The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein tyrosine phosphatases (RPTPs) receive cues from the extracellular environment and are well placed to influence cell signaling. However, the direct events downstream of these receptors have been challenging to resolve. We report here that the homophilic receptor PTPRK is stabilized at cell-cell contacts in epithelial cells. By combining interaction studies, quantitative tyrosine phosphoproteomics, proximity labeling and dephosphorylation assays we identify high confidence PTPRK substrates. PTPRK directly and selectively dephosphorylates at least five substrates, including Afadin, PARD3 and δ-catenin family members, which are all important cell-cell adhesion regulators. In line with this, loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics. Thus, identifying PTPRK substrates provides insight into its downstream signaling and a potential molecular explanation for its proposed tumor suppressor function.

[1]  M. Peifer,et al.  Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment , 2017, Development.

[2]  Samuel Bouyain,et al.  Receptor‐type tyrosine phosphatase ligands: looking for the needle in the haystack , 2013, The FEBS journal.

[3]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[4]  S. Knapp,et al.  The crystal structure of human receptor protein tyrosine phosphatase κ phosphatase domain 1 , 2006 .

[5]  N. Tonks,et al.  Protein tyrosine phosphatases: from genes, to function, to disease , 2006, Nature Reviews Molecular Cell Biology.

[6]  Sarah Edkins,et al.  Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease , 2011, Nature Genetics.

[7]  A. Whetton,et al.  SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1. , 2009, Molecular cell.

[8]  J. Olsen,et al.  Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling. , 2018, Cell reports.

[9]  H. Clark,et al.  The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation , 2016, Nature Communications.

[10]  José A. Dianes,et al.  2016 update of the PRIDE database and its related tools , 2016, Nucleic Acids Res..

[11]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[12]  D. Rotin,et al.  The Second Catalytic Domain of Protein Tyrosine Phosphatase δ (PTPδ) Binds to and Inhibits the First Catalytic Domain of PTPς , 1998, Molecular and Cellular Biology.

[13]  M. Moran,et al.  Protein‐phosphotyrosine proteome profiling by superbinder‐SH2 domain affinity purification mass spectrometry, sSH2‐AP‐MS , 2017, Proteomics.

[14]  M. Sowa,et al.  A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin , 2016, Scientific Reports.

[15]  D. Barford,et al.  Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Brian Burke,et al.  A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells , 2012, The Journal of cell biology.

[17]  P. Bastiaens,et al.  Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling , 2018, Science Signaling.

[18]  Huadong Liu,et al.  Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder. , 2016, Nature chemical biology.

[19]  Gavin J. Wright Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions† †This article is part of a Molecular BioSystems themed issue on Computational and Systems Biology. , 2009, Molecular bioSystems.

[20]  Zhenhuan Guo,et al.  E-cadherin interactome complexity and robustness resolved by quantitative proteomics , 2014, Science Signaling.

[21]  P G Drake,et al.  Structural and Evolutionary Relationships among Protein Tyrosine Phosphatase Domains , 2001, Molecular and Cellular Biology.

[22]  Lino C. Gonzalez,et al.  TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia , 2016, Neuron.

[23]  Melanie A. Huntley,et al.  Recurrent R-spondin fusions in colon cancer , 2012, Nature.

[24]  B. Neel,et al.  Identification of Major Binding Proteins and Substrates for the SH2-Containing Protein Tyrosine Phosphatase SHP-1 in Macrophages , 1998, Molecular and Cellular Biology.

[25]  W. Choi,et al.  Receptor-type tyrosine-protein phosphatase κ directly targets STAT3 activation for tumor suppression in nasal NK/T-cell lymphoma. , 2015, Blood.

[26]  Gerard Manning,et al.  Genomics and evolution of protein phosphatases , 2017, Science Signaling.

[27]  A. Ullrich,et al.  Furin-, ADAM 10-, and γ-Secretase-Mediated Cleavage of a Receptor Tyrosine Phosphatase and Regulation of β-Catenin's Transcriptional Activity , 2006, Molecular and Cellular Biology.

[28]  M. Matsuda,et al.  abLIM3 is a novel component of adherens junctions with actin-binding activity. , 2010, European journal of cell biology.

[29]  Amber L. Couzens,et al.  The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data , 2013, Nature Methods.

[30]  Amber L. Couzens,et al.  Phenotypic and Interaction Profiling of the Human Phosphatases Identifies Diverse Mitotic Regulators. , 2016, Cell reports.

[31]  Goberdhan P Dimri,et al.  Modeling breast cancer-associated c-Src and EGFR overexpression in human MECs: c-Src and EGFR cooperatively promote aberrant three-dimensional acinar structure and invasive behavior. , 2007, Cancer research.

[32]  P. Villagrasa,et al.  Specific Phosphorylation of p120-Catenin Regulatory Domain Differently Modulates Its Binding to RhoA , 2006, Molecular and Cellular Biology.

[33]  Albert B. Reynolds,et al.  A core function for p120-catenin in cadherin turnover , 2003, The Journal of cell biology.

[34]  S. Brady-Kalnay,et al.  Regulation of development and cancer by the R2B subfamily of RPTPs and the implications of proteolysis. , 2015, Seminars in cell & developmental biology.

[35]  Sara Fahs,et al.  Approaches to Study Phosphatases. , 2016, ACS chemical biology.

[36]  M. Hayman,et al.  Molecular Mechanism for a Role of SHP2 in Epidermal Growth Factor Receptor Signaling , 2003, Molecular and Cellular Biology.

[37]  W. Franke,et al.  Identification and localization of a neurally expressed member of the plakoglobin/armadillo multigene family. , 1997, Differentiation; research in biological diversity.

[38]  N. Brown,et al.  Drosophila p120-catenin is crucial for endocytosis of the dynamic E-cadherin–Bazooka complex , 2016, Journal of Cell Science.

[39]  Weixian Lu,et al.  Structure of a Tyrosine Phosphatase Adhesive Interaction Reveals a Spacer-Clamp Mechanism , 2007, Science.

[40]  J. Todd,et al.  The chromosome 6q22.33 region is associated with age at diagnosis of type 1 diabetes and disease risk in those diagnosed under 5 years of age , 2017, Diabetologia.

[41]  Yarden Katz,et al.  A single-cell survey of the small intestinal epithelium , 2017, Nature.

[42]  Devin K. Schweppe,et al.  Architecture of the human interactome defines protein communities and disease networks , 2017, Nature.

[43]  J. den Hertog,et al.  Dimerization In Vivo and Inhibition of the Nonreceptor Form of Protein Tyrosine Phosphatase Epsilon , 2003, Molecular and Cellular Biology.

[44]  M. Tremblay,et al.  Substrate-trapping techniques in the identification of cellular PTP targets. , 2005, Methods.

[45]  A. Emili,et al.  A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome. , 2017, Molecular cell.

[46]  Jayanta Debnath,et al.  Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. , 2003, Methods.

[47]  H. Nam,et al.  Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45 , 2005, The Journal of experimental medicine.

[48]  Gareth W. Fearnley,et al.  Detection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells. , 2015, Methods in molecular biology.

[49]  K. Green,et al.  Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains , 2003, Journal of Cell Science.

[50]  Bryan M. Zhao,et al.  High-resolution crystal structures of the D1 and D2 domains of protein tyrosine phosphatase epsilon for structure-based drug design. , 2018, Acta crystallographica. Section D, Structural biology.

[51]  A. Rokas,et al.  The Molecular Evolution of the p120-Catenin Subfamily and Its Functional Associations , 2010, PloS one.

[52]  A. Bennett,et al.  Receptor Protein Tyrosine Phosphatase-Receptor Tyrosine Kinase Substrate Screen Identifies EphA2 as a Target for LAR in Cell Migration , 2013, Molecular and Cellular Biology.

[53]  N. Tonks,et al.  A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells* , 2015, The Journal of Biological Chemistry.

[54]  Mathias J Friedrich,et al.  A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes , 2017, Nature Genetics.

[55]  Wen Hwa Lee,et al.  Large-Scale Structural Analysis of the Classical Human Protein Tyrosine Phosphatome , 2009, Cell.

[56]  S. Brady-Kalnay,et al.  Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation , 1993, The Journal of cell biology.

[57]  Viktória Hudacsek,et al.  [Genome engineering using the CRISPR-Cas9 system and applications in cancer research]. , 2018, Magyar onkologia.

[58]  Frederick Y. Wu,et al.  Transforming Growth Factor β (TGF-β)-Smad Target Gene Protein Tyrosine Phosphatase Receptor Type Kappa Is Required for TGF-β Function , 2005, Molecular and Cellular Biology.

[59]  Abdullahi Umar Ibrahim,et al.  Genome Engineering Using the CRISPR Cas9 System , 2019 .

[60]  Jesse K. Placone,et al.  RAP2 Mediates Mechano-responses of Hippo Pathway , 2018, Nature.

[61]  José A. Dianes,et al.  2016 update of the PRIDE database and its related tools , 2016, Nucleic Acids Res..

[62]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[63]  D. Rotin,et al.  The second catalytic domain of protein tyrosine phosphatase delta (PTP delta) binds to and inhibits the first catalytic domain of PTP sigma. , 1998, Molecular and cellular biology.

[64]  Bin Zhang,et al.  PhosphoSitePlus, 2014: mutations, PTMs and recalibrations , 2014, Nucleic Acids Res..

[65]  J. Hardcastle,et al.  Colorectal cancer , 1993, Europe Against Cancer European Commission Series for General Practitioners.

[66]  J. Yates Large-Scale Phosphoproteomics , 2019, Proteomics for Biological Discovery.

[67]  J. Thornton,et al.  Elucidating Human Phosphatase-Substrate Networks , 2013, Science Signaling.

[68]  Robert A. Weinberg,et al.  EMT in cancer , 2018, Nature Reviews Cancer.

[69]  A. Reynolds,et al.  EGFR signaling to p120-catenin through phosphorylation at Y228 , 2004, Journal of Cell Science.

[70]  V. Grachtchouk,et al.  Receptor-type Protein-tyrosine Phosphatase-κ Regulates Epidermal Growth Factor Receptor Function* , 2005, Journal of Biological Chemistry.

[71]  R. Okamoto,et al.  Regulation of Platelet-derived Growth Factor Receptor Activation by Afadin through SHP-2 , 2007, Journal of Biological Chemistry.

[72]  H. Nam,et al.  Crystal Structure of the Tandem Phosphatase Domains of RPTP LAR , 1999, Cell.

[73]  T. Hunter Tyrosine phosphorylation: thirty years and counting. , 2009, Current opinion in cell biology.

[74]  M. Tenhagen,et al.  p120-catenin in cancer – mechanisms, models and opportunities for intervention , 2013, Journal of Cell Science.

[75]  J. Schlessinger,et al.  Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding , 1994, Molecular and cellular biology.

[76]  A. Reynolds,et al.  Receptor Protein-tyrosine Phosphatase RPTPμ Binds to and Dephosphorylates the Catenin p120 ctn * , 2000, The Journal of Biological Chemistry.

[77]  M. Gresser,et al.  Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate* , 1997, The Journal of Biological Chemistry.

[78]  D. Barford,et al.  Visualization of the Cysteinyl-phosphate Intermediate of a Protein-tyrosine Phosphatase by X-ray Crystallography* , 1998, The Journal of Biological Chemistry.

[79]  S. Dalal,et al.  Plakophilin3 increases desmosome assembly, size and stability by increasing expression of desmocollin2. , 2018, Biochemical and biophysical research communications.

[80]  M. T. Brown,et al.  Regulation, substrates and functions of src. , 1996, Biochimica et biophysica acta.

[81]  R. Beijersbergen,et al.  Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase. , 1993, The Journal of biological chemistry.

[82]  J. Sap,et al.  Homophilic Interactions Mediated by Receptor Tyrosine Phosphatases μ and κ. A CRITICAL ROLE FOR THE NOVEL EXTRACELLULAR MAM DOMAIN (*) , 1995, The Journal of Biological Chemistry.

[83]  A. Rotter,et al.  Genomic structure and alternative splicing of murine R2B receptor protein tyrosine phosphatases (PTPκ, μ, ρ and PCP-2) , 2004, BMC Genomics.

[84]  R. Aguiar,et al.  Protein tyrosine phosphatase receptor-type O truncated (PTPROt) regulates SYK phosphorylation, proximal B-cell-receptor signaling, and cellular proliferation. , 2006, Blood.

[85]  Aaron S. Gajadhar,et al.  Early signaling dynamics of the epidermal growth factor receptor , 2016, Proceedings of the National Academy of Sciences.

[86]  A. Żarnecki Global analysis of , 1999, hep-ph/9904334.

[87]  Nadia Martinez-Martin,et al.  Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions , 2017, Journal of immunology research.

[88]  Dmitri D. Pervouchine,et al.  The human transcriptome across tissues and individuals , 2015, Science.

[89]  G. Mosayebi,et al.  Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA) as a Candidate Subunit Cholera Vaccine , 2017, Journal of immunology research.

[90]  R. Coffey,et al.  p120-catenin controls contractility along the vertical axis of epithelial lateral membranes , 2016, Journal of Cell Science.

[91]  Yigong Shi Serine/Threonine Phosphatases: Mechanism through Structure , 2009, Cell.

[92]  Xun Li,et al.  The human DEPhOsphorylation database DEPOD: a 2015 update , 2014, Nucleic Acids Res..

[93]  A. Rust,et al.  Insertional mutagenesis identifies multiple networks of co-operating genes driving intestinal tumorigenesis , 2011, Nature Genetics.

[94]  Christian Siebold,et al.  Molecular analysis of receptor protein tyrosine phosphatase μ‐mediated cell adhesion , 2006, The EMBO journal.

[95]  N. Nicola,et al.  The molecular regulation of Janus kinase (JAK) activation. , 2014, The Biochemical journal.

[96]  Y. Asmann,et al.  A Transposon-Based Genetic Screen in Mice Identifies Genes Altered in Colorectal Cancer , 2009, Science.

[97]  Y. Shintani,et al.  The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane. , 2008, Experimental cell research.

[98]  Genee Y. Lee,et al.  Three-dimensional culture models of normal and malignant breast epithelial cells , 2007, Nature Methods.

[99]  I. Thorey,et al.  A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood , 2014, PloS one.

[100]  Peter J. Verveer,et al.  EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation , 2003, Nature Cell Biology.

[101]  W. Alkema,et al.  BioVenn – a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams , 2008, BMC Genomics.

[102]  Marco Y. Hein,et al.  The Perseus computational platform for comprehensive analysis of (prote)omics data , 2016, Nature Methods.