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The PDZ Protein MPP2 Interacts with C-Src in Epithelial Cells

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The PDZ Protein MPP2 Interacts with C-Src in Epithelial Cells Author's personal copy EXPERIMENTAL CELL RESEARCH 315 (2009) 2888 – 2 8 9 8 available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article The PDZ protein MPP2 interacts with c-Src in epithelial cells Martin Baumgartnera,⁎,1, Andreas Weissa,2,3, Thorsten Fritziusa,3,4, Jochen Heinricha, Karin Moellinga,b aInstitute of Medical Virology, University of Zürich, Zürich, Switzerland bInstitute of Advanced Study, Berlin, Germany ARTICLEINFORMATIONABSTRACT Article Chronology: c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and Received 7 August 2008 cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and Revised version received 21 July 2009 through protein–protein interactions. The interaction of c-Src with PDZ proteins was recently Accepted 31 July 2009 identified as novel mechanism to restrict c-Src function. The objective of this study was to identify Available online 6 August 2009 and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Keywords: Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) PDZ family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of Dlg MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not MAGUK known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 Src associated with the cytoskeleton in filamentous structures, which partially co-localised with Breast cancer cells microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted Cytoskeleton increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively Phosphorylation regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function. © 2009 Elsevier Inc. All rights reserved. ⁎ Corresponding author. Department of Clinical Research and Veterinary Public Health, Division of Molecular Pathobiology, University of Bern, Bern, Switzerland. Fax: +41 31 631 26 58. E-mail address: [email protected] (M. Baumgartner). Abbreviations: Dlg, Discs large; L27, Lin-2/Lin-7 domain; FERM, (four point 1, ezrin, radxin, moesin); IF, Immunofluorescence; MAGUK, Membrane- Associated Guanylate Kinase; MPP2, Membrane Protein Palmitoylated 2; PDZ, Postsynaptic density-95/Discs large 1/Zonula Occludens-1; SH, Src homology; Wb, Western blot 1 Current address: Department of Clinical Research and Veterinary Public Health, Division of Molecular Pathobiology, University of Bern, Bern, Switzerland. 2 Current address: Oncology Drug Discovery, Novartis Institutes for BioMedical Research, Basel, Switzerland. 3 These authors contributed equally. 4 Current address: Department of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, Basel, Switzerland. 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.07.028 Author's personal copy EXPERIMENTAL CELL RESEARCH 315 (2009) 2888 – 2 8 9 8 2889 DME medium (Gibco) containing 10% foetal calf serum (FCS). Introduction Starvation medium was DME medium without FCS. Chicken c-Src amino acid numbering is used throughout this article. c-Src regulates a wide range of cellular functions including cell proliferation and cell migration [1,2]. Proper regulation of Src kinases Constructs and expression vectors is important and the C-terminal deletion in the viral oncogene v-Src of the Rous Sarcoma Virus leads to deregulated Src activity and Human MPP2 isoform 2 was obtained as cDNA clone (RZPD ID: oncogenic transformation [1,2]. Deregulation of c-Src is believed to IRAKp961N0662Q2, Germany) and subcloned into pFLAG-CMV-2 play an important role in human tumourigenesis by promoting (SIGMA). Truncated MPP2 constructs were generated by PCR and proliferation, survival and migration of tumour cells [3]. The subcloned into the EcoRI and SalI sites in pFLAG-CMV-2. Single regulation of c-Src is complex and involves the reversible phosphor- point mutations were introduced by site directed mutagenesis ylation of Y416 and Y527 as well as protein–protein interactions (Stratagene). The TAT codon coding for Y was replaced with GCT through its SH2 and SH3 domains [4]. Elimination of the PSD95/Dlg/ (Ala) or GAT (Asp), respectively and the sequences were verified ZO-1 (PDZ) ligand sequence in c-Src increases the potential of c-Src by sequencing. HA-tagged c-Src constructs are described else- to promote an invasive phenotype in epithelial cells [5]. With the where [5,6]. For all overexpression experiments, FLAG-tagged adherens junction PDZ protein AF-6 one candidate protein has been MPP2 and HA-tagged Src proteins were used. identified to inhibit c-Src function [6], but additional PDZ proteins are likely implicated in regulating c-Src function. PDZ array Scaffolding proteins of the Membrane-Associated Guanylate Kinase (MAGUK) family tether adhesion molecules, receptors and TranSignal PDZ arrays I–IV (Panomics) were screened for components of intracellular signalling pathways to spatially organise interaction with Src by incubating the arrays with cell extracts multi-component signalling complexes [7]. MAGUKs contain a containing overexpressed HA-c-Src as described recently [10]. catalytically inactive guanylate kinase (GuK) domain, a Src-homol- ogy 3 (SH3) domain and one or several PSD95/Dlg/ZO-1 (PDZ) Cell transfection domains. Membrane Protein Palmitoylated 2 (MPP2) has previously been isolated in a screen for cDNAs subjected to allelic losses on HEK293 and COS-7 cells were transfected using JetPEI (Polyplus) chromosome 17q12–q21, a genomic region frequently altered in according to the manufacturer's instructions. 24 h after transfec- breast cancer cells[8]. MPP2 has two L27 domains at its N-terminus in tion, growth medium containing 10% FCS was replaced with fresh addition to the conserved PDZ-SH3-GuK module. MPP2 is structurally growth medium or with starvation medium without FCS. 48 h after similar to Discs large (Dlg), one of the founding and well- transfection, cells were lysed and lysates were processed for characterised members of the MAGUK family [9]. MAGUK proteins immunoprecipitation analysis. Alternatively, cells were fraction- including Dlg are considered to act as tumour suppressor proteins in ated as described below. Where indicated, EGF was used at a vertebrates because of their essential structural and regulatory role in concentration of 50 ng/ml. tissue organisation and maintenance and in cell signalling. Here we identified the interaction between the MAGUK protein Cell lysis, Western blot (Wb) and antibodies MPP2 and c-Src. Localisation and function of MPP2 in cells are unknown. We investigated the function of MPP2 and its interac- Cells were lysed in modified radio-immunoprecipitation assay tion with c-Src. We characterised the localisation of endogenous (RIPA) buffer containing 150 mM NaCl, 50 mM Tris (pH7.4), 1 mM MPP2 in human breast epithelial cells and of overexpressed MPP2 EDTA (pH 8.0), 1.0% NP40, 0.25% Na-Deoxycholate, 2 mM Na- in HEK293 cells. We detected MPP2 in the cytoskeleton, where it Vanadate, 25 mM NaF and protease inhibitor cocktail (Roche). Wb partially co-localised with microtubules and c-Src. The c-Src kinase was carried out using standard procedures. Cellular fractionations activity allowed MPP2 phosphorylation and increased the inter- were performed either by using FractionPREP (BioVISION, http:// action between MPP2 and c-Src. Conversely, MPP2 negatively www.biovision.com/) according to the manufacturer's instructions regulated c-Src kinase activity and prevented disorganisation of or by ultracentrifugation as described recently [5]. Antibodies used the actin cytoskeleton in lamellipodia of cells overexpressing c-Src. were the following: mouse mc anti-Src clone GD11 and anti- Our data thus indicate that MPP2 and c-Src constitute a protein phospho-Y 4G10 (Upstate); anti-Na+/K+ ATPase (468.8) (Abcam); complex to restrict c-Src activity and function in cells. rabbit pc anti-Src2, anti-focal adhesion kinases C-20, anti-cortactin H-191 and anti-Ephrin-B1 C-18 (Santa Cruz); anti-phospho-Src family (pY416) (Cell Signaling Technology). MPP2 is a custom-made Materials and methods (Eurogentec) rabbit polyclonal antibody raised against a 15-mer peptide in the C-terminus of human MPP2 and was used at a Cells concentration of 250 ng/ml in Wbs and 2.5 μg/ml for IF. The MCF-10A cells were obtained from the American Type Culture Immunofluorescence microscopy Collection and grown in complete growth medium (DMEM/F12 (Gibco) with 2 mM L-glutamine supplemented with 20 ng/ml Cells were fixed/permeabilised in 100% MetOH at −20 °C for epidermal growth factor (EGF, Sigma), 100 ng/ml cholera toxin 3 min, treated with 0.5% TX-100 for 5 min and blocked with 10% (Sigma), 10 ng/ml insulin (Sigma), 500 ng/ml hydrocortisone FCS in PBS. Primary antibodies were used at 1/200 dilution in 5% (Sigma) and 5% horse serum). Starvation medium is DME-F12 FCS in PBS. Secondary anti-mouse or anti-rabbit antibodies
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