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First published online 9 October 2007
doi: 10.1242/jcs.011320

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AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane

Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nuremberg, Glückstr. 6, 91054 Erlangen, Germany

View larger version (38K): [in this window] [in a new window]   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 -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 -galactosidase units of representative experiments. Binding sites #1 and 2 produced similar high -galactosidase values upon interaction with APC-ARM when tested in the DNA-binding-domain vector (not shown).

View larger version (36K): [in this window] [in a new window]   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%.

View larger version (54K): [in this window] [in a new window]   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.

View larger version (71K): [in this window] [in a new window]   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--tubulin immunofluorescence; (c') GFP fluorescence. Broken lines indicate the edge of colonies.

View larger version (48K): [in this window] [in a new window]   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 -catenin-stained samples in 20 optical fields using the 40x objective. Results show the mean±s.d. of at least two independent experiments.