APC is a multifunctional tumor suppressor protein that negatively controls Wnt signaling, but also regulates cell adhesion and migration by interacting with the plasma membrane and the microtubule cytoskeleton. Although the molecular basis for the microtubule association of APC is well understood, molecular mechanisms that underlie its plasma membrane localization have remained elusive. We show here that APC is recruited to the plasma membrane by binding to APC membrane recruitment 1 (AMER1), a novel membrane-associated protein that interacts with the ARM repeat domain of APC. The N-terminus of AMER1 contains two distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2]-binding domains, which mediate its localization to the plasma membrane. Overexpression of AMER1 increases APC levels and redirects APC from microtubule ends to the plasma membrane of epithelial cells. Conversely, siRNA-mediated knockdown of AMER1 reduces the overall levels of APC, promotes its association with microtubule ends in cellular protrusions and disturbs intercellular junctions. These data indicate that AMER1 controls the subcellular distribution of APC between membrane- and microtubule-associated pools, and might thereby regulate APC-dependent cellular morphogenesis, cell migration and cell-cell adhesion.

Introduction

The tumor suppressor gene APC is mutated at an early stage in the development of most sporadic and inherited colorectal tumors (Polakis, 2000). Wild-type APC negatively regulates the Wnt signaling pathway by promoting the proteasomal degradation of β-catenin, thereby preventing TCF/β-catenin dependent target gene transcription (Lustig and Behrens, 2003). Evidence from a large number of studies indicates that aberrant activation of the Wnt pathway resulting from truncating mutations of APC is a central event in colorectal tumor formation (Polakis, 2000; Schneikert and Behrens, 2007). Besides its role in the Wnt pathway, APC has been implicated in a variety of cellular functions based on its association with regulators and components of the cytoskeleton. Most prominently, APC interacts with and stabilizes microtubules, thereby regulating diverse biological processes such as axon outgrowth, cell migration and mitosis (Hanson and Miller, 2005; Zumbrunn et al., 2001). Several of these functions appear to be altered by tumor-associated APC mutations, indicating that the tumor suppressor activity of APC extends beyond the regulation of Wnt signaling (Fodde, 2003).

Functional evidence in different cellular contexts suggests that the microtubule plus-end association of APC is required for the establishment of cell polarity during directed cell migration (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 can directly associate with microtubules, and an adjacent domain interacts with the microtubule-associated protein EB1 (MAPRE1) (Askham et al., 2000; Smith et al., 1994). APC appears to regulate microtubule dynamics as well as the interplay of microtubules with the actin network through interactions with cytoskeletal regulators such as the Rho effector mammalian Diaphanous-related (mDia), the Rac-specific guanine nucleotide exchange factor Asef (Arhgef4), and the Rac effector IQGAP1, the latter two associating with the 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 sites within the cell (reviewed in Bienz and Hamada, 2004; Hanson and Miller, 2005). It can be detected in cortical clusters at the basal plasma membrane, in association with microtubules and at the tips of cellular protrusions characterized by microtubule ends in migrating cells (Barth et al., 2002; Reilein and Nelson, 2005). At cellular protrusions, APC can be anchored to the membrane via interaction of its C-terminal PDZ-binding motif with the peripheral membrane protein DLG1 (Etienne-Manneville et al., 2005; Mimori-Kiyosue et al., 2007). The N-terminal ARM repeats and the β-catenin-binding domains of APC also play a role in the localization of APC at the tip structures (Sharma et al., 2006). Outside of these microtubule-associated clusters, APC has a more global distribution at the plasma membrane. The ARM repeat domain was shown to be involved in the lateral membrane targeting of APC both in Drosophila and mammalian epithelial cells (Hamada and Bienz, 2002; Langford et al., 2006; McCartney et al., 1999; McCartney et al., 2006). This localization can be promoted by destruction of microtubules, suggesting that APC can exchange between the microtubule-associated and the lateral plasma membrane pools (Rosin-Arbesfeld et al., 2001). Missense mutations or deletions of individual amino acids in the ARM repeats of Drosophila epithelial APC (E-APC)/APC2 abolish the membrane localization of E-APC and result in defects of cadherin/catenin-based cell-cell adhesion and in the tethering of mitotic spindles to cortical actin (Hamada and Bienz, 2002; McCartney et al., 2001). The mechanisms by which APC associates with the plasma membrane outside microtubule-associated clusters are largely unknown. In particular, molecules capable of linking APC to the plasma membrane have not yet been identified, precluding the in-depth analysis of the APC function in this compartment. Here, we report the identification and characterization of the AMER proteins, which link APC to the plasma membrane thereby preventing association of this protein with microtubule ends.

Fig. 1.

Structure of AMER1, AMER1(short) and AMER2, and their interaction with the ARM repeats of APC. (A) Scheme of human AMER1, AMER1(short) and AMER2. The APC-interacting sequences are indicated by gray shading. In AMER1(short), the C-terminal amino acids that are different from AMER1 are indicated in black. (B) Amino acid sequence of human AMER1 and AMER1(short). APC-interacting sequences are shaded, glutamic acid-rich and proline-rich sequences are underlined, and the REA repeats are in italics. For AMER1(short), only the amino acids from position 786 onwards, which are different to AMER1, are shown. This sequence is encoded by a separate 3′ exon (see text for details). (C, upper panel) Interaction of murine Amer1 sequences #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 ARM repeats are indicated by shaded boxes. Values represent β-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 β-galactosidase units of representative experiments. Binding sites #1 and 2 produced similar high β-galactosidase values upon interaction with APC-ARM when tested in the DNA-binding-domain vector (not shown).

Fig. 1.

Structure of AMER1, AMER1(short) and AMER2, and their interaction with the ARM repeats of APC. (A) Scheme of human AMER1, AMER1(short) and AMER2. The APC-interacting sequences are indicated by gray shading. In AMER1(short), the C-terminal amino acids that are different from AMER1 are indicated in black. (B) Amino acid sequence of human AMER1 and AMER1(short). APC-interacting sequences are shaded, glutamic acid-rich and proline-rich sequences are underlined, and the REA repeats are in italics. For AMER1(short), only the amino acids from position 786 onwards, which are different to AMER1, are shown. This sequence is encoded by a separate 3′ exon (see text for details). (C, upper panel) Interaction of murine Amer1 sequences #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 ARM repeats are indicated by shaded boxes. Values represent β-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 β-galactosidase units of representative experiments. Binding sites #1 and 2 produced similar high β-galactosidase values upon interaction with APC-ARM when tested in the DNA-binding-domain vector (not shown).

Results

AMER1 binds to the ARM repeat domain of APC

We identified the novel protein AMER1 as an APC interaction partner in a yeast two-hybrid screen using the ARM repeat domain of human APC (APC-ARM, amino acids 308-789, Fig. 1C) as a bait. We isolated three independent sequences (#1-3) of Amer1, which are all located in the central region of the protein but do not overlap and share no amino acid sequence similarity (Fig. 1A,B). The sequences interacted with similar efficiency with the ARM repeats of APC-ARM (Fig. 1C, upper panel). Interestingly, an asparagine-to-lysine substitution in the second ARM repeat of APC-ARM abolished or significantly reduced its interaction with the AMER1 sequences (Fig. 1C, upper panel). The corresponding mutation of Drosophila E-APC has been shown to disrupt its membrane localization (Hamada and Bienz, 2002).

The coding sequences of both human and mouse AMER1 reside on single exons located on syntenic regions of the X chromosomes of both species (Xq11.1 and XC3, respectively). AMER1 consists of 1135 amino acids. It lacks known functional protein domains, but contains conspicuous glutamic acid-rich and proline-rich stretches as well as a series of arginine-glutamic acid-alanine (REA) repeats (Fig. 1B). A human cDNA clone (FLJ39827) annotated in the NCBI Reference Sequence (RefSeq) database is identical to the AMER1 coding sequence up to base pair 2356, after which 59 coding base pairs, present on a downstream exon, are added by splicing (supplementary material Fig. S1A). This isoform lacks a major part of the third APC-interacting fragment and was named AMER1(short) (Fig. 1A,B).

AMER1 is ubiquitously expressed in human tissues similar to APC (supplementary material Fig. S1B). Flag-tagged and endogenous AMER1 protein ran at the same position of about 190 kDa in anti-AMER1 western blots (supplementary material Fig. S1C). Moreover, expression of the endogenous 190 kDa protein was reduced after treatment with siRNA oligonucleotides against AMER1, thus proving the authenticity of the 190 kDa band as AMER1. AMER1 was detected in lysates from several human cancer cell lines by western blotting (supplementary material Fig. S1C).

The NCBI RefSeq database contains another protein sequence with high similarity to AMER1 (FAM123A), which we named AMER2 (Fig. 1A). The coding sequence of AMER2 is located on chromosome 13. According to the database, two isoforms 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 sharing 26% amino acid identity with AMER1. Interestingly, this isoform contains stretches with 100% amino acid identity to AMER1 within two regions analogous to the APC-binding sites #1 and #2 (supplementary material Fig. S2B). These regions of AMER2 interacted with APC-ARM in yeast (Fig. 1C, lower panel). By BLAST searches, homologs of AMER1 and AMER2 were found in mouse, chicken, Xenopus and zebrafish, but not in Drosophila.

AMER1 forms complexes with APC in mammalian cells

In SW480 colon carcinoma cells, endogenous (truncated) APC (APCmut) as well as exogenous wild-type APC (APC) could be specifically co-immunoprecipitated with Flag-tagged AMER1 (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 presence of endogenous AMER1-APC complexes (Fig. 2C). In line with the yeast two-hybrid data (cf. Fig. 1C), the APC-ARMN507K mutant showed diminished interaction with AMER1 and AMER1(short) in co-immunoprecipitation experiments as compared with wild-type APC-ARM (Fig. 2D). APC-ARM co-immunoprecipitated with AMER1 fragments containing at least one of the APC-interacting domains [i.e. with AMER1, AMER1(2-839), AMER1(2-601) and AMER1(319-716)] but not 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 interacts with APC via three independent binding domains, and that the two proteins form endogenous complexes in mammalian cells. APC was also co-immunoprecipitated with EGFP-tagged AMER2 in 293T cells (supplementary material Fig. S2C).

AMER1 controls APC levels

Cells transiently expressing AMER1 showed increased levels of co-expressed APC (Fig. 2A), as well as APC-ARM (Fig. 2E), as compared with control transfectants, whereas the amount of a co-expressed unrelated protein, FKBP8, remained unchanged (Fig. 2E, lower panel and lower scheme). AMER1 fragments lacking APC interaction domains, such as AMER1(2-321), AMER1(531-716) and AMER1(833-1135), did not increase APC-ARM levels, indicating that AMER1 requires direct interaction for stabilization of APC (Fig. 2E). Importantly, the amounts of endogenous APC were also significantly increased in MDCK cell clones stably expressing EGFP-tagged AMER1 as compared to EGFP-expressing controls, or untransfected MDCK cells (Fig. 2F and data not shown). Conversely, knockdown of AMER1 in 293T cells by different siRNA oligonucleotides led to a reduction of endogenous APC protein levels (Fig. 2G), whereas mRNA levels of APC were not changed (data not shown).

AMER1 localizes to the plasma membrane and recruits APC

In MCF-7 cells, EGFP-tagged AMER1 was localized at the plasma membrane, whereas EGFP was diffusely distributed (Fig. 3A, upper panels). Flag-tagged AMER1 also localized to the membrane as determined by immunofluorescence staining, which required permeabilization of fixed cells, indicating that AMER1 associates with the cytoplasmic side of the plasma membrane (data not shown). Similarly, EGFP- or Flag-tagged AMER2 localized to the plasma membrane of MCF-7 cells (Fig. 3A, and data not shown). AMER proteins were enriched in but not restricted to lateral membranes. AMER1 fragments comprising amino acids 2-601 and 2-209 associated with the plasma membrane similar to full-length AMER1 (Fig. 3A, upper panel), whereas deletion fragments of AMER1 lacking the N-terminus, such as AMER1(207-839), did not localize to the plasma membrane but were distributed throughout the cell. Thus, the N-terminal domain of AMER1, containing amino acids 2-209, is necessary and sufficient for the membrane localization of this protein.

Importantly, both AMER1 and AMER1(short), as well as AMER2, recruited co-expressed APC from filamentous structures, which probably represented microtubules (Munemitsu et al., 1994; Smith et al., 1994), to the plasma membrane (Fig. 3A, lower panels and data not shown). AMER1-mediated membrane recruitment of APC required the APC-binding sites and the membrane docking N-terminus of AMER1, because the mutants AMER1(2-209) and AMER1(207-839), which lack these domains, respectively, did not recruit APC to the plasma membrane. Interestingly, in AMER1(2-209)-expressing cells, APC was frequently associated with the tips of microtubules, in contrast to filamentous staining in control cells. The C-terminal half of AMER1 was not required for APC recruitment, as demonstrated by AMER1(2-601) (Fig. 3A). Both AMER1 and AMER1(short) also recruited the ARM repeat domain of APC alone (APC-ARM) to the plasma membrane. The N507K substitution of APC-ARM barely reduced the recruitment by AMER1 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.

Fig. 2.

Interaction between AMER1 and APC. (A) Co-immunoprecipitation of wild-type APC (APC) and endogenous mutant APC (APCmut) with Flag-tagged AMER1 after transient transfections of SW480 cells, as indicated. Western blottings were performed using anti-APC Ab1 (for APCmut) and Ab2 (for APC), and anti-Flag antibodies. The double band for Flag-AMER1 is observed in some but not all experiments (cf. B) and might result from incomplete denaturation of the protein in the gel sample buffer. (B) Co-immunoprecipitation of endogenous AMER1 and Flag-tagged AMER1 with EGFP-tagged APC-ARM after transient transfections of 293T cells, as indicated. Western blottings were performed using anti-AMER1 or anti-GFP antibodies. (C) Co-immunoprecipitation of endogenous AMER1 with APC from lysates of nontransfected 293T and SW480 cells. Immunoprecipitations were performed with anti-GFP antibody as a control or with anti-APC antibody Ali followed by western blotting using the anti-AMER1 antibody or Ali. (D) Co-immunoprecipitation of EGFP-tagged APC-ARM or APC-ARMN507K with Flag-tagged AMER1 or AMER1(short) after transient transfections of 293T cells as indicated. Western blottings were performed using anti-GFP or anti-Flag antibodies. (E) Co-immunoprecipitation of Flag-tagged APC-ARM with EGFP-tagged AMER1 and AMER1 deletion fragments after transient transfections of 293T cells as indicated. Western blottings were performed using anti-Flag or anti-GFP antibodies. The bottom blot shows levels of the Flag-FKBP8 control protein in lysates of 293T cells after co-expression with the indicated AMER1 constructs. The scheme below shows the structure of the deletion mutants of AMER1 and quantification from separate experiments of the fold change of APC-ARM or FKBP8 protein levels (as a control) after co-transfection with the 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 different siRNA oligonucleotides against AMER1 (siAMER1a, c). The numbers below the lanes indicate relative protein levels normalized to Pan-cadherin, with siGFP controls set to 100%.

Fig. 2.

Interaction between AMER1 and APC. (A) Co-immunoprecipitation of wild-type APC (APC) and endogenous mutant APC (APCmut) with Flag-tagged AMER1 after transient transfections of SW480 cells, as indicated. Western blottings were performed using anti-APC Ab1 (for APCmut) and Ab2 (for APC), and anti-Flag antibodies. The double band for Flag-AMER1 is observed in some but not all experiments (cf. B) and might result from incomplete denaturation of the protein in the gel sample buffer. (B) Co-immunoprecipitation of endogenous AMER1 and Flag-tagged AMER1 with EGFP-tagged APC-ARM after transient transfections of 293T cells, as indicated. Western blottings were performed using anti-AMER1 or anti-GFP antibodies. (C) Co-immunoprecipitation of endogenous AMER1 with APC from lysates of nontransfected 293T and SW480 cells. Immunoprecipitations were performed with anti-GFP antibody as a control or with anti-APC antibody Ali followed by western blotting using the anti-AMER1 antibody or Ali. (D) Co-immunoprecipitation of EGFP-tagged APC-ARM or APC-ARMN507K with Flag-tagged AMER1 or AMER1(short) after transient transfections of 293T cells as indicated. Western blottings were performed using anti-GFP or anti-Flag antibodies. (E) Co-immunoprecipitation of Flag-tagged APC-ARM with EGFP-tagged AMER1 and AMER1 deletion fragments after transient transfections of 293T cells as indicated. Western blottings were performed using anti-Flag or anti-GFP antibodies. The bottom blot shows levels of the Flag-FKBP8 control protein in lysates of 293T cells after co-expression with the indicated AMER1 constructs. The scheme below shows the structure of the deletion mutants of AMER1 and quantification from separate experiments of the fold change of APC-ARM or FKBP8 protein levels (as a control) after co-transfection with the 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 different siRNA oligonucleotides against AMER1 (siAMER1a, c). The numbers below the lanes indicate relative protein levels normalized to Pan-cadherin, with siGFP controls set to 100%.

Because AMER1 lacks any obvious membrane-anchoring domains, we next investigated whether it would bind directly to membrane lipids. Recombinant GST-tagged AMER1(2-285), but not GST-AMER1(260-545), bound to phosphatidylinositol mono-, bis- and tris-phosphate species, and to 3-sulfogalactosylceramide (sulfatide) in vitro (Fig. 3B). Interestingly, GST-AMER1 fragments comprising amino acids 2-142 or 143-209 also bound to membrane lipids, indicating that AMER1 contains two lipid-binding sites (Fig. 3B). Phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] is the predominant phosphoinositide at the plasma membrane (Di Paolo and De Camilli, 2006) and therefore is a good candidate for a physiological membrane link for AMER1. We analyzed whether cleavage of PtdIns(4,5)P2 by phospholipase C (PLC) affects AMER1 localization in cells. Membrane localization of ectopically expressed full-length AMER1 and APC, and of the deletion fragments AMER1(2-142) and AMER1(143-209), in MCF-7 cells was completely abolished upon treatment with ionomycin, which induces Ca2+ influx and thereby activates PLC (Varnai and Balla, 1998) (Fig. 3Cb,b′,g,j). AMER1 and APC localized again to the plasma membrane when Ca2+ was chelated with EGTA after ionomycin treatment (Fig. 3Cc,c′,h,k). Relocalization at the membrane was prevented by treatment of cells with wortmannin, which, at high concentrations, 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 of AMER1 from the plasma membrane (Fig. 3Ce). These results suggest that AMER1 localizes to the plasma membrane by direct interaction with PtdIns(4,5)P2 via two distinct binding domains at the N-terminus.

Fig. 3.

AMER1 localizes to the membrane via binding to PtdIns(4,5)P2. (A) Double staining of EGFP, EGFP-AMER1, EGFP-AMER1 deletion constructs as in Fig. 2E or EGFP-AMER2 (upper panels, GFP fluorescence), and APC (lower panels, antibody Ali immunofluorescence) in MCF-7 cells transiently transfected as indicated above the panels. Arrowheads denote the membrane, and arrows the filamentous localizations. (B) Membrane lipid-binding assays of AMER1 deletion mutants. Membrane lipid strips were incubated with the indicated recombinant GST-AMER1 fusion proteins, revealing two phosphoinositide binding sites. DAG, diacylglycerol; PA, phosphatidic acid; PS/PE/PC/PG, phosphatidyl-serine/-ethanolamine/-choline/-glycerol. (C) Localization of AMER1 (a-e) and APC (a′-c′) or AMER1 deletion mutants (f-k) in transiently transfected MCF7 cells. 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 ionomycin treatment (Iono), or with ionomycin treatment followed by EGTA treatment (c,c′). Staining of EGFP-AMER1 in cells treated with wortmannin (Wm) prior to ionomycin/EGTA treatment (d) or with neomycin (Neo) prior to ionomycin (e). Staining of EGFP-AMER1(2-142) (f-h) or EGFP-AMER1(143-209) (i-k) in cells treated with ionomycin/EGTA as indicated.

Fig. 3.

AMER1 localizes to the membrane via binding to PtdIns(4,5)P2. (A) Double staining of EGFP, EGFP-AMER1, EGFP-AMER1 deletion constructs as in Fig. 2E or EGFP-AMER2 (upper panels, GFP fluorescence), and APC (lower panels, antibody Ali immunofluorescence) in MCF-7 cells transiently transfected as indicated above the panels. Arrowheads denote the membrane, and arrows the filamentous localizations. (B) Membrane lipid-binding assays of AMER1 deletion mutants. Membrane lipid strips were incubated with the indicated recombinant GST-AMER1 fusion proteins, revealing two phosphoinositide binding sites. DAG, diacylglycerol; PA, phosphatidic acid; PS/PE/PC/PG, phosphatidyl-serine/-ethanolamine/-choline/-glycerol. (C) Localization of AMER1 (a-e) and APC (a′-c′) or AMER1 deletion mutants (f-k) in transiently transfected MCF7 cells. 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 ionomycin treatment (Iono), or with ionomycin treatment followed by EGTA treatment (c,c′). Staining of EGFP-AMER1 in cells treated with wortmannin (Wm) prior to ionomycin/EGTA treatment (d) or with neomycin (Neo) prior to ionomycin (e). Staining of EGFP-AMER1(2-142) (f-h) or EGFP-AMER1(143-209) (i-k) in cells treated with ionomycin/EGTA as indicated.

Fig. 4.

AMER1 controls the distribution of APC between microtubules and the plasma membrane. (A) Localization of APC (a,b) in MDCK cells stably expressing EGFP (a,a′) or EGFP-tagged AMER1 (b,b′). (a,b) Immunofluorescence stainings using the anti-APC antibody Ali; (a′,b′) corresponding EGFP fluorescence. Notice the membrane association of EGFP-AMER1, which is not observed for EGFP. Arrowheads point to APC at cytoplasmic clusters at cellular protrusions in the EGFP transfectants (a) and to colocalization of AMER1 and APC at the plasma membrane in the EGFP-AMER1 transfectants (b,b′). Insets in upper panels represent higher magnifications. (B) Immunofluorescence staining of APC (anti-M-APC) in MDCK cells treated with solvent (DMSO), nocodazole (Noco), or nocodazole followed by ionomycin (Noco/Iono). Arrowheads indicate lateral plasma membranes. (C) Immunofluorescence staining of APC (anti-M-APC) in MCF-7 cells treated with siRNA against either GFP as a control (siGFP), AMER1 (siAMER1c), or AMER1 and APC (siAMER1c+siAPC). Arrowheads indicate tips of cellular protrusions. (D) Staining of transiently transfected APC (a-c), microtubules (a′,b′, `MT') or AMER1 (c′) in MCF-7 cells transiently transfected with APC together with siGFP (a,a′), siAMER1c (b,b′), or siAMER1c and the siAMER1c-insensitive EGFP-rAMER1 expression construct (c,c′). (a,a′;b,b′;c,c′) Double stainings. (a-c) Anti-M-APC immunofluorescence; (a′,b′) anti-α-tubulin immunofluorescence; (c′) GFP fluorescence. Broken lines indicate the edge of colonies.

Fig. 4.

AMER1 controls the distribution of APC between microtubules and the plasma membrane. (A) Localization of APC (a,b) in MDCK cells stably expressing EGFP (a,a′) or EGFP-tagged AMER1 (b,b′). (a,b) Immunofluorescence stainings using the anti-APC antibody Ali; (a′,b′) corresponding EGFP fluorescence. Notice the membrane association of EGFP-AMER1, which is not observed for EGFP. Arrowheads point to APC at cytoplasmic clusters at cellular protrusions in the EGFP transfectants (a) and to colocalization of AMER1 and APC at the plasma membrane in the EGFP-AMER1 transfectants (b,b′). Insets in upper panels represent higher magnifications. (B) Immunofluorescence staining of APC (anti-M-APC) in MDCK cells treated with solvent (DMSO), nocodazole (Noco), or nocodazole followed by ionomycin (Noco/Iono). Arrowheads indicate lateral plasma membranes. (C) Immunofluorescence staining of APC (anti-M-APC) in MCF-7 cells treated with siRNA against either GFP as a control (siGFP), AMER1 (siAMER1c), or AMER1 and APC (siAMER1c+siAPC). Arrowheads indicate tips of cellular protrusions. (D) Staining of transiently transfected APC (a-c), microtubules (a′,b′, `MT') or AMER1 (c′) in MCF-7 cells transiently transfected with APC together with siGFP (a,a′), siAMER1c (b,b′), or siAMER1c and the siAMER1c-insensitive EGFP-rAMER1 expression construct (c,c′). (a,a′;b,b′;c,c′) Double stainings. (a-c) Anti-M-APC immunofluorescence; (a′,b′) anti-α-tubulin immunofluorescence; (c′) GFP fluorescence. Broken lines indicate the edge of colonies.

AMER1 controls the distribution of APC between microtubules and the plasma membrane

In MDCK clones stably expressing AMER1, endogenous APC delocalized from focal cellular protrusions that had been previously characterized as microtubule ends (Nathke et al., 1996; Rosin-Arbesfeld et al., 2001) and became recruited to the plasma membrane (Fig. 4Aa,b). As has been shown previously, disruption of microtubules by nocodazole treatment resulted in membrane association of endogenous APC in MDCK cells [Fig. 4B (cf. Rosin-Arbesfeld et al., 2001)]. Treatment with ionomycin, which blocks AMER1 membrane association (cf. Fig. 3C), prevented the nocodazole-induced membrane localization of APC (Fig. 4B), which is in line with APC being linked to the membrane by endogenous AMER1 in these cells.

Next, we analyzed whether loss of endogenous AMER1 in MCF-7 cells by RNAi would promote microtubule association of APC. Transient transfection of the siAMER1c oligonucleotide in MCF-7 cells resulted in an overall downregulation of AMER1 mRNA by about 30%, which corresponds to the transfection efficiency obtained in these cells (supplementary material Fig. S5A). After transient knockdown of AMER1 with siAMER1c, 13.2% (n=657) of cells at the edges of colonies showed staining of APC in tips of cellular protrusions, often corresponding to microtubule ends (Nathke et al., 1996). This staining was specific because it was abolished by concomitant treatment with siAPC (Fig. 4C, supplementary material Fig. S5B). In cells treated with control siRNA (siGFP), only 3.9% of cells (n=751) showed such tip staining. Alterations in the membrane association of APC could not be reliably determined, because the membrane staining with the anti-M-APC serum appeared to be nonspecific because it was not affected by siRNA against APC, in contrast to APC staining at the protrusions. Transiently transfected APC localized to filamentous structures partially overlapping with microtubules in most of the siGFP-treated MCF-7 control cells (Fig. 4Da,a′ and Fig. 3A). In 25.5% of transfected cells at the edge of colonies, APC was found at the tips of cellular protrusions (n=106). This localization was markedly increased to 56.8% of the cells in siAMER1c-treated cultures (Fig. 4D,b; n=111). Moreover, in these structures APC overlapped with the ends of microtubules, which were frequently reoriented perpendicular to the membrane indicating a rearrangement of the microtubular network (Fig. 4Db′). To verify that tip localization of APC was caused by the specific depletion of AMER1, we additionally expressed a mutated AMER1 cDNA resistant to knockdown by siAMER1c (rAMER1, supplementary material Fig. S5C,D). Tip localization of APC was completely abolished in rAMER1-expressing cells (n=30), and APC was exclusively observed at the cell membrane co-localizing with rAMER1 (Fig. 4Dc,c′). These data demonstrate that silencing of endogenous AMER1 promotes association of APC with the tips of cellular protrusions, in which it overlaps with microtubule ends.

AMER1 controls intercellular junctions together with APC

MCF-7 cells are epithelial and show the classical cobblestone pattern when stained for E-cadherin or β-catenin (Fig. 5A). When AMER1 was transiently knocked down by transfection with the siAMER1c oligonucleotide, the cell junctions of MCF-7 cells were frequently disrupted, resulting in `gaps' between cells (Fig. 5A,B). This was most apparent at sites at which multiple cells met (e.g. at tricellular junctions). Interestingly, membrane association of E-cadherin, β-catenin and other junctional proteins was still preserved (Fig. 5A and data not shown). The additional knockdown of APC led to a more severe disruption of cellular junctions, with cells frequently showing complete detachment from each other (Fig. 5A,B), whereas knockdown of APC alone had only a minor effect (Fig. 5B). These data indicate a role for AMER1 in maintaining the integrity of intercellular junctions, probably by mediating the membrane localization of APC.

Discussion

In this paper, we provide a novel molecular mechanism by which APC can be recruited to the plasma membrane by interacting with the AMER1 and AMER2 proteins. When overexpressed, AMER proteins recruited both exogenous and endogenous APC to the plasma membrane and prevented its interaction with microtubules. Depletion of PtdIns(4,5)P2 by ionomycin treatment, which abolishes AMER1 localization at plasma membranes, also reduced the association of APC with the membrane. Endogenous AMER1-APC complexes were identified, and knockdown of AMER1 increased the fraction of APC associated with microtubule ends. Finally, a point mutation, which was shown to reduce membrane association of Drosophila E-APC, in the ARM repeat domain also diminished the AMER1-APC interaction.

Fig. 5.

AMER1 controls intercellular junctions together with APC. (A) Immunofluorescence staining of E-cadherin in MCF-7 cells treated with a control siRNA against GFP (siGFP), siRNA against AMER1 (siAMER1c), or siAMER1c together with an siRNA against APC (siAMER1c+siAPC). Arrowheads indicate disrupted cell junctions. (B) Relative number of gaps between MCF-7 cells transfected with siRNA oligonucleotides as indicated below the bars. Gaps were counted in E-cadherin- and β-catenin-stained samples in 20 optical fields using the 40× objective. Results show the mean±s.d. of at least two independent experiments.

Fig. 5.

AMER1 controls intercellular junctions together with APC. (A) Immunofluorescence staining of E-cadherin in MCF-7 cells treated with a control siRNA against GFP (siGFP), siRNA against AMER1 (siAMER1c), or siAMER1c together with an siRNA against APC (siAMER1c+siAPC). Arrowheads indicate disrupted cell junctions. (B) Relative number of gaps between MCF-7 cells transfected with siRNA oligonucleotides as indicated below the bars. Gaps were counted in E-cadherin- and β-catenin-stained samples in 20 optical fields using the 40× objective. Results show the mean±s.d. of at least two independent experiments.

AMER1 was localized at the plasma membrane, and the N-terminus of AMER1 is necessary and sufficient for this localization. We could identify two distinct sites (amino acids 2-142 and 143-209) that mediate membrane association and directly bind PtdIns(4,5)P2. These sites have high isoelectric points (pI=9.3 and 10.55, respectively; ProtParam) compared with full-length AMER1 (pI=4.77), contain highly conserved basic and aromatic residues and are particularly enriched in lysine residues (11.3 and 14.9%, respectively, compared with 4.1% in full-length AMER1; supplementary material Fig. S2B). Basic and aromatic amino acids have been shown to mediate PtdIns(4,5)P2 binding in other proteins (Kagan and Medzhitov, 2006). The majority of these lysine residues are conserved in AMER2 (supplementary material Fig. S4), which can also interact with the plasma membrane. We could further show that activation of phospholipase C by ionomycin-induced Ca2+ influx leads to the delocalization of AMER1 and APC from the plasma membrane, probably because of PtdIns(4,5)P2 cleavage. These data strongly support a role for PtdIns(4,5)P2 as the physiological membrane anchor for AMER1. AMER1 was not restricted to a specific membrane compartment in the cell lines used in this study, which is in line with its association with membrane lipids. However, local PtdIns(4,5)P2 concentrations are regulated by signaling events and are significantly enriched at the apical plasma membrane in polarized epithelial cells (Martin-Belmonte et al., 2007; McLaughlin et al., 2002). Such changes might influence AMER1 localization and thus control the subcellular distribution of APC.

AMER1 directly interacted with the ARM repeat domain of APC via three separate binding sites, which were identified in our initial yeast two-hybrid screen. The sites interact with similar efficiency with APC yet do not share any obvious sequence similarity. Other known interaction partners of the ARM repeat domain of APC, such as Asef, KAP3 (KIFAP3) and IQGAP1, also show no sequence similarity to each other or to AMER1, indicating that the ARM repeats can contact various partners using different binding modes. This is similar to β-catenin, in which the ARM repeats can interact with various interaction partners using distinct amino acids (von Kries et al., 2000). It is possible that the presence of multiple APC-binding domains in AMER1 increases the overall affinity of the interaction. Moreover, the differences in sequence between these domains might allow for individual modulation of APC binding by biochemical modifications or competing interactions. Interestingly, the third APC-interaction domain of AMER1 retained significant capacity to interact with the N507K mutant of the ARM repeat domain in the yeast two-hybrid assays. In AMER1(short), this domain is largely missing and membrane recruitment of the N507K mutant by AMER1(short) was reduced, whereas it was largely preserved in AMER1, at least under conditions of overexpression. This indicates that, depending on the expression of the AMER1 isoform, APC might be differentially recruited to the plasma membrane.

Two main cellular functions have been assigned to peripheral APC. First, several reports have indicated a role for APC in the polarization of migrating cells via its association with 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 treatment increased the fraction of cells harboring APC at microtubules in cellular protrusions, which was reverted by expression of an siRNA-resistant cDNA of AMER1. Plasma membrane recruitment of APC by AMER1 might therefore block cell polarization and counteract cell migration. Second, it was suggested that plasma membrane association of APC controls cell-cell adhesion and adherens junction formation (Faux et al., 2004; Hamada and Bienz, 2002). Mutations within the ARM repeat domain leading to reduced membrane association of Drosophila E-APC results in the disruption of cell junctions (Hamada and Bienz, 2002). However, this might result from a dominant action of the mutants, because null mutations did not affect 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 the cytoskeleton and thereby affect cellular junctions (Watanabe et al., 2004). We observed disturbance of the integrity of cell junctions in MCF-7 cells treated with the siAMER1c oligonucleotide, which could result from diminished membrane localization of APC. Interestingly, the effect was predominantly seen at vertices in which more than two cells contact each other. Possibly, adherens junctions at these locations are more vulnerable than at the lateral domains of two neighboring cells. AMER1 might compete with cytoskeletal regulators, such as Asef (Kawasaki et al., 2003), for binding to APC, and loss of AMER1 would allow signaling by these APC complexes, leading to the observed effects. Alternatively, localization of APC at the plasma membrane might be directly required for cell junction formation (e.g. by promoting β-catenin recruitment to cadherins), as was suggested previously (Hamada and Bienz, 2002). Because both E-cadherin and β-catenin membrane localization were not strongly altered in siAMER1-treated cells, such a function is rather unlikely in our experimental setting. The fact that simultaneous knockdown of APC aggravated the junctional defects of the AMER1 knockdown suggests a direct and positive role of both proteins in junction formation and/or maintenance. The identification of AMER1 as a plasma membrane link of APC will allow us to analyze more specifically the function of this important tumor suppressor in polarized cell migration and cell-cell adhesion.

After this paper had been submitted, a report described WTX as a negative regulator of the Wnt signaling pathway, which forms complexes with APC, Axin, β-catenin and β-TRCP (Major et al., 2007). WTX is identical to AMER1, indicating that the β-catenin destruction complex as a whole might become recruited to the plasma membrane, which might affect Wnt signaling. WTX/AMER1 is mutated in Wilms tumors, leading to truncations or deletions of the complete gene (Rivera et al., 2007). It was suggested that loss of WTX leads to aberrant activation of Wnt signaling and thereby promotes tumorigenesis (Major et al., 2007). From our data, these mutations might additionally contribute to tumorigenesis by abolishing functions of APC at the plasma membrane and activating those at microtubules.

Materials and Methods

DNA constructs and transfections

For EGFP-tagging, cDNAs were inserted into pEGFP-C3 (Clontech); for GST-tagging they were inserted into pGEX-4T3 (Amersham Pharmacia Biotech); and for Flag-tagging into pcDNA-Flag, which was derived from pcDNA3.1 (Invitrogen) by inserting the Flag peptide coding sequence into the multiple cloning site. APC cDNA fragments were obtained by PCR amplification using pCMV-APC (Smith et al., 1994) as a template, which was also used for expression of human full-length APC. The N507K mutant of APC-ARM was created by PCR mutagenesis. Full-length human AMER1 was cloned by replacing the 3′ coding sequence of clone FLJ39827 (NITE Biological Resource Center, Chiba, Japan) at nt 2073 with the corresponding coding sequence of AMER1 obtained by reverse transcriptase (RT)-PCR from mRNA of 293T cells (cf. supplementary material Fig. S1A). Deletion mutants of AMER1 were generated by restriction digests or PCR amplification. rAMER1 cDNA was generated by PCR mutagenesis exchanging three nucleotides of the targeting sequence of siAMER1c without changing the amino acid sequence. Full-length AMER2 transcript variant 1 was obtained by RT-PCR from mRNA of 293T cells. APC-binding sites #1 and #2 of AMER2 were obtained by RT-PCR from mRNA of Caki cells. Transient transfections of plasmids were performed using ESCORT IV (Sigma), and of siRNA using TransIT-TKO (Mirus, Madison, WI, USA).

siRNA oligonucleotides

The sequences of the siRNA oligonucleotides are: siAPC, 5′-GACGUUGCGAGAAGUUGGAdTdT-3′; siGFP, 5′-GCUACCUGUUCCAUGGCCAdTdT-3′ (Eurogentec, Seraing, Belgium). siAMER1a, siAMER1b and siAMER1c were purchased from Dharmacon (catalog numbers D-016075-01, D-016075-02 and D-016075-03).

Cell culture and drug treatments

Cells were cultured in 10% CO2 at 37°C in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. For stable expression of EGFP-AMER1 and EGFP, MDCK cells were transfected with pEGFP-AMER1 or pEGFP-C3, respectively, and subsequently selected in medium containing 1 mg/ml geneticin (G418, Invitrogen). For drug treatments, cells were incubated for 5 minutes at 37°C with 10 μM ionomycin (Calbiochem), followed by 10 minutes at 37°C with 2 mM EGTA in culture medium where indicated, or cells were incubated for 30 minutes at 37°C with 10 μM wortmannin, or 10 mM neomycin (Calbiochem) in culture medium prior to ionomycin treatment. For microtubule disruption, cells were incubated with 10 μg/ml nocodazole (Sigma) for 15 minutes on ice followed by 60 minutes at 37°C, or with solvent containing medium as controls.

Antibodies

The AMER1-specific monoclonal mouse antibody was raised against amino acids 2-285 of recombinant human AMER1 generated as a GST fusion in bacteria. The anti-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 was a gift of Frank Edlich and Gunter Fischer (Max-Planck Research Unit for Enzymology of Protein Folding, Halle, Germany). Commercial antibodies were obtained from Calbiochem (APC Ab-1 and Ab-2), Sigma (β-Actin: clone AC-15; rabbit anti-Flag polyclonal antibody; mouse anti-GST monoclonal antibody; rabbit anti-Pan-cadherin serum), Roche (GFP, mixture of clones 7.1 and 13.1), Serotec (α-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 HRP conjugates for western blotting.

Yeast two-hybrid screen

Yeast two-hybrid and β-galactosidase assays were performed in the L40 yeast strain using pBTM116 as a bait vector and a mouse embryonic day 10.5 cDNA library in pVP16 as described previously (Behrens et al., 1998).

Preparation of protein lysates, immunoprecipitation and western blotting

Cells were washed three times with PBS and lysed in Triton X-100 buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT and 1 mM PMSF) at 4°C for 10 minutes. Lysates were cleared at 16,000 g for 10 minutes at 4°C. For co-immunoprecipitation, lysates were incubated for 4 hours at 4°C with the appropriate antibody and protein A/G-Sepharose beads (Santa Cruz Biotechnology), 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 the AIDA image analyzer software v. 3.52 (Raytest, Straubenhardt, Germany).

Lipid-binding assays

Expression of GST-AMER1 fragments in pGEX-4T3 was induced in Escherichia coli BL21 with 0.2 mM IPTG for 3 hours at 37°C. GST-AMER1 fusion proteins were freshly purified before the experiments using glutathione Sepharose 4B beads (Amersham) as described previously (Frangioni and Neel, 1993). Beads were washed three times in ice-cold PBS, and GST-AMER1 fusion proteins were eluted with glutathione elution buffer (20 mM Tris-HCl, pH 7.5, 8 mM glutathione and 5 mM DTT) and quantified by SDS-PAGE/Coomassie staining. Membrane Lipid Strips (Echelon) were incubated with the GST fusion proteins at a concentration of 1 μg/ml at 4°C overnight and detected by a mouse anti-GST antibody, according to the manufacturer's instructions.

Immunofluorescence microscopy

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

Acknowledgements

We thank Thomas Winkler for help in antibody generation, Inke Näthke for providing anti-APC antibodies, Eva Kohler for the FKBP8 expression construct and Angela Döbler for secretarial assistance. This work was supported by a grant of the Marohn Stiftung of the University of Erlangen-Nuremberg.

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Supplementary information