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Purification and characterization of the human protein tyrosine phosphatase, PTPμ, from a baculovirus expression system

  • Protein Phosphatases
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Abstract

The receptor like PTPase, PTPμ, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTPμ (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment *80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTPμ was membrane associated while the truncated form was recovered in the soluble fraction. PTPμ preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a Km of 400 nM and a Vmax of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTPμ. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn++ and Mn++ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.

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References

  1. Fischer E, Krebs E: Commentary on ‘the phosphorylase b to a converting enzyme of rabbit skeletal muscle”. Biochim Biophys Acta 1000: 297–301, 1989

    PubMed  Google Scholar 

  2. Hardie DG: Protein phosphorylation, a practical approach. IRL Press, 1993

  3. Hunter T: Oncogene products in the cytoplasm: the protein kinases. In: RA Weinberg (ed.) Oncogenes and Molecular Origins of Cancer. Cold Spring Harbor Laboratory Press, New York, 1989, pp 147–173

    Google Scholar 

  4. Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203–212, 1990

    PubMed  Google Scholar 

  5. Charbonneau H, Tonks NK: 1002 protein phosphatases? Annu Rev Cell Biol 8: 463–493, 1992

    Google Scholar 

  6. Gebbink M, Van Etten I, Hateboer G, Suijkerbuijk R, Beijersbergen R, Van Kessel A, Moolenaar W: Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase. FEBS Lett 290: 123–130, 1991

    PubMed  Google Scholar 

  7. Beckman G, Bork P: An adhesive domain detected in functionally diverse receptors. TIBS 18: 40–41, 1993

    PubMed  Google Scholar 

  8. Jiang W, Gorben C, Flannery A, Beynon R, Grant G, Bond J: The α subunit of meprin A: molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the α and β subunits. J Biol Chem 267: 9185–9193, 1992

    PubMed  Google Scholar 

  9. Takagi S, Hirata T, Agata K, Mochii M, Eguchi G, Fuisawa H: The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors. Neuron 7:295–307, 1991

    PubMed  Google Scholar 

  10. Jiang Y, Wang H, D'Eustachio P, Musacchio J, Schlessinger J, Sap J: Cloning and characterization of R-PTP-κ, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region. Mol Cell Biol 13: 2942–2951, 1993

    PubMed  Google Scholar 

  11. Summers M, Smith G: A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agriculture Experimental Station, 1987

  12. Flint A, Gebbink M, Franza B, Hill D, Tonks NK: Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: Identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation EMBO J 12: 1937–1946, 1993

    PubMed  Google Scholar 

  13. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72: 248–254, 1976

    PubMed  Google Scholar 

  14. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970

    PubMed  Google Scholar 

  15. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76: 4350–4354, 1979

    PubMed  Google Scholar 

  16. Tonks NK, Diltz CD, Fischer EH: Characterization of the major protein tyrosine phosphatases of human placenta. J Biol Chem 265: 6731–6737, 1988

    Google Scholar 

  17. Tonks NK, Diltz CD, Fischer EH: CD45, an integral membrane protein tyrosine phosphatase. J Biol Chem 265: 10674–10680, 1990

    PubMed  Google Scholar 

  18. Klarlund J: Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell 41: 707–717, 1985

    PubMed  Google Scholar 

  19. LaForgia S, Morse B, Levy J, Barnea G, Cannizzaro LA, Li F, Nowell PC, Boghosian-Sell L, Glick J, Weston A, Harris CC, Drabkin H, Patterson D, Croce CM, Schlessinger J, Huebner K: Receptor protein-tyrosine phosphatase γ is a candidate tumor suppressor gene at human chromosome region 3p21. Proc Natl Acad Sci USA 88: 5036–5040, 1991

    PubMed  Google Scholar 

  20. Koretzky GA, Picus J, Schultz T, Weiss A: Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci USA 88: 2037–2041, 1991

    PubMed  Google Scholar 

  21. Koretzky GA, Picus J, Thomas ML, Weiss A: Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. Nature 346: 66–68, 1990

    PubMed  Google Scholar 

  22. Perkins LA, Larsen I, Perrimon N:corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinasetorso. Cell 70: 225–236, 1992

    PubMed  Google Scholar 

  23. Strausfeld U, Labbe JC, Fesquet Cavadore JC, Picard A, Sadhu K, Russell P, Dorée M: Dephosphorylation and activation of a p34cdc2 cyclin-B complexin vitro by human cdc25 protein. Nature 351: 242–245, 1991

    PubMed  Google Scholar 

  24. Lee MS, Ogg S, Xu M, Parker LL, Donaghue DJ, Maller JL, Pinica-Worms H: cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2. Mol Cell Biol 3: 73–84, 1992

    Google Scholar 

  25. Feng G-S, Hui C-C, Pawson T: SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science 259: 1607–1611, 1993

    PubMed  Google Scholar 

  26. Gu M, York JD, Warshawsky L, Majerus PW: Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein. Proc Natl Acad Sci USA 88: 5867–5871, 1991

    PubMed  Google Scholar 

  27. Yang Q, Tonks NK: Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4.1, ezrin, and talin. Proc Natl Acad Sci USA 88: 5949–5953, 1991

    PubMed  Google Scholar 

  28. McLaughlin S, Dixon JE: Alternative splicing gives rise to a nuclear protein tyrosine phosphatase in Drosophila. J Biol Chem 268: 6839–6842, 1993

    PubMed  Google Scholar 

  29. Cool DE, Tonks NK, Charbonneau H, Fischer EH, Krebs EG: Expression of a human T-cell protein-tyrosine-phosphatase in baby hamster kidney cells. Proc Natl Acad Sci USA 87: 7280–7284, 1990

    PubMed  Google Scholar 

  30. Frangioni JV, Beahm PH, Shifrin V, Jost CA, Neel BG: The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell 68: 545–560, 1992

    PubMed  Google Scholar 

  31. Ostergaard HL, Trowbridge IS: Negative regulation of CD45 protein tyrosine phosphatase activity by ionomycin in T cells. Science 253: 1423–1425, 1991

    PubMed  Google Scholar 

  32. Hoffmann I, Clarke PR, Marcote MJ, Karsenti E, Draetta G: Phosphorylation and activation of human cdc25-C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J 12: 53–63, 1993

    PubMed  Google Scholar 

  33. Izumi T, Walker DH, Maller JL: Periodic changes in phosphorylation of theXenopus cdc25 phosphatase regulate its activity. Mol Biol Cell 3: 927–939, 1992

    PubMed  Google Scholar 

  34. Stamenkovic I, Sgroi D, Aruffo A, Sy MS, Anderson T: The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45 RO on T cells and α2–6 sialyltransferase, CD75 on B cells. Cell 66: 1133–1144, 1991

    PubMed  Google Scholar 

  35. Powell LD, Sgroi D, Sjoberg ER, Stamenkovic I, Varki A: Natural ligands of the B cell adhesion molecule CD22β carry N-linked oligosaccharides with α-2,6-linked sialic acids that are required for recognition. J Biol Chem 268: 7019–7027, 1993

    PubMed  Google Scholar 

  36. Sgroi D, Varki A, Braesch-Andersen S, Stamenkovic I: CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin. J Biol Chem 268: 7011–7018, 1993

    PubMed  Google Scholar 

  37. Tonks NK, Diltz CD, Fischer EH: CD45, an integral membrane protein tyrosine phosphatase. J Biol Chem 265: 10674–10680, 1990

    PubMed  Google Scholar 

  38. Tan X, Stover DR, Walsh KA: Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45. J Biol Chem 268: 6835–6838, 1993

    PubMed  Google Scholar 

  39. Desai DM, Sap J, Schlessinger J, Weiss A: Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase. Cell 73: 1–20, 1993

    PubMed  Google Scholar 

  40. Streuli M, Kreuger NX, Hall LR, Schlossman SF, Saito H: A new member of the immunoglobulin superfamily that has a cytoplasmic region homologous to the leukocyte common antigen. J Exp Med 168: 1523–1530, 1988

    PubMed  Google Scholar 

  41. Streuli M, Krueger NX, Tsai AYM, Saito H: A family of receptor-linked protein tyrosine phosphatases in humans andDrosophila. Proc Natl Acad Sci USA 86: 8698–8702, 1989

    PubMed  Google Scholar 

  42. Tian S-S, Tsoulfas P, Zinn K: Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in theDrosophila embryo. Cell 67: 675–685, 1991

    PubMed  Google Scholar 

  43. Yang X, Seow KT, Bahri SM, Oon SH, Chia W: TwoDrosophila receptor-like tyrosine phosphatase genes are expressed in a subset of developing axons and pioneer neurons in the embryonic CNS. Cell 67: 661–673, 1991

    PubMed  Google Scholar 

  44. Edelman GM, Crossin KL: Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem 60: 155–190, 1991

    PubMed  Google Scholar 

  45. Tonks NK, Charbonneau H, Diltz CD, Kumar S, Cicirelli MF, Krebs EG, Walsh KA, Fischer EH: Protein tyrosine phosphatases: structure, properties and role in signal transduction. Adv Prot Phosphatases 5: 149–180, 1989

    Google Scholar 

  46. Pallen CJ, Tong PH: Elevation of membrane tyrosine phosphatase activity in density-dependent growth-arrested fibroblasts. Proc Natl Acad Sci USA 88: 6996–7000, 1991

    PubMed  Google Scholar 

  47. Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H: Cloning and expression of theret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene 3: 571–578, 1988

    PubMed  Google Scholar 

  48. Rescigno J, Mansukhani A, Basilico C: A putative receptor tyrosine kinase with unique structural topology. Oncogene 6: 1909–1913, 1991

    PubMed  Google Scholar 

  49. O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B:axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 11: 5016–5031, 1991

    PubMed  Google Scholar 

  50. Pulido D, Campuzano S, Koda T, Modotell J, Barbacid M:Dtrk, a Drosophila gene related to thetrk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J 11: 391–404, 1992

    PubMed  Google Scholar 

  51. Brady-Kalnay S, Flint AJ, Tonks NK: Homophilic binding of the receptor-type protein tyrosine phosphatase PTPμ mediates cell-cell aggregation. J Cell Biol 122: 961–972, 1993

    PubMed  Google Scholar 

  52. Burgoon M, Grumet M, Mauro V, Edelman G, Cunningham B: Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: Origin of the polypeptides and relation to the Ig superfamily. J Cell Biol 112: 1017–1029, 1991

    PubMed  Google Scholar 

  53. Faissner A, Teplow D, Kubler D, Keilhauer G, Kinzel V, Schachner M: Biosynthesis and membrane topography of the neural cell adhesion molecule L1. EMBO J 4: 3105–3113, 1985

    PubMed  Google Scholar 

  54. Faiz Kayyem J, Roman J, de la Rosa E, Schwarz U, Dreyer W: Bravo/Nr-CAM is closely related to the cell adhesion molecules L1 and Ng-CAM and has a similar heterodimer structure. J Cell Biol 118: 1259–1270, 1992

    PubMed  Google Scholar 

  55. Streuli M, Krueger N, Ariniello P, Tang M, Munro J, Blattler W, Adler D, Disteche C, Saito H: Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region. EMBO J 11: 897–907, 1992

    PubMed  Google Scholar 

  56. Tonks NK, Yang O, Flint A, Gebbink M, Franza B, Hill D, Sun H, Brady-Kalnay S: Protein tyrosine phosphatases: the problems of a growing family. Cold Spring Harbor Symposia on Quantitative Biology, 57, 1992

  57. Nagafuchi A, Takeichi M: Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J 7: 3679–3684, 1988

    PubMed  Google Scholar 

  58. Nagafuchi A, Takeichi M: Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin. Cell Regulation 1: 37–44, 1989

    PubMed  Google Scholar 

  59. Ozawa M, Ringwald M, Kemler R: Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. PNAS 87: 4246–4250, 1990

    PubMed  Google Scholar 

  60. Ozawa M, Baribault H, Kemler R: The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J 8: 1711–1717, 1989

    PubMed  Google Scholar 

  61. Kintner C: Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain. Cell 69: 225–236, 1992

    PubMed  Google Scholar 

  62. Volberg T, Geiger B, Dror R, Zick Y: Modulation of intercellular adherens type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chicken cells. Cell Regulation 2: 105–120, 1992

    Google Scholar 

  63. Tsukita S, Oishi K, Akiyama T, Yamanashi Y, Yamamoto T, Tsukita S: Specific proto-oncogene tyrosine kinases ofsrc family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J Cell Biol 113: 867–879, 1991

    PubMed  Google Scholar 

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Brady-Kalnay, S.M., Tonks, N.K. Purification and characterization of the human protein tyrosine phosphatase, PTPμ, from a baculovirus expression system. Mol Cell Biochem 127, 131–141 (1993). https://doi.org/10.1007/BF01076764

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