SUPPLEMENTARY INFORMATION “Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation” Kristina Tanneberger, Astrid S. Pfister, Katharina Brauburger, Jean Schneikert, Michel V. Hadjihannas, Vitezslav Kriz, Gunnar Schulte, Vitezslav Bryja and Jürgen Behrens SUPPLEMENTARY FIGURES Supplementary Figure S1 (A, B) Amer1 is required for phosphorylation of endogenous LRP6 at Ser1490 and Thr1479. Cells were transfected with the indicated siRNAs and incubated with Wnt3A for 1 hour. Membrane fractions from HEK293T cells (A) and whole cell lysates from SW480 cells (B) were analysed by Western blotting. (C) Overexpression of Amer1 promotes phosphorylation of endogenous LRP6 at Ser1490. HEK293T cells were transfected with EGFP-Amer1 and treated with Wnt3A for 20 minutes. Membrane fractions were analysed by Western blotting. 2 Supplementary Figure S2 (A) Sequence comparison of the two N-terminal PtdIns(4,5)P2 binding sites of Amer1 from different species and of Amer2 (Grohmann et al, 2007). Highly conserved basic and aromatic residues are highlighted in red. Red boxes indicate lysine residues that have been mutated to alanine in this study. (B) Phospholipid-binding assays of Amer1 mutants. Membrane lipid strips were incubated with the indicated GST-Amer1 fusion proteins revealing a severely diminished ability of Amer1(7µLys) to bind to phosphatidylinositol lipids. The Coomassie staining below shows relative amounts of purified proteins used. (C) Fluorescence micrographs of MCF-7 cells transfected with EGFP-tagged Amer1 constructs as indicated above the panels. Scale bar is 20 µm. 3 Supplementary Figure S3 (A) Schematic representation of Amer1 splice variants Amer1-S1 and Amer1-S2 (Jenkins et al, 2009). In Amer1-S2 amino acids 50-326 are deleted in frame. The localization of target sequences of Amer1 siRNAs is depicted. siAmer1a targets both Amer1 splice variants while siAmer1-S1 and siAmer1-S2 specifically target Amer1-S1 and Amer1-S2, respectively. The two N-terminal membrane localization domains (M1, M2) are highlighted by light grey and the three APC interaction domains (A1-3) by dark grey shading (Grohmann et al, 2007). (B) Fluorescence micrographs of MCF-7 cells transfected with EGFP-tagged Amer1 splice variants as indicated above the panels. Scale bar is 20 µm. (C) Effect of Amer1 splice variants on LRP6 phosphorylation when overexpressed in HEK293T cells stably expressing VSVGLRP6. (D) Specific knockdown of Amer1 splice variants after transient transfection of Flag-tagged Amer1 together with the indicated siRNAs into HEK293T cells. 4 Supplementary Figure S4 Knockdown of PI4KIIα prevents Wnt-induced plasma membrane recruitment of Amer1. HEK293T cells were transfected with two different siRNAs, incubated with Wnt3A for 1 hour and membrane fractions were analysed by Western blotting. AntiPan-Cadherin Western blot is shown as loading control for membrane fractions. 5 Supplementary Figure S5 (A) Co-immunoprecipitation of endogenous Axin and Amer1 from lysates of SW480 cells. Immunoprecipitations were performed with mouse anti-Amer1 or control IgG antibodies. (B, C) Amer1 leads to plasma membrane recruitment of endogenous Axin (B) or Conductin (C). Amer1 mutants Amer1(7µLys) and NES-Amer1(7µLys), which are defective in membrane localization, are not able to recruit Axin/Conductin. (B) HEK293T cells were transfected with the indicated EGFP-Amer1 constructs and membrane fractions were analysed by Western blotting. VSVG-LRP6 stably expressed in these cells is shown as loading control for membrane fractions. (C) Double staining of EGFP-tagged Amer1 and the indicated Amer1 mutants (left panels, GFP fluorescence) and Conductin (middle panels, anti-Conductin immunofluorescence) in SW480 cells. Scale bar is 20 µm. 6 Supplementary Figure S6 (A, B) Co-immunoprecipitation of Flag-tagged Axin (A) or Conductin (B) with EGFPtagged Amer1 or Amer1 deletion mutants after immunoprecipitation with anti-GFP antibodies in HEK293T cells. (C) Schematic representation of Amer1 deletion mutants and their ability to bind to the indicated interaction partners. For the Amer1 scheme see Supplementary Figure S3A. 7 Supplementary Figure S7 8 (A) Structure of the LRP6 intracellular domain highlighting the five PPPSPxS motifs (labelled A-E) and adjacent Ser/Thr clusters, which are represented by the grey and red boxes, respectively. The sequence of PPPSPxS motif A and the two flanking CK1 clusters (cluster 1 and 2) is shown above. Red amino acids represent CK1 phosphorylation sites. Residues Thr1479 and Ser1490 detected by phospho-specific antibodies are indicated. Schematic representation of LRP6 deletion mutants and their ability to bind to Amer1 as shown in (B) is indicated. LRP6 mutants have been described in Davidson et al, 2005. Scheme adapted from Davidson et al, 2009. (B) Co-immunoprecipitation of Flag-tagged LRP6 mutants as detailed in (A) with EGFPtagged Amer1 after immunoprecipitation with anti-GFP antibodies in HEK293T cells. (C) Overexpression of Axin does not promote the interaction between Amer1 and LRP6. HEK293T cells stably expressing VSVG-LRP6 were transfected with FlagAmer1 with or without YFP-Axin and anti-Flag immunprecipitations were performed. Supplementary Figure S8 (A, B) Amer1 interacts with CK1γ. (A) Flag-Amer1 co-immunoprecipitated with EYFPCK1γ, but not with a CK1γ mutant lacking the C-terminal membrane association motif (EYFP-CK1γΔC). (B) Co-immunoprecipitation of Flag-CK1γ with EGFP-Amer1 after immunoprecipitation with anti-GFP antibodies in HEK293T cells. 9 Supplementary Figure S9 Fluorescence micrographs of MCF-7 cells expressing the indicated EGFP-tagged fusion constructs. For the Amer1 scheme see Supplementary Figure S3A. Scale bar is 20 µm. Supplementary Figure S10 Knockdown of APC does not affect LRP6 phosphorylation at Ser1490. HEK293 cells stably expressing either an APC shRNA (293iAPC) or control shRNA (293control) (Schneikert and Behrens, 2006) were incubated with Wnt3A for 1 hour. Membrane fractions (M) and whole cell lysates (WCL) were analysed by Western blotting. 10 Supplementary Figure S11 Overexpression of Amer1 represses a TCF/β-catenin dependent transcriptional reporter. HEK293T cells stably expressing a β-catenin responsive firefly luciferase (Major et al, 2007) were transfected with RFP-Wnt3A, RFP-daLRP6 and EGFPAmer1 as indicated. Fold changes of luciferase activity are presented relative to control transfected cell. Error bars indicate standard deviations. 11 SUPPLEMENTARY MATERIALS AND METHODS Cell culture and transfection All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (PAA Laboratories, Pasching, Austria) at 37°C in a humidified atmosphere of 10% CO2. Plasmid transfections were performed using either polyethylenimin (Sigma) for HEK293T and SW480 cells or TransIT-TKO (Mirus, Madison, WI, USA) for MCF-7 cells. siRNAs were transfected using Oligofectamine (Invitrogen) according to the manufacturer’s instructions. Plasmids and siRNAs The following plasmids have been described previously: pEGFP-Amer1, pcDNAFlag-Amer1 (Grohmann et al, 2007); mRFP-daLRP6, mRFP-Wnt3A, mYFP-Axin, mYFP-Conductin, mCFP-Axin, mCFP-Conductin, mCFP-Dvl2 (Krieghoff et al, 2006); pcDNA3.1-Flag, pcDNA-Flag-Conductin, GSK3β (Behrens et al, 1998). LRP6-EYFP, Fz8-EYFP, MESD, LRP6ΔE(1-4)Δ87, EYFP-CK1γ, EYFP-CK1γΔC, Flag-LRP6ΔE(1-4)Δ127, Flag-LRP6ΔE(1-4), Flag-LRP6ΔE(1-4)Δ162 and FlagFlag- LDLRΔN-miniC were kindly provided by C. Niehrs, VSVG-LRP6, VSVG-LRP6m10 and pcDNA-Flag-Axin L396Q by X. He and pcDNA-Flag-Axin by A. Kikuchi. Deletion mutants of Amer1 were generated by restriction digests or PCR amplification. The sequences of the siRNA oligonucleotides are: siGFP, 5’-GCTACCTGTTCCATGGCCA-3’; siLuc, 5’-CTTACGCTGAGTACTTCGA-3’; siAmer1a, 5’-GGGAGTACCCGTGAACAAA-3’; siAmer1b, 5’-CCTCTATGCCCAAGCCAAA-3’; siAmer1-S1, 5’- 12 CCACCAGCTACTGAGAAAA-3’; siAmer1-S2, 5’-GGCCCAGGTTGTGGTGACA-3’; siPI4KIIα, 5’-GGATCATTGCTGTCTTCAA-3’ (Pan et al, 2008); siPI4KIIα-2, 5’GGAAGAGGACCTATATGAA-3’ (Pan et al, 2008). All siRNA oligonucleotides were purchased from Eurogentec. Preparation of protein lysates, immunoprecipitation and Western blotting For cytoplasmic or whole cell lysates cells were lysed for 10 minutes at 4°C in hypotonic buffer (25 mM Tris-HCl, pH 8, 1 mM EDTA, 10 mM NaF, 1 mM DTT and 1 mM PMSF) or Triton-X-100 buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM DTT and 1 mM PMSF), respectively. Lysates were cleared at 16,000 g for 10 minutes. For immunoprecipitation, lysates were incubated for 4 hours at 4°C with the appropriate antibody and Protein A/G PLUS agarose beads (Santa Cruz Biotechnology Inc.) or with Anti-FLAG M2 affinity gel beads (Sigma). Immunoprecipitates were collected, washed four times with low salt NET buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 10 mM NaF) and eluted with SDS sample buffer. For Western blotting (Lustig et al, 2002) proteins were visualized with a luminoimager (LAS-3000, Fuji) using Enhanced Chemiluminescence reagent (Perkin Elmer) and quantified using the AIDA image analyser software v. 3.52 (Raytest, Straubenhardt, Germany). Antibodies The mouse monoclonal antibody against Conductin has been described before (Lustig et al, 2002). Rabbit anti-pT1479 and rabbit anti-PI4KIIα were kindly provided by C. Niehrs and P. De Camilli, respectively. Commercial antibodies were obtained from Abcam (mouse anti-LRP6 (1C10)), Cell Signaling (rabbit anti-Axin1 (C76H11), rabbit anti-GSK3β (27C10), rabbit anti-LRP6 (C5C7), rabbit anti-pLRP6 (Ser1490)), 13 R&D Systems (sheep anti-CK1γ), Roche (mouse anti-GFP, mixture of clones 7.1 and 13.1), Santa Cruz Biotechnology Inc. (rabbit anti-β-catenin (H102)), Serotec (rat antiα-tubulin, clone YL1/2) and Sigma (rabbit anti-Flag, mouse anti-Flag, mouse anti-GST, rabbit anti-Pan-Cadherin). Secondary antibodies coupled to horseradish peroxidase or Cy3 were purchased from Jackson ImmunoResearch. RT-PCR Total cellular RNA was isolated with the RNeasy mini kit (Qiagen) and possible genomic contaminations were removed by treatment with DNase I. Single stranded cDNA was synthesized from 1 µg total cellular RNA using the AffinityScript QPCR cDNA Synthesis Kit (Stratagene) according to the manufacturer’s instructions. 14 SUPPLEMENTARY REFERENCES Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D Birchmeier W (1998) Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 280: 596-599 Davidson G, Shen J, Huang YL, Su Y, Karaulanov E, Bartscherer K, Hassler C, Stannek P, Boutros M, Niehrs C (2009) Cell cycle control of wnt receptor activation. Dev Cell 17: 788-799 Davidson G, Wu W, Shen J, Bilic J, Fenger U, Stannek P, Glinka A, Niehrs C (2005) Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438: 867-872 Grohmann A, Tanneberger K, Alzner A, Schneikert J, Behrens J (2007) AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane. 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