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Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

  • A. R. Escalona-Montaño (a1), R. Pérez-Montfort (a2), N. Cabrera (a2), R. Mondragón-Flores (a3), D. E. Vélez-Ramírez (a4), J. N. Gómez-Sandoval (a5), L. Gutiérrez-Kobeh (a1), I. Becker (a1) and M. M. Aguirre-García (a1)...


The main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes of Leishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2 and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinant LmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinant LmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in different Leishmania species; the LmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

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This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Corresponding author

Author for correspondence: M.M. Aguirre-García, E-mail:


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Aguirre-Garcia, MM, Escalona-Montano, AR, Bakalara, N, Perez-Torres, A, Gutierrez-Kobeh, L and Becker, I (2006) Leishmania major: detection of membrane-bound protein tyrosine phosphatase. Parasitology 132, 641649.
Andreeva, AV and Kutuzov, MA (2004) Widespread presence of ‘bacterial-like’ PPP phosphatases in eukaryotes. BMC Evolutionary Biology 4, 47.
Arnold, K, Bordoli, L, Kopp, J and Schwede, T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195201.
Biasini, M, Bienert, S, Waterhouse, A, Arnold, K, Studer, G, Schmidt, T, Kiefer, F, Gallo Cassarino, T, Bertoni, M, Bordoli, L and Schwede, T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research 42, W252W258.
Bordoli, L, Kiefer, F, Arnold, K, Benkert, P, Battey, J and Schwede, T (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nature Protocols 4, 113.
Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248254.
Brenchley, R, Tariq, H, McElhinney, H, Szoor, B, Huxley-Jones, J, Stevens, R, Matthews, K and Tabernero, L (2007) The TriTryp phosphatome: analysis of the protein phosphatase catalytic domains. BMC Genomics 8, 434.
Buchan, DW, Minneci, F, Nugent, TC, Bryson, K and Jones, DT (2013) Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Research 41, W349W357.
Burns, JM Jr, Parsons, M, Rosman, DE and Reed, SG (1993) Molecular cloning and characterization of a 42-kDa protein phosphatase of Leishmania chagasi . The Journal of Biological Chemistry 268, 1715517161.
Cohen, P (2002) The origins of protein phosphorylation. Nature Cell Biology 4, E127E130.
Das, AK, Helps, NR, Cohen, PT and Barford, D (1996) Crystal structure of the protein serine/threonine phosphatase 2C at 2·0 A resolution. The EMBO Journal. 15, 67986809.
Dissing, J, Dahl, O and Svensmark, O (1979) Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography. Biochimica et Biophys Acta 569, 159176.
Gilbert, LA, Ravindran, S, Turetzky, JM, Boothroyd, JC and Bradley, PJ (2007) Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryotic Cell 6, 7383.
Gomez de Leon, CT, Diaz Martin, RD, Mendoza Hernandez, G, Gonzalez Pozos, S, Ambrosio, JR and Mondragon Flores, R (2014) Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites. Journal of Proteomics 111, 8699.
Guex, N, Peitsch, MC and Schwede, T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30 (Suppl. 1), S162S173.
Ivens, AC, Peacock, CS, Worthey, EA, Murphy, L, Aggarwal, G, Berriman, M, Sisk, E, Rajandream, MA, Adlem, E, Aert, R, Anupama, A, Apostolou, Z, Attipoe, P, Bason, N, Bauser, C, Beck, A, Beverley, SM, Bianchettin, G, Borzym, K, Bothe, G, Bruschi, CV, Collins, M, Cadag, E, Ciarloni, L, Clayton, C, Coulson, RM, Cronin, A, Cruz, AK, Davies, RM, De Gaudenzi, J, Dobson, DE, Duesterhoeft, A, Fazelina, G, Fosker, N, Frasch, AC, Fraser, A, Fuchs, M, Gabel, C, Goble, A, Goffeau, A, Harris, D, Hertz-Fowler, C, Hilbert, H, Horn, D, Huang, Y, Klages, S, Knights, A, Kube, M, Larke, N, Litvin, L, Lord, A, Louie, T, Marra, M, Masuy, D, Matthews, K, Michaeli, S, Mottram, JC, Muller-Auer, S, Munden, H, Nelson, S, Norbertczak, H, Oliver, K, O'Neil, S, Pentony, M, Pohl, TM, Price, C, Purnelle, B, Quail, MA, Rabbinowitsch, E, Reinhardt, R, Rieger, M, Rinta, J, Robben, J, Robertson, L, Ruiz, JC, Rutter, S, Saunders, D, Schafer, M, Schein, J, Schwartz, DC, Seeger, K, Seyler, A, Sharp, S, Shin, H, Sivam, D, Squares, R, Squares, S, Tosato, V, Vogt, C, Volckaert, G, Wambutt, R, Warren, T, Wedler, H, Woodward, J, Zhou, S, Zimmermann, W, Smith, DF, Blackwell, JM, Stuart, KD, Barrell, B and Myler, PJ (2005) The genome of the kinetoplastid parasite, Leishmania major . Science 309, 436442.
Kennelly, PJ (2001) Protein phosphatases – a phylogenetic perspective. Chemical Reviews 101, 22912312.
Kumar, KA, Garcia, CR, Chandran, VR, Van Rooijen, N, Zhou, Y, Winzeler, E and Nussenzweig, V (2007) Exposure of plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity. Molecular and Biochemical Parasitology 156, 3240.
Lammers, T and Lavi, S (2007) Role of type 2C protein phosphatases in growth regulation and in cellular stress signaling. Critical Reviews in Biochemistry and Molecular Biology 42, 437461.
Mamoun, CB, Sullivan, DJ Jr, Banerjee, R and Goldberg, DE (1998) Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum . The Journal of Biological Chemistry 273, 1124111247.
Melo, F, Sanchez, R and Sali, A (2002) Statistical potentials for fold assessment. Protein Science: a Publication of the Protein Society 11, 430448.
Mitula, F, Tajdel, M, Ciesla, A, Kasprowicz-Maluski, A, Kulik, A, Babula-Skowronska, D, Michalak, M, Dobrowolska, G, Sadowski, J and Ludwikow, A (2015) Arabidopsis ABA-activated kinase MAPKKK18 is regulated by protein phosphatase 2C ABI1 and the ubiquitin-proteasome pathway. Plant & Cell Physiology 56, 23512367.
Montfort, I, Perez-Tamayo, R, Perez-Montfort, R, Gonzalez Canto, A and Olivos, A (1994) Purification and immunologic characterization of a 30-kDa cysteine proteinase of Entamoeba histolytica . Parasitology Research 80, 607613.
Moorhead, GB, Trinkle-Mulcahy, L and Ulke-Lemee, A (2007) Emerging roles of nuclear protein phosphatases. Nature Reviews Molecular Cell Biology 8, 234244.
Mukhopadhyay, S, Kapatral, V, Xu, W and Chakrabarty, AM (1999) Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa . Journal of Bacteriology 181, 66156622.
Obuchowski, M, Madec, E, Delattre, D, Boel, G, Iwanicki, A, Foulger, D and Seror, SJ (2000) Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family. Journal of Bacteriology 182, 56345638.
Prestes, EB, Bayer-Santos, E, Hermes Stoco, P, Sincero, TC, Wagner, G, Umaki, A, Fragoso, SP, Bordignon, J, Steindel, M and Grisard, EC (2012) Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum. Memorias do Instituto Oswaldo Cruz 107, 713719.
Pullen, KE, Ng, HL, Sung, PY, Good, MC, Smith, SM and Alber, T (2004) An alternate conformation and a third metal in PstP/Ppp, the M. tuberculosis PP2C-family Ser/Thr protein phosphatase. Structure 12, 19471954.
Rajagopal, L, Clancy, A and Rubens, CE (2003) A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence. The Journal of Biological Chemistry 278, 1442914441.
Ruppert, U, Irmler, A, Kloft, N and Forchhammer, K (2002) The novel protein phosphatase PphA from synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII. Molecular Microbiology 44, 855864.
Schlicker, C, Fokina, O, Kloft, N, Grune, T, Becker, S, Sheldrick, GM and Forchhammer, K (2008) Structural analysis of the PP2C phosphatase tPphA from Thermosynechococcus elongatus: a flexible flap subdomain controls access to the catalytic site. Journal of Molecular Biology 376, 570581.
Schweighofer, A, Hirt, H and Meskiene, I (2004) Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Science 9, 236243.
Shen, MY and Sali, A (2006) Statistical potential for assessment and prediction of protein structures. Protein Science: a Publication of the Protein Society 15, 25072524.
Su, J and Forchhammer, K (2013) Determinants for substrate specificity of the bacterial PP2C protein phosphatase tPphA from Thermosynechococcus elongatus . The FEBS Journal 280, 694707.
Su, J, Schlicker, C and Forchhammer, K (2011) A third metal is required for catalytic activity of the signal-transducing protein phosphatase M tPphA. The Journal of Biological Chemistry 286, 1348113488.
Wang, B, Zhang, P and Wei, Q (2008) Recent progress on the structure of Ser/Thr protein phosphatases. Science in China. Series C, Life Sciences/Chinese Academy of Sciences 51, 487494.



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