Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl– ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores.
References
[1]
Murray CJL, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, et al. (2012) Global malaria mortality between 1980 and 2010: a systematic analysis. The Lancet 379: 413–431.
[2]
Kostich M, English J, Madison V, Gheyas F, Wang L, et al. (2002) Human members of the eukaryotic protein kinase family. Genome Biol 3: RESEARCH0043.
[3]
Solyakov L, Halbert J, Alam MM, Semblat JP, Dorin-Semblat D, et al. (2011) Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat Commun 2: 565.
[4]
Treeck M, Sanders JL, Elias JE, Boothroyd JC (2011) The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries. Cell Host Microbe 10: 410–419.
[5]
Agarwal S, Kern S, Halbert J, Przyborski JM, Baumeister S, et al. (2011) Two nucleus-localized CDK-like kinases with crucial roles for malaria parasite erythrocytic replication are involved in phosphorylation of splicing factor. J Cell Biochem 112: 1295–1310.
[6]
Dixit A, Singh PK, Sharma GP, Malhotra P, Sharma P (2010) PfSRPK1, a novel splicing-related kinase from Plasmodium falciparum. J Biol Chem 285: 38315–38323.
[7]
Philip N, Haystead TA (2007) Characterization of a UBC13 kinase in Plasmodium falciparum. Proc Natl Acad Sci U S A 104: 7845–7850.
[8]
Rached FB, Ndjembo-Ezougou C, Chandran S, Talabani H, Yera H, et al. (2012) Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites. Biol Cell 104: 34–47.
[9]
Zhang M, Fennell C, Ranford-Cartwright L, Sakthivel R, Gueirard P, et al. (2010) The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. J Exp Med 207: 1465–1474.
[10]
Tewari R, Straschil U, Bateman A, Bohme U, Cherevach I, et al. (2010) The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 8: 377–387.
[11]
McRobert L, Taylor CJ, Deng W, Fivelman QL, Cummings RM, et al. (2008) Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase. PLoS Biol 6: e139.
[12]
Billker O, Dechamps S, Tewari R, Wenig G, Franke-Fayard B, et al. (2004) Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 117: 503–514.
[13]
Tewari R, Dorin D, Moon R, Doerig C, Billker O (2005) An atypical mitogen-activated protein kinase controls cytokinesis and flagellar motility during male gamete formation in a malaria parasite. Mol Microbiol 58: 1253–1263.
[14]
Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, et al. (2005) Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell 121: 675–687.
[15]
Reininger L, Billker O, Tewari R, Mukhopadhyay A, Fennell C, et al. (2005) A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites. J Biol Chem 280: 31957–31964.
[16]
Reininger L, Tewari R, Fennell C, Holland Z, Goldring D, et al. (2009) An essential role for the Plasmodium Nek-2 Nima-related protein kinase in the sexual development of malaria parasites. J Biol Chem 284: 20858–20868.
[17]
Moon RW, Taylor CJ, Bex C, Schepers R, Goulding D, et al. (2009) A cyclic GMP signalling module that regulates gliding motility in a malaria parasite. PLoS Pathog 5: e1000599.
[18]
Siden-Kiamos I, Ecker A, Nyback S, Louis C, Sinden RE, et al. (2006) Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion. Mol Microbiol 60: 1355–1363.
[19]
Doerig C, Billker O, Pratt D, Endicott J (2005) Protein kinases as targets for antimalarial intervention: Kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes. Biochim Biophys Acta 1754: 132–150.
[20]
Yokoyama D, Saito-Ito A, Asao N, Tanabe K, Yamamoto M, et al. (1998) Modulation of the growth of Plasmodium falciparum in vitro by protein serine/threonine phosphatase inhibitors. Biochem Biophys Res Commun 247: 18–23.
[21]
Kutuzov MA, Andreeva AV (2002) Protein Ser/Thr phosphatases with kelch-like repeat domains. Cell Signal 14: 745–750.
[22]
Wilkes JM, Doerig C (2008) The protein-phosphatome of the human malaria parasite Plasmodium falciparum. BMC Genomics 9: 412.
[23]
Adams J, Kelso R, Cooley L (2000) The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol 10: 17–24.
[24]
Imaizumi T, Schultz TF, Harmon FG, Ho LA, Kay SA (2005) FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science 309: 293–297.
[25]
Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, et al. (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 13: 76–86.
[26]
Mata J, Nurse P (1997) tea1 and the microtubular cytoskeleton are important for generating global spatial order within the fission yeast cell. Cell 89: 939–949.
[27]
Schmid MF, Agris JM, Jakana J, Matsudaira P, Chiu W (1994) Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin. J Cell Biol 124: 341–350.
[28]
Budhwar R, Fang G, Hirsch JP (2011) Kelch repeat proteins control yeast PKA activity in response to nutrient availability. Cell Cycle 10: 767–770.
[29]
Harashima T, Anderson S, Yates JR, 3rd, Heitman J (2006) The kelch proteins Gpb1 and Gpb2 inhibit Ras activity via association with the yeast RasGAP neurofibromin homologs Ira1 and Ira2. Mol Cell 22: 819–830.
[30]
Mora-Garcia S, Vert G, Yin Y, Cano-Delgado A, Cheong H, et al. (2004) Nuclear protein phosphatases with Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis. Genes Dev 18: 448–460.
[31]
Li JL, Baker DA (1998) A putative protein serine/threonine phosphatase from Plasmodium falciparum contains a large N-terminal extension and five unique inserts in the catalytic domain. Mol Biochem Parasitol 95: 287–295.
[32]
Canning EU, Sinden RE (1973) The organization of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei. Parasitology 67: 29–40.
[33]
Bannister LH, Hopkins JM, Dluzewski AR, Margos G, Williams IT, et al. (2003) Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is translocated within micronemes along subpellicular microtubules during merozoite development. J Cell Sci 116: 3825–3834.
[34]
Moorhead GB, De Wever V, Templeton G, Kerk D (2009) Evolution of protein phosphatases in plants and animals. Biochem J 417: 401–409.
[35]
Melnikova I, Golden J (2004) Targeting protein kinases. Nat Rev Drug Discov 3: 993–994.
[36]
McConnell JL, Wadzinski BE (2009) Targeting protein serine/threonine phosphatases for drug development. Mol Pharmacol 75: 1249–1261.
[37]
Kutuzov MA, Andreeva AV (2008) Protein Ser/Thr phosphatases of parasitic protozoa. Mol Biochem Parasitol 161: 81–90.
[38]
Hall N, Karras M, Raine JD, Carlton JM, Kooij TW, et al. (2005) A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science 307: 82–86.
[39]
Bushell ES, Ecker A, Schlegelmilch T, Goulding D, Dougan G, et al. (2009) Paternal effect of the nuclear formin-like protein MISFIT on Plasmodium development in the mosquito vector. PLoS Pathog 5: e1000539.
[40]
Raibaud A, Lupetti P, Paul RE, Mercati D, Brey PT, et al. (2001) Cryofracture electron microscopy of the ookinete pellicle of Plasmodium gallinaceum reveals the existence of novel pores in the alveolar membranes. J Struct Biol 135: 47–57.
[41]
Tremp AZ, Dessens JT (2011) Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape. J Biol Chem 286: 5383–5391.
[42]
Tremp AZ, Khater EI, Dessens JT (2008) IMC1b is a putative membrane skeleton protein involved in cell shape, mechanical strength, motility, and infectivity of malaria ookinetes. J Biol Chem 283: 27604–27611.
[43]
Green JL, Rees-Channer RR, Howell SA, Martin SR, Knuepfer E, et al. (2008) The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. J Biol Chem 283: 30980–30989.
[44]
Hirai M, Arai M, Kawai S, Matsuoka H (2006) PbGCbeta is essential for Plasmodium ookinete motility to invade midgut cell and for successful completion of parasite life cycle in mosquitoes. J Biochem 140: 747–757.
[45]
Szoor B, Ruberto I, Burchmore R, Matthews KR (2010) A novel phosphatase cascade regulates differentiation in Trypanosoma brucei via a glycosomal signaling pathway. Genes Dev 24: 1306–1316.
[46]
Szoor B, Wilson J, McElhinney H, Tabernero L, Matthews KR (2006) Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes. J Cell Biol 175: 293–303.
[47]
Kim TW, Guan S, Burlingame AL, Wang ZY (2011) The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2. Mol Cell 43: 561–571.
[48]
Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, et al. (2008) Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol Syst Biol 4: 193.
[49]
Janse CJ, Franke-Fayard B, Mair GR, Ramesar J, Thiel C, et al. (2006) High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol 145: 60–70.
[50]
Hegge S, Kudryashev M, Smith A, Frischknecht F (2009) Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infection. Biotechnol J 4: 903–913.
[51]
Mair GR, Braks JA, Garver LS, Wiegant JC, Hall N, et al. (2006) Regulation of sexual development of Plasmodium by translational repression. Science 313: 667–669.