that we also found γ-tubulin Fig. 7. Analysis of APC interaction with potential binding partners in the associating with APC in the 60S fraction. APC has been membrane fraction (P100). Confluent HCT116 cells were lysed and fractionated γ as described above. The distributions of APC and its potential interaction previously shown to colocalize with -tubulin near the partners in the cytosolic fraction (S100) and the membrane fraction (P100) were nucleus in MDCK cells, where APC preferentially localized analyzed by western blot. The membrane pellet (P100) was resuspended in 1% to one of the two centrosomes [37]. Even though the 60S TX100 and cleared by centrifugation and both the solubilized sample and fraction of proteins isolated in our experiments after differen- insoluble pellet were collected and analyzed. (TL: Total Lysate, PNF: Post tial centrifugation could not contain intact centrosomes, large Nuclear Fraction, S1: S100, P: P100, I: Insoluble P100, S2: Solubilized P100). γ The solubilized P100 sample was then subject to immunoprecipitation as amounts of -tubulin exist in the soluble pool in mammalian described above and the immunoprecipitates were analyzed by western blotting cells as two discrete complexes [39], a large γ-tubulin ring (IgG: Rabbit IgG Control, IP: APC antibody (C-20) immunoprecipitate, B.P.: complex (γ-TuRC, 25–32S) and a small γ-tubulin small immunoprecipitation with blocking peptide, sn: supernatant after immunopre- complex (γ-TuSC, 6–9.8S) [41]. γ-TuRC can interact with a cipitation). Note that the IP and B.P. samples shown are 5X amount comparing subset of well-formed microtubule minus ends or with tubulin to the supernatant (sn). Arrowhead: The black dot between two lanes is not a protein band and resulted from a blemish on the nitrocellulose. subunits. The ring complex has an approximate molecular weight 2000 kDa and contains eight proteins in addition tο 10–13 γ-tubulin molecules per complex [42]. from the bulk of the α-tubulin in the velocity gradient Most of the γ-tubulin distributed around 25–40S during the demonstrates that the 60S APC complex does not simply velocity sizing analysis in our study is probably part of this associate with all microtubules or tubulin polymers in the cell. ring complex. Even though 60S APC does not copurify with Our observation is in concordance with the previously reported this ring complex, it is possible that the 60S APC complex colocalization studies where endogenous APC was shown to could represent an association of APC and other proteins with bind to only a subset of microtubules [6]. the ring complex to give rise to a larger complex. A variety of 128 S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130

has been proposed that IQGAP1 links APC to actin filaments for cell polarization and directional migration. The presence of IQGAP1 in the 60S APC fraction raises the possibility that this distinct fraction of APC has the potential to interact with actin cytoskeletal network through its association with IQGAP1. It seemed that only a small fraction of the total IQGAP1 copurified with APC. Because of the difficulties in calculating the stoichiometry of APC–IQGAP1 interaction due to the differences in antibody sensitivity and detection methods, it is difficult to say whether this protein is a major binding partner of 60S APC complex. Moreover, no actin is present in the 60S APC fraction. However it remains possible that this 60S APC represents a complex that links microtubule with actin network through IQGAP1 as previously described [28]. It is also important to note that the interactions between APC and IQGAP1, α-tubulin and γ-tubulin were also observed in 20S fraction (Fig. 9). This could imply that the 60S APC complex is formed from the 20S complex plus additional proteins. However, the interaction between β-catenin and APC was only observed in the 20S fraction but not in the 60S complex (Figs. 2b and 9), indicating that they are probably distinct protein complexes and may have different roles in the cell. Our data suggest that APC in the 60S complex identified in HCT116 cells is a discrete high molecular weight protein complex. This distinct APC complex could be a pool of APC that plays a role in cytoskeletal regulation or it may have some novel function in the cell yet to be identified.

Fig. 8. Analysis of APC interaction with potential binding partners in the cytosolic fraction (S100). Confluent HCT116 cells were lysed and fractionated as described above. The distributions of APC and potential interaction partners in the cytosolic fraction (S100) and the membrane fraction (P100) were analyzed by western blot (TL: Total Lysate, PNF: Post Nuclear Fraction, S: S100, P: P100). The total cytosolic fraction (S100) was subject to immuno- precipitation with anti-APC Ab or APC Ab preincubated with 100M excess blocking peptide. Immunoprecipitates were analyzed by western blotting (IgG: Rabbit IgG Control, IP: APC antibody (C-20) immunoprecipitate, B.P.: immunoprecipitation with blocking peptide, sn: supernatant after immunopre- cipitation). Note that the IP and B.P. samples shown are 5X amount comparing to the supernatant (sn). genetic and biochemical experiments have shown that γ- tubulin is required for microtubule nucleation [38] and half of the microtubules formed in the presence of γ-TuRC in vitro Fig. 9. Analysis of APC interaction with potential binding partners in the 20S appears to have one complex at their minus end [43]. fraction. Confluent HCT116 cells were lysed and fractionated as described above. The 20S fraction was subject to immunoprecipitation with anti-APC Considering that the 60S APC complex is present at very low Ab or APC Ab preincubated with 100 M excess blocking peptide. Immuno- levels in these cells and interacts with a selective fraction of α precipitates were analyzed by western blotting (IgG: Rabbit IgG Control, IP: and γ-tubulin, it is tempting to speculate that this discrete APC antibody (C-20) immunoprecipitate, B.P.: immunoprecipitation with fraction of APC play a role in microtubule nucleation at the blocking peptide, sn: supernatant after immunoprecipitation). In the APC blot, minus end. the IP and B.P. samples were loaded in equal proportions to the supernatant (sn) samples in order to show the efficiency of immunoprecipitation; in the Recently APC has also been shown to interact with other blots the IP and B.P. samples were loaded at 10 times the proportion of IQGAP1, a protein involved in cytoskeletal organization [28]. the supernatant (sn) samples to better detect the small amounts of co- Activated Rac1 and Cdc42 can recruit APC/IQGAP1 and it immunoprecipitating proteins. S. Mahadevaiyer et al. / Biochimica et Biophysica Acta 1773 (2007) 120–130 129

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