3738 Short Report

IntroductionThe tumor suppressor gene APC is mutated at an early stagein the development of most sporadic and inherited colorectaltumors (Polakis, 2000). Wild-type APC negatively regulatesthe Wnt signaling pathway by promoting the proteasomaldegradation of �-catenin, thereby preventing TCF/�-catenindependent target gene transcription (Lustig and Behrens,2003). Evidence from a large number of studies indicates thataberrant activation of the Wnt pathway resulting fromtruncating mutations of APC is a central event in colorectaltumor formation (Polakis, 2000; Schneikert and Behrens,2007). Besides its role in the Wnt pathway, APC has beenimplicated in a variety of cellular functions based on itsassociation with regulators and components of thecytoskeleton. Most prominently, APC interacts with andstabilizes microtubules, thereby regulating diverse biologicalprocesses such as axon outgrowth, cell migration and mitosis(Hanson and Miller, 2005; Zumbrunn et al., 2001). Several ofthese functions appear to be altered by tumor-associated APCmutations, indicating that the tumor suppressor activity of APCextends beyond the regulation of Wnt signaling (Fodde, 2003).

Functional evidence in different cellular contexts suggeststhat the microtubule plus-end association of APC is requiredfor the establishment of cell polarity during directed cellmigration (Etienne-Manneville et al., 2005; Kawasaki et al.,2003; Nathke et al., 1996; Watanabe et al., 2004; Wen et al.,2004). A basic domain in the C-terminal third of APC candirectly associate with microtubules, and an adjacent domain

interacts with the microtubule-associated protein EB1(MAPRE1) (Askham et al., 2000; Smith et al., 1994). APCappears to regulate microtubule dynamics as well as theinterplay of microtubules with the actin network throughinteractions with cytoskeletal regulators such as the Rhoeffector mammalian Diaphanous-related (mDia), the Rac-specific guanine nucleotide exchange factor Asef (Arhgef4),and the Rac effector IQGAP1, the latter two associating withthe N-terminal armadillo (ARM) repeat domain of APC(Kawasaki et al., 2000; Watanabe et al., 2004; Wen et al.,2004).

APC is connected to the plasma membrane at different siteswithin the cell (reviewed in Bienz and Hamada, 2004; Hansonand Miller, 2005). It can be detected in cortical clusters at thebasal plasma membrane, in association with microtubules andat the tips of cellular protrusions characterized by microtubuleends in migrating cells (Barth et al., 2002; Reilein and Nelson,2005). At cellular protrusions, APC can be anchored to themembrane via interaction of its C-terminal PDZ-binding motifwith the peripheral membrane protein DLG1 (Etienne-Manneville et al., 2005; Mimori-Kiyosue et al., 2007). The N-terminal ARM repeats and the �-catenin-binding domains ofAPC also play a role in the localization of APC at the tipstructures (Sharma et al., 2006). Outside of these microtubule-associated clusters, APC has a more global distribution at theplasma membrane. The ARM repeat domain was shown to beinvolved in the lateral membrane targeting of APC both inDrosophila and mammalian epithelial cells (Hamada and

APC is a multifunctional tumor suppressor protein thatnegatively controls Wnt signaling, but also regulates celladhesion and migration by interacting with the plasmamembrane and the microtubule cytoskeleton. Although themolecular basis for the microtubule association of APC iswell understood, molecular mechanisms that underlie itsplasma membrane localization have remained elusive. Weshow here that APC is recruited to the plasma membraneby binding to APC membrane recruitment 1 (AMER1), anovel membrane-associated protein that interacts withthe ARM repeat domain of APC. The N-terminus ofAMER1 contains two distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2]-binding domains, whichmediate its localization to the plasma membrane.Overexpression of AMER1 increases APC levels and

redirects APC from microtubule ends to the plasmamembrane of epithelial cells. Conversely, siRNA-mediatedknockdown of AMER1 reduces the overall levels of APC,promotes its association with microtubule ends in cellularprotrusions and disturbs intercellular junctions. Thesedata indicate that AMER1 controls the subcellulardistribution of APC between membrane- and microtubule-associated pools, and might thereby regulate APC-dependent cellular morphogenesis, cell migration and cell-cell adhesion.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/21/3738/DC1

Key words: APC, Cytoskeleton, Tumor suppressor

Summary

AMER1 regulates the distribution of the tumorsuppressor APC between microtubules and theplasma membraneAnnette Grohmann, Kristina Tanneberger, Astrid Alzner, Jean Schneikert and Jürgen Behrens*Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nuremberg, Glückstr. 6, 91054 Erlangen, Germany*Author for correspondence (e-mail: jbehrens@molmed.uni-erlangen.de)

Accepted 6 August 2007Journal of Cell Science 120, 3738-3747 Published by The Company of Biologists 2007doi:10.1242/jcs.011320

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3739AMER1 regulates the distribution of APC

Bienz, 2002; Langford et al., 2006; McCartney et al., 1999;McCartney et al., 2006). This localization can be promoted bydestruction of microtubules, suggesting that APC can exchangebetween the microtubule-associated and the lateral plasmamembrane pools (Rosin-Arbesfeld et al., 2001). Missensemutations or deletions of individual amino acids in the ARMrepeats of Drosophila epithelial APC (E-APC)/APC2 abolishthe membrane localization of E-APC and result in defects ofcadherin/catenin-based cell-cell adhesion and in the tetheringof mitotic spindles to cortical actin (Hamada and Bienz, 2002;McCartney et al., 2001). The mechanisms by which APCassociates with the plasma membrane outside microtubule-associated clusters are largely unknown. In particular,

molecules capable of linking APC to the plasma membranehave not yet been identified, precluding the in-depth analysisof the APC function in this compartment. Here, we report theidentification and characterization of the AMER proteins,which link APC to the plasma membrane thereby preventingassociation of this protein with microtubule ends.

ResultsAMER1 binds to the ARM repeat domain of APCWe identified the novel protein AMER1 as an APCinteraction partner in a yeast two-hybrid screen using theARM repeat domain of human APC (APC-ARM, amino acids308-789, Fig. 1C) as a bait. We isolated three independent

Fig. 1. Structure of AMER1, AMER1(short) and AMER2, and theirinteraction with the ARM repeats of APC. (A) Scheme of humanAMER1, AMER1(short) and AMER2. The APC-interacting sequencesare indicated by gray shading. In AMER1(short), the C-terminal aminoacids that are different from AMER1 are indicated in black. (B) Aminoacid sequence of human AMER1 and AMER1(short). APC-interactingsequences are shaded, glutamic acid-rich and proline-rich sequencesare underlined, and the REA repeats are in italics. For AMER1(short),only the amino acids from position 786 onwards, which are different toAMER1, are shown. This sequence is encoded by a separate 3� exon(see text for details). (C, upper panel) Interaction of murine Amer1sequences #1-3 with the human APC ARM repeat region (APC-ARM)as well as with an asparagine-to-lysine substitution mutant (APC-ARMN507K) in quantitative yeast two-hybrid assays. The seven ARMrepeats are indicated by shaded boxes. Values represent �-galactosidase units of representative experiments. (C, lower panel)Interaction of human AMER2 APC-binding sites #1 and 2 with APC-ARM in quantitative yeast two-hybrid assays. Empty DNA-binding-domain vector was used as a control. Values represent �-galactosidaseunits of representative experiments. Binding sites #1 and 2 producedsimilar high �-galactosidase values upon interaction with APC-ARMwhen tested in the DNA-binding-domain vector (not shown).

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sequences (#1-3) of Amer1, which are all located in thecentral region of the protein but do not overlap and share noamino acid sequence similarity (Fig. 1A,B). The sequencesinteracted with similar efficiency with the ARM repeats ofAPC-ARM (Fig. 1C, upper panel). Interestingly, anasparagine-to-lysine substitution in the second ARM repeatof APC-ARM abolished or significantly reduced itsinteraction with the AMER1 sequences (Fig. 1C, upperpanel). The corresponding mutation of Drosophila E-APChas been shown to disrupt its membrane localization (Hamadaand Bienz, 2002).

The coding sequences of both human and mouse AMER1reside on single exons located on syntenic regions of the Xchromosomes of both species (Xq11.1 and XC3, respectively).AMER1 consists of 1135 amino acids. It lacks knownfunctional protein domains, but contains conspicuous glutamicacid-rich and proline-rich stretches as well as a series ofarginine-glutamic acid-alanine (REA) repeats (Fig. 1B). Ahuman cDNA clone (FLJ39827) annotated in the NCBIReference Sequence (RefSeq) database is identical to theAMER1 coding sequence up to base pair 2356, after which 59coding base pairs, present on a downstream exon, are added bysplicing (supplementary material Fig. S1A). This isoform lacksa major part of the third APC-interacting fragment and wasnamed AMER1(short) (Fig. 1A,B).

AMER1 is ubiquitously expressed in human tissues similarto APC (supplementary material Fig. S1B). Flag-tagged andendogenous AMER1 protein ran at the same position of about190 kDa in anti-AMER1 western blots (supplementarymaterial Fig. S1C). Moreover, expression of the endogenous190 kDa protein was reduced after treatment with siRNAoligonucleotides against AMER1, thus proving the authenticityof the 190 kDa band as AMER1. AMER1 was detected inlysates from several human cancer cell lines by westernblotting (supplementary material Fig. S1C).

The NCBI RefSeq database contains another proteinsequence with high similarity to AMER1 (FAM123A), whichwe named AMER2 (Fig. 1A). The coding sequence of AMER2is located on chromosome 13. According to the database, twoisoforms of AMER2 are generated by alternative splicing(transcript variants 1 and 2; supplementary material Fig. S2A).We cloned transcript variant 1, which encodes a protein sharing26% amino acid identity with AMER1. Interestingly, thisisoform contains stretches with 100% amino acid identity toAMER1 within two regions analogous to the APC-bindingsites #1 and #2 (supplementary material Fig. S2B). Theseregions of AMER2 interacted with APC-ARM in yeast (Fig.1C, lower panel). By BLAST searches, homologs of AMER1and AMER2 were found in mouse, chicken, Xenopus andzebrafish, but not in Drosophila.

AMER1 forms complexes with APC in mammalian cellsIn SW480 colon carcinoma cells, endogenous (truncated) APC(APCmut) as well as exogenous wild-type APC (APC) couldbe specifically co-immunoprecipitated with Flag-taggedAMER1 (Fig. 2A). Reciprocally, endogenous as well as Flag-tagged AMER1 were co-immunoprecipitated with EGFP-tagged APC-ARM in 293T cells (Fig. 2B). Importantly,AMER1 was also co-immunoprecipitated with APC from non-transfected 293T or SW480 cells, demonstrating the presenceof endogenous AMER1-APC complexes (Fig. 2C). In line with

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the yeast two-hybrid data (cf. Fig. 1C), the APC-ARMN507K

mutant showed diminished interaction with AMER1 andAMER1(short) in co-immunoprecipitation experiments ascompared with wild-type APC-ARM (Fig. 2D). APC-ARMco-immunoprecipitated with AMER1 fragments containing atleast one of the APC-interacting domains [i.e. with AMER1,AMER1(2-839), AMER1(2-601) and AMER1(319-716)] butnot with fragments lacking such domains [i.e. with AMER1(2-321), AMER1(531-716) and AMER1(833-1135)] (Fig. 2E).Together, our results show that AMER1 specifically interactswith APC via three independent binding domains, and that thetwo proteins form endogenous complexes in mammalian cells.APC was also co-immunoprecipitated with EGFP-taggedAMER2 in 293T cells (supplementary material Fig. S2C).

AMER1 controls APC levelsCells transiently expressing AMER1 showed increased levelsof co-expressed APC (Fig. 2A), as well as APC-ARM (Fig.2E), as compared with control transfectants, whereas theamount of a co-expressed unrelated protein, FKBP8, remainedunchanged (Fig. 2E, lower panel and lower scheme). AMER1fragments lacking APC interaction domains, such asAMER1(2-321), AMER1(531-716) and AMER1(833-1135),did not increase APC-ARM levels, indicating that AMER1requires direct interaction for stabilization of APC (Fig. 2E).Importantly, the amounts of endogenous APC were alsosignificantly increased in MDCK cell clones stably expressingEGFP-tagged AMER1 as compared to EGFP-expressingcontrols, or untransfected MDCK cells (Fig. 2F and data notshown). Conversely, knockdown of AMER1 in 293T cells bydifferent siRNA oligonucleotides led to a reduction ofendogenous APC protein levels (Fig. 2G), whereas mRNAlevels of APC were not changed (data not shown).

AMER1 localizes to the plasma membrane and recruitsAPCIn MCF-7 cells, EGFP-tagged AMER1 was localized at theplasma membrane, whereas EGFP was diffusely distributed(Fig. 3A, upper panels). Flag-tagged AMER1 also localized tothe membrane as determined by immunofluorescence staining,which required permeabilization of fixed cells, indicating thatAMER1 associates with the cytoplasmic side of the plasmamembrane (data not shown). Similarly, EGFP- or Flag-taggedAMER2 localized to the plasma membrane of MCF-7 cells(Fig. 3A, and data not shown). AMER proteins were enrichedin but not restricted to lateral membranes. AMER1 fragmentscomprising amino acids 2-601 and 2-209 associated with theplasma membrane similar to full-length AMER1 (Fig. 3A,upper panel), whereas deletion fragments of AMER1 lackingthe N-terminus, such as AMER1(207-839), did not localize tothe plasma membrane but were distributed throughout the cell.Thus, the N-terminal domain of AMER1, containing aminoacids 2-209, is necessary and sufficient for the membranelocalization of this protein.

Importantly, both AMER1 and AMER1(short), as well asAMER2, recruited co-expressed APC from filamentousstructures, which probably represented microtubules(Munemitsu et al., 1994; Smith et al., 1994), to the plasmamembrane (Fig. 3A, lower panels and data not shown).AMER1-mediated membrane recruitment of APC required theAPC-binding sites and the membrane docking N-terminus of

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AMER1, because the mutants AMER1(2-209) andAMER1(207-839), which lack these domains, respectively, didnot recruit APC to the plasma membrane. Interestingly, inAMER1(2-209)-expressing cells, APC was frequently

associated with the tips of microtubules, in contrast tofilamentous staining in control cells. The C-terminal half ofAMER1 was not required for APC recruitment, asdemonstrated by AMER1(2-601) (Fig. 3A). Both AMER1 and

Fig. 2. Interaction between AMER1and APC. (A) Co-immunoprecipitationof wild-type APC (APC) andendogenous mutant APC (APCmut)with Flag-tagged AMER1 aftertransient transfections of SW480 cells,as indicated. Western blottings wereperformed using anti-APC Ab1 (forAPCmut) and Ab2 (for APC), and anti-Flag antibodies. The double band forFlag-AMER1 is observed in some butnot all experiments (cf. B) and mightresult from incomplete denaturation ofthe protein in the gel sample buffer.(B) Co-immunoprecipitation ofendogenous AMER1 and Flag-taggedAMER1 with EGFP-tagged APC-ARM after transient transfections of293T cells, as indicated. Westernblottings were performed using anti-AMER1 or anti-GFP antibodies.(C) Co-immunoprecipitation ofendogenous AMER1 with APC fromlysates of nontransfected 293T andSW480 cells. Immunoprecipitationswere performed with anti-GFPantibody as a control or with anti-APCantibody Ali followed by westernblotting using the anti-AMER1antibody or Ali. (D) Co-immunoprecipitation of EGFP-taggedAPC-ARM or APC-ARMN507K withFlag-tagged AMER1 orAMER1(short) after transienttransfections of 293T cells asindicated. Western blottings wereperformed using anti-GFP or anti-Flagantibodies. (E) Co-immunoprecipitation of Flag-taggedAPC-ARM with EGFP-taggedAMER1 and AMER1 deletionfragments after transient transfectionsof 293T cells as indicated. Westernblottings were performed using anti-Flag or anti-GFP antibodies. Thebottom blot shows levels of the Flag-FKBP8 control protein in lysates of293T cells after co-expression with theindicated AMER1 constructs. Thescheme below shows the structure ofthe deletion mutants of AMER1 andquantification from separateexperiments of the fold change ofAPC-ARM or FKBP8 protein levels(as a control) after co-transfection withthe indicated AMER1 constructs,relative to EGFP transfection. (F) Western blotting for APC (antibody Ali) in stable clones of MDCK cells expressing EGFP (EGFP#1,EGFP#2) or EGFP-tagged AMER1 (EGFP-AMER1#1, EGFP-AMER1#2). (G) Western blotting for APC (antibody Ali), AMER1, Pan-cadherin and FKBP8 from lysates of 293T cells transiently transfected with siRNA oligonucleotides against GFP (as a control), or two differentsiRNA oligonucleotides against AMER1 (siAMER1a, c). The numbers below the lanes indicate relative protein levels normalized to Pan-cadherin, with siGFP controls set to 100%.

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AMER1(short) also recruited the ARM repeat domain of APCalone (APC-ARM) to the plasma membrane. The N507Ksubstitution of APC-ARM barely reduced the recruitment byAMER1 but impaired the recruitment by AMER1(short)(supplementary material Fig. S3). Notice that AMER1(short)lacks a large part of the third APC-binding domain of AMER1.

Because AMER1 lacks any obvious membrane-anchoringdomains, we next investigated whether it would bind directlyto membrane lipids. Recombinant GST-tagged AMER1(2-

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285), but not GST-AMER1(260-545), bound tophosphatidylinositol mono-, bis- and tris-phosphate species,and to 3-sulfogalactosylceramide (sulfatide) in vitro (Fig. 3B).Interestingly, GST-AMER1 fragments comprising amino acids2-142 or 143-209 also bound to membrane lipids, indicatingthat AMER1 contains two lipid-binding sites (Fig. 3B).Phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] is thepredominant phosphoinositide at the plasma membrane (DiPaolo and De Camilli, 2006) and therefore is a good candidate

Fig. 3. AMER1 localizes to the membranevia binding to PtdIns(4,5)P2. (A) Doublestaining of EGFP, EGFP-AMER1, EGFP-AMER1 deletion constructs as in Fig. 2Eor EGFP-AMER2 (upper panels, GFPfluorescence), and APC (lower panels,antibody Ali immunofluorescence) inMCF-7 cells transiently transfected asindicated above the panels. Arrowheadsdenote the membrane, and arrows thefilamentous localizations. (B) Membranelipid-binding assays of AMER1 deletionmutants. Membrane lipid strips wereincubated with the indicated recombinantGST-AMER1 fusion proteins, revealingtwo phosphoinositide binding sites. DAG,diacylglycerol; PA, phosphatidic acid;PS/PE/PC/PG, phosphatidyl-serine/-ethanolamine/-choline/-glycerol.(C) Localization of AMER1 (a-e) andAPC (a�-c�) or AMER1 deletion mutants(f-k) in transiently transfected MCF7cells. Double staining of EGFP-AMER1(a-c, GFP fluorescence) and APC (a�-c�,anti-M-APC immunofluorescence) with(b,b�) and without (a,a�) prior ionomycintreatment (Iono), or with ionomycintreatment followed by EGTA treatment(c,c�). Staining of EGFP-AMER1 in cellstreated with wortmannin (Wm) prior toionomycin/EGTA treatment (d) or withneomycin (Neo) prior to ionomycin (e).Staining of EGFP-AMER1(2-142) (f-h) orEGFP-AMER1(143-209) (i-k) in cellstreated with ionomycin/EGTA asindicated.

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for a physiological membrane link for AMER1. We analyzedwhether cleavage of PtdIns(4,5)P2 by phospholipase C (PLC)affects AMER1 localization in cells. Membrane localization ofectopically expressed full-length AMER1 and APC, and of thedeletion fragments AMER1(2-142) and AMER1(143-209), inMCF-7 cells was completely abolished upon treatment withionomycin, which induces Ca2+ influx and thereby activatesPLC (Varnai and Balla, 1998) (Fig. 3Cb,b�,g,j). AMER1 andAPC localized again to the plasma membrane when Ca2+ waschelated with EGTA after ionomycin treatment (Fig.3Cc,c�,h,k). Relocalization at the membrane was prevented bytreatment of cells with wortmannin, which, at highconcentrations, inhibits phosphatidylinositol 4-kinases and

thereby prevents resynthesis of PtdIns(4,5)P2 (Nakanishi et al.,1995) (Fig. 3Cd). Furthermore, pretreatment with neomycin,which affects the turnover of PtdIns(4,5)P2 (Gabev et al.,1989), impaired the ionomycin-induced dissociation ofAMER1 from the plasma membrane (Fig. 3Ce). These resultssuggest that AMER1 localizes to the plasma membrane bydirect interaction with PtdIns(4,5)P2 via two distinct bindingdomains at the N-terminus.

AMER1 controls the distribution of APC betweenmicrotubules and the plasma membraneIn MDCK clones stably expressing AMER1, endogenous APCdelocalized from focal cellular protrusions that had beenpreviously characterized as microtubule ends (Nathke et al.,1996; Rosin-Arbesfeld et al., 2001) and became recruited tothe plasma membrane (Fig. 4Aa,b). As has been shownpreviously, disruption of microtubules by nocodazole treatmentresulted in membrane association of endogenous APC inMDCK cells [Fig. 4B (cf. Rosin-Arbesfeld et al., 2001)].Treatment with ionomycin, which blocks AMER1 membraneassociation (cf. Fig. 3C), prevented the nocodazole-inducedmembrane localization of APC (Fig. 4B), which is in line withAPC being linked to the membrane by endogenous AMER1 inthese cells.

Next, we analyzed whether loss of endogenous AMER1 inMCF-7 cells by RNAi would promote microtubule associationof APC. Transient transfection of the siAMER1c

oligonucleotide in MCF-7 cells resulted in an overalldownregulation of AMER1 mRNA by about 30%, whichcorresponds to the transfection efficiency obtained inthese cells (supplementary material Fig. S5A). Aftertransient knockdown of AMER1 with siAMER1c, 13.2%(n=657) of cells at the edges of colonies showed staining

Fig. 4. AMER1 controls the distribution of APC betweenmicrotubules and the plasma membrane. (A) Localization ofAPC (a,b) in MDCK cells stably expressing EGFP (a,a�) orEGFP-tagged AMER1 (b,b�). (a,b) Immunofluorescencestainings using the anti-APC antibody Ali; (a�,b�)corresponding EGFP fluorescence. Notice the membraneassociation of EGFP-AMER1, which is not observed forEGFP. Arrowheads point to APC at cytoplasmic clusters atcellular protrusions in the EGFP transfectants (a) and tocolocalization of AMER1 and APC at the plasma membrane inthe EGFP-AMER1 transfectants (b,b�). Insets in upper panelsrepresent higher magnifications. (B) Immunofluorescencestaining of APC (anti-M-APC) in MDCK cells treated withsolvent (DMSO), nocodazole (Noco), or nocodazole followedby ionomycin (Noco/Iono). Arrowheads indicate lateralplasma membranes. (C) Immunofluorescence staining of APC(anti-M-APC) in MCF-7 cells treated with siRNA againsteither GFP as a control (siGFP), AMER1 (siAMER1c), orAMER1 and APC (siAMER1c+siAPC). Arrowheads indicatetips of cellular protrusions. (D) Staining of transientlytransfected APC (a-c), microtubules (a�,b�, ‘MT’) or AMER1(c�) in MCF-7 cells transiently transfected with APC togetherwith siGFP (a,a�), siAMER1c (b,b�), or siAMER1c and thesiAMER1c-insensitive EGFP-rAMER1 expression construct(c,c�). (a,a�;b,b�;c,c�) Double stainings. (a-c) Anti-M-APCimmunofluorescence; (a�,b�) anti-�-tubulinimmunofluorescence; (c�) GFP fluorescence. Broken linesindicate the edge of colonies.

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of APC in tips of cellular protrusions, often corresponding tomicrotubule ends (Nathke et al., 1996). This staining wasspecific because it was abolished by concomitant treatmentwith siAPC (Fig. 4C, supplementary material Fig. S5B). Incells treated with control siRNA (siGFP), only 3.9% of cells(n=751) showed such tip staining. Alterations in the membraneassociation of APC could not be reliably determined, becausethe membrane staining with the anti-M-APC serum appearedto be nonspecific because it was not affected by siRNA againstAPC, in contrast to APC staining at the protrusions. Transientlytransfected APC localized to filamentous structures partiallyoverlapping with microtubules in most of the siGFP-treatedMCF-7 control cells (Fig. 4Da,a� and Fig. 3A). In 25.5% oftransfected cells at the edge of colonies, APC was found at thetips of cellular protrusions (n=106). This localization wasmarkedly increased to 56.8% of the cells in siAMER1c-treatedcultures (Fig. 4D,b; n=111). Moreover, in these structures APCoverlapped with the ends of microtubules, which werefrequently reoriented perpendicular to the membraneindicating a rearrangement of the microtubular network (Fig.4Db�). To verify that tip localization of APC was caused by thespecific depletion of AMER1, we additionally expressed amutated AMER1 cDNA resistant to knockdown by siAMER1c(rAMER1, supplementary material Fig. S5C,D). Tiplocalization of APC was completely abolished in rAMER1-expressing cells (n=30), and APC was exclusively observed atthe cell membrane co-localizing with rAMER1 (Fig. 4Dc,c�).These data demonstrate that silencing of endogenous AMER1promotes association of APC with the tips of cellularprotrusions, in which it overlaps with microtubule ends.

AMER1 controls intercellular junctions together withAPCMCF-7 cells are epithelial and show the classical cobblestonepattern when stained for E-cadherin or �-catenin (Fig. 5A).When AMER1 was transiently knocked down by transfectionwith the siAMER1c oligonucleotide, the cell junctions ofMCF-7 cells were frequently disrupted, resulting in ‘gaps’between cells (Fig. 5A,B). This was most apparent at sites atwhich multiple cells met (e.g. at tricellular junctions).Interestingly, membrane association of E-cadherin, �-cateninand other junctional proteins was still preserved (Fig. 5A anddata not shown). The additional knockdown of APC led to amore severe disruption of cellular junctions, with cellsfrequently showing complete detachment from each other (Fig.5A,B), whereas knockdown of APC alone had only a minoreffect (Fig. 5B). These data indicate a role for AMER1 inmaintaining the integrity of intercellular junctions, probably bymediating the membrane localization of APC.

DiscussionIn this paper, we provide a novel molecular mechanism bywhich APC can be recruited to the plasma membrane byinteracting with the AMER1 and AMER2 proteins. Whenoverexpressed, AMER proteins recruited both exogenous andendogenous APC to the plasma membrane and prevented itsinteraction with microtubules. Depletion of PtdIns(4,5)P2 byionomycin treatment, which abolishes AMER1 localization atplasma membranes, also reduced the association of APC withthe membrane. Endogenous AMER1-APC complexes wereidentified, and knockdown of AMER1 increased the fraction

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of APC associated with microtubule ends. Finally, a pointmutation, which was shown to reduce membrane associationof Drosophila E-APC, in the ARM repeat domain alsodiminished the AMER1-APC interaction.

AMER1 was localized at the plasma membrane, and the N-terminus of AMER1 is necessary and sufficient for thislocalization. We could identify two distinct sites (amino acids2-142 and 143-209) that mediate membrane association anddirectly bind PtdIns(4,5)P2. These sites have high isoelectricpoints (pI=9.3 and 10.55, respectively; ProtParam) comparedwith full-length AMER1 (pI=4.77), contain highly conservedbasic and aromatic residues and are particularly enriched inlysine residues (11.3 and 14.9%, respectively, compared with4.1% in full-length AMER1; supplementary material Fig. S2B).Basic and aromatic amino acids have been shown to mediatePtdIns(4,5)P2 binding in other proteins (Kagan and Medzhitov,2006). The majority of these lysine residues are conserved inAMER2 (supplementary material Fig. S4), which can alsointeract with the plasma membrane. We could further show thatactivation of phospholipase C by ionomycin-induced Ca2+

influx leads to the delocalization of AMER1 and APC from theplasma membrane, probably because of PtdIns(4,5)P2 cleavage.These data strongly support a role for PtdIns(4,5)P2 as the

Fig. 5. AMER1 controls intercellular junctions together with APC.(A) Immunofluorescence staining of E-cadherin in MCF-7 cellstreated with a control siRNA against GFP (siGFP), siRNA againstAMER1 (siAMER1c), or siAMER1c together with an siRNA againstAPC (siAMER1c+siAPC). Arrowheads indicate disrupted celljunctions. (B) Relative number of gaps between MCF-7 cellstransfected with siRNA oligonucleotides as indicated below the bars.Gaps were counted in E-cadherin- and �-catenin-stained samples in20 optical fields using the 40� objective. Results show themean±s.d. of at least two independent experiments.

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physiological membrane anchor for AMER1. AMER1 was notrestricted to a specific membrane compartment in the cell linesused in this study, which is in line with its association withmembrane lipids. However, local PtdIns(4,5)P2 concentrationsare regulated by signaling events and are significantly enrichedat the apical plasma membrane in polarized epithelial cells(Martin-Belmonte et al., 2007; McLaughlin et al., 2002). Suchchanges might influence AMER1 localization and thus controlthe subcellular distribution of APC.

AMER1 directly interacted with the ARM repeat domain ofAPC via three separate binding sites, which were identified inour initial yeast two-hybrid screen. The sites interact withsimilar efficiency with APC yet do not share any obvioussequence similarity. Other known interaction partners of theARM repeat domain of APC, such as Asef, KAP3 (KIFAP3)and IQGAP1, also show no sequence similarity to each otheror to AMER1, indicating that the ARM repeats can contactvarious partners using different binding modes. This is similarto �-catenin, in which the ARM repeats can interact withvarious interaction partners using distinct amino acids (vonKries et al., 2000). It is possible that the presence of multipleAPC-binding domains in AMER1 increases the overall affinityof the interaction. Moreover, the differences in sequencebetween these domains might allow for individual modulationof APC binding by biochemical modifications or competinginteractions. Interestingly, the third APC-interaction domain ofAMER1 retained significant capacity to interact with theN507K mutant of the ARM repeat domain in the yeast two-hybrid assays. In AMER1(short), this domain is largelymissing and membrane recruitment of the N507K mutant byAMER1(short) was reduced, whereas it was largely preservedin AMER1, at least under conditions of overexpression. Thisindicates that, depending on the expression of the AMER1isoform, APC might be differentially recruited to the plasmamembrane.

Two main cellular functions have been assigned toperipheral APC. First, several reports have indicated a role forAPC in the polarization of migrating cells via its associationwith the cytoskeleton (Etienne-Manneville et al., 2005;Kawasaki et al., 2003; Kroboth et al., 2007; Nathke et al., 1996;Watanabe et al., 2004; Wen et al., 2004). siAMER1 treatmentincreased the fraction of cells harboring APC at microtubulesin cellular protrusions, which was reverted by expression of ansiRNA-resistant cDNA of AMER1. Plasma membranerecruitment of APC by AMER1 might therefore block cellpolarization and counteract cell migration. Second, it wassuggested that plasma membrane association of APC controlscell-cell adhesion and adherens junction formation (Faux et al.,2004; Hamada and Bienz, 2002). Mutations within the ARMrepeat domain leading to reduced membrane association ofDrosophila E-APC results in the disruption of cell junctions(Hamada and Bienz, 2002). However, this might result from adominant action of the mutants, because null mutations did notaffect cell junctions (McCartney et al., 2006). Furthermore,ARM repeat mutations also diminish the interaction with Asef,which could have an impact on the regulation of thecytoskeleton and thereby affect cellular junctions (Watanabe etal., 2004). We observed disturbance of the integrity of celljunctions in MCF-7 cells treated with the siAMER1coligonucleotide, which could result from diminishedmembrane localization of APC. Interestingly, the effect was

predominantly seen at vertices in which more than two cellscontact each other. Possibly, adherens junctions at theselocations are more vulnerable than at the lateral domains of twoneighboring cells. AMER1 might compete with cytoskeletalregulators, such as Asef (Kawasaki et al., 2003), for binding toAPC, and loss of AMER1 would allow signaling by these APCcomplexes, leading to the observed effects. Alternatively,localization of APC at the plasma membrane might be directlyrequired for cell junction formation (e.g. by promoting �-catenin recruitment to cadherins), as was suggested previously(Hamada and Bienz, 2002). Because both E-cadherin and �-catenin membrane localization were not strongly altered insiAMER1-treated cells, such a function is rather unlikely in ourexperimental setting. The fact that simultaneous knockdown ofAPC aggravated the junctional defects of the AMER1knockdown suggests a direct and positive role of both proteinsin junction formation and/or maintenance. The identificationof AMER1 as a plasma membrane link of APC will allow usto analyze more specifically the function of this importanttumor suppressor in polarized cell migration and cell-celladhesion.

After this paper had been submitted, a report describedWTX as a negative regulator of the Wnt signaling pathway,which forms complexes with APC, Axin, �-catenin and �-TRCP (Major et al., 2007). WTX is identical to AMER1,indicating that the �-catenin destruction complex as a wholemight become recruited to the plasma membrane, which mightaffect Wnt signaling. WTX/AMER1 is mutated in Wilmstumors, leading to truncations or deletions of the completegene (Rivera et al., 2007). It was suggested that loss of WTXleads to aberrant activation of Wnt signaling and therebypromotes tumorigenesis (Major et al., 2007). From our data,these mutations might additionally contribute to tumorigenesisby abolishing functions of APC at the plasma membrane andactivating those at microtubules.

Materials and MethodsDNA constructs and transfectionsFor EGFP-tagging, cDNAs were inserted into pEGFP-C3 (Clontech); for GST-tagging they were inserted into pGEX-4T3 (Amersham Pharmacia Biotech); and forFlag-tagging into pcDNA-Flag, which was derived from pcDNA3.1 (Invitrogen) byinserting the Flag peptide coding sequence into the multiple cloning site. APCcDNA fragments were obtained by PCR amplification using pCMV-APC (Smith etal., 1994) as a template, which was also used for expression of human full-lengthAPC. The N507K mutant of APC-ARM was created by PCR mutagenesis. Full-length human AMER1 was cloned by replacing the 3� coding sequence of cloneFLJ39827 (NITE Biological Resource Center, Chiba, Japan) at nt 2073 with thecorresponding coding sequence of AMER1 obtained by reverse transcriptase (RT)-PCR from mRNA of 293T cells (cf. supplementary material Fig. S1A). Deletionmutants of AMER1 were generated by restriction digests or PCR amplification.rAMER1 cDNA was generated by PCR mutagenesis exchanging three nucleotidesof the targeting sequence of siAMER1c without changing the amino acid sequence.Full-length AMER2 transcript variant 1 was obtained by RT-PCR from mRNA of293T cells. APC-binding sites #1 and #2 of AMER2 were obtained by RT-PCR frommRNA of Caki cells. Transient transfections of plasmids were performed usingESCORT IV (Sigma), and of siRNA using TransIT-TKO (Mirus, Madison, WI,USA).

siRNA oligonucleotidesThe sequences of the siRNA oligonucleotides are: siAPC, 5�-GACGUUGCG -AGAAGUUGGAdTdT-3�; siGFP, 5�-GCUACCUGU UC CAUGGCCAdTdT-3�(Eurogentec, Seraing, Belgium). siAMER1a, siAMER1b and siAMER1c werepurchased from Dharmacon (catalog numbers D-016075-01, D-016075-02 and D-016075-03).

Cell culture and drug treatmentsCells were cultured in 10% CO2 at 37°C in DMEM supplemented with 10% FCS

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and 1% penicillin/streptomycin. For stable expression of EGFP-AMER1 and EGFP,MDCK cells were transfected with pEGFP-AMER1 or pEGFP-C3, respectively, andsubsequently selected in medium containing 1 mg/ml geneticin (G418, Invitrogen).For drug treatments, cells were incubated for 5 minutes at 37°C with 10 �Mionomycin (Calbiochem), followed by 10 minutes at 37°C with 2 mM EGTA inculture medium where indicated, or cells were incubated for 30 minutes at 37°Cwith 10 �M wortmannin, or 10 mM neomycin (Calbiochem) in culture mediumprior to ionomycin treatment. For microtubule disruption, cells were incubated with10 �g/ml nocodazole (Sigma) for 15 minutes on ice followed by 60 minutes at 37°C,or with solvent containing medium as controls.

AntibodiesThe AMER1-specific monoclonal mouse antibody was raised against amino acids2-285 of recombinant human AMER1 generated as a GST fusion in bacteria. Theanti-APC antibody Ali and the rabbit anti-M-APC serum were a gift from Inke S.Näthke (University of Dundee, Dundee, UK). The rabbit anti-FKBP8 antibody wasa gift of Frank Edlich and Gunter Fischer (Max-Planck Research Unit forEnzymology of Protein Folding, Halle, Germany). Commercial antibodies wereobtained from Calbiochem (APC Ab-1 and Ab-2), Sigma (�-Actin: clone AC-15;rabbit anti-Flag polyclonal antibody; mouse anti-GST monoclonal antibody; rabbitanti-Pan-cadherin serum), Roche (GFP, mixture of clones 7.1 and 13.1), Serotec (�-tubulin, clone YL1/2) and TaKaRa (mouse anti-E-cadherin monoclonal antibody,clone HECD-1). Secondary antibodies (Jackson ImmunoResearch, Cambridgeshire,UK) were Cy2, Cy3 or Cy5 conjugates for immunofluorescence and HRPconjugates for western blotting.

Yeast two-hybrid screenYeast two-hybrid and �-galactosidase assays were performed in the L40 yeast strainusing pBTM116 as a bait vector and a mouse embryonic day 10.5 cDNA library inpVP16 as described previously (Behrens et al., 1998).

Preparation of protein lysates, immunoprecipitation andwestern blottingCells were washed three times with PBS and lysed in Triton X-100 buffer (20 mMTris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT and1 mM PMSF) at 4°C for 10 minutes. Lysates were cleared at 16,000 g for 10 minutesat 4°C. For co-immunoprecipitation, lysates were incubated for 4 hours at 4°C withthe appropriate antibody and protein A/G-Sepharose beads (Santa CruzBiotechnology), or with anti-FLAG M2 affinity gel beads (Sigma).Immunoprecipitates were collected, washed four times in Triton X-100 buffer,eluted with SDS sample buffer and subjected to western blotting (Lustig et al.,2002). Proteins were visualized using Enhanced Chemiluminescence reagent(Perkin Elmer) and a LuminoImager (LAS-3000, Fuji), and quantified using theAIDA image analyzer software v. 3.52 (Raytest, Straubenhardt, Germany).

Lipid-binding assaysExpression of GST-AMER1 fragments in pGEX-4T3 was induced in Escherichiacoli BL21 with 0.2 mM IPTG for 3 hours at 37°C. GST-AMER1 fusion proteinswere freshly purified before the experiments using glutathione Sepharose 4B beads(Amersham) as described previously (Frangioni and Neel, 1993). Beads werewashed three times in ice-cold PBS, and GST-AMER1 fusion proteins were elutedwith glutathione elution buffer (20 mM Tris-HCl, pH 7.5, 8 mM glutathione and 5mM DTT) and quantified by SDS-PAGE/Coomassie staining. Membrane LipidStrips (Echelon) were incubated with the GST fusion proteins at a concentration of1 �g/ml at 4°C overnight and detected by a mouse anti-GST antibody, accordingto the manufacturer’s instructions.

Immunofluorescence microscopyImmunofluorescence staining was performed as described previously using 0.5%Triton X-100 for cell permeabilization (Behrens et al., 1996). Photographs weretaken with a CCD camera (Visitron, Munich, Germany) on a Zeiss Axioplan 2microscope (Zeiss, Oberkochen, Germany, 63� objective) and MetaMorph software(Molecular Devices). Images were processed using Adobe Photoshop CS software.

We thank Thomas Winkler for help in antibody generation, InkeNäthke for providing anti-APC antibodies, Eva Kohler for the FKBP8expression construct and Angela Döbler for secretarial assistance.This work was supported by a grant of the Marohn Stiftung of theUniversity of Erlangen-Nuremberg.

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