Malaria pp 241-257 | Cite as

Experimental Tools for the Study of Protein Phosphorylation in Plasmodium

  • Dominique Dorin-Semblat
  • Andrew R. Bottrill
  • Lev Solyakov
  • Andrew Tobin
  • Christian DoerigEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 923)


The central role played by protein phosphorylation in the regulation of eukaryotic cellular processes calls for detailed investigations of this phenomenon in malaria parasites. Here, we describe protocols to measure the activity of protein kinases (using either recombinant proteins or native enzymes purified from parasite extracts), and outline procedures to identify phosphorylation sites on parasite proteins following a mass spectrometry approach.

Key words

Phosphorylation Protein kinase Kinase assay Phosphoproteomics 



This work was supported by a joint project award from the Wellcome Trust to AT and CD (090313). Work in the CD laboratory was also supported by Inserm, EPFL, the FP6 (SIGMAL and ANTIMAL projects, BioMalPar Network of Excellence), and FP7 (MALSIG project and EviMalar network of Excellence) programmes of the European Commission.


  1. 1.
    Ward P et al (2004) Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genomics 5:79PubMedCrossRefGoogle Scholar
  2. 2.
    Schneider AG, Mercereau-Puijalon O (2005) A new Apicomplexa-specific protein kinase family: multiple members in Plasmodium falciparum, all with an export signature. BMC Genomics 6:30PubMedCrossRefGoogle Scholar
  3. 3.
    Tewari R et al (2010) The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 8:377–387PubMedCrossRefGoogle Scholar
  4. 4.
    Solyakov L et al (2011) Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat Commun 2:565PubMedCrossRefGoogle Scholar
  5. 5.
    Billker O et al (2004) Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 117:503–514PubMedCrossRefGoogle Scholar
  6. 6.
    McRobert L et al (2008) Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase. PLoS Biol 6:e139PubMedCrossRefGoogle Scholar
  7. 7.
    Reininger L et al (2005) A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites. J Biol Chem 280:31957–31964PubMedCrossRefGoogle Scholar
  8. 8.
    Reininger L 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 284:20858–20868PubMedCrossRefGoogle Scholar
  9. 9.
    Siden-Kiamos I et al (2006) Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion. Mol Microbiol 60:1355–1363PubMedCrossRefGoogle Scholar
  10. 10.
    Tewari R et al (2005) An atypical mitogen-activated protein kinase controls cytokinesis and flagellar motility during male gamete formation in a malaria parasite. Mol Microbiol 58:1253–1263PubMedCrossRefGoogle Scholar
  11. 11.
    Abdi A et al (2010) SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum. Cell Mol Life Sci 67:3355–3369PubMedCrossRefGoogle Scholar
  12. 12.
    Agarwal 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 112:1295–1310PubMedCrossRefGoogle Scholar
  13. 13.
    Dorin-Semblat D et al (2007) Functional ­characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics. Mol Microbiol 65:1170–1180PubMedCrossRefGoogle Scholar
  14. 14.
    Dorin-Semblat D et al (2011) Plasmodium falciparum NIMA-related kinase Pfnek-1: sex-specificity and assessment of essentiality for the erythrocytic asexual cycle. Microbiology 157:2785–2794PubMedCrossRefGoogle Scholar
  15. 15.
    Dorin-Semblat D et al (2008) Disruption of the PfPK7 gene impairs schizogony and sporogony in the human malaria parasite Plasmodium falciparum. Eukaryot Cell 7:279–285PubMedCrossRefGoogle Scholar
  16. 16.
    Fennell C et al (2009) PfeIK1, a eukaryotic initiation factor 2 alpha kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation. Malar J 8:99PubMedCrossRefGoogle Scholar
  17. 17.
    Holland Z et al (2009) Functional analysis of protein kinase CK2 of the human malaria parasite Plasmodium falciparum. Eukaryot Cell 8:388–397PubMedCrossRefGoogle Scholar
  18. 18.
    Rangarajan R et al (2005) A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei. EMBO Rep 6:464–469PubMedCrossRefGoogle Scholar
  19. 19.
    Reininger L et al (2011) An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony. Mol Microbiol 79:205–221PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang M et al (2010) The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the ­mosquito salivary glands. J Exp Med 207:1465–1474PubMedCrossRefGoogle Scholar
  21. 21.
    Philip N, Haystead TA (2007) Characterization of a UBC13 kinase in Plasmodium falciparum. Proc Natl Acad Sci USA 104:7845–7850PubMedCrossRefGoogle Scholar
  22. 22.
    Merckx A et al (2009) Anion channels in Plasmodium falciparum-infected erythrocytes and protein kinase A. Trends Parasitol 25:139–144PubMedCrossRefGoogle Scholar
  23. 23.
    Keller A et al (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74:5383–5392PubMedCrossRefGoogle Scholar
  24. 24.
    Nesvizhskii AI et al (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658PubMedCrossRefGoogle Scholar
  25. 25.
    Doerig C (2004) Protein kinases as targets for anti-parasitic chemotherapy. Biochim Biophys Acta 1697:155–168PubMedCrossRefGoogle Scholar
  26. 26.
    Billker O et al (2009) Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5:612–622PubMedCrossRefGoogle Scholar
  27. 27.
    Green JL et al (2008) The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. J Biol Chem 283:30980–30989PubMedCrossRefGoogle Scholar
  28. 28.
    Kato N et al (2008) Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. Nat Chem Biol 4:347–356PubMedCrossRefGoogle Scholar
  29. 29.
    Ranjan R et al (2009) Dissection of mechanisms involved in the regulation of Plasmodium falciparum calcium-dependent protein kinase 4. J Biol Chem 284:15267–15276PubMedCrossRefGoogle Scholar
  30. 30.
    Le Roch K et al (2000) Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue. J Biol Chem 275:8952–8958PubMedCrossRefGoogle Scholar
  31. 31.
    Li Z et al (2001) Influence of human p16(INK4) and p21(CIP1) on the in vitro activity of recombinant Plasmodium falciparum cyclin-dependent protein kinases. Biochem Biophys Res Commun 288:1207–1211PubMedCrossRefGoogle Scholar
  32. 32.
    Merckx A et al (2003) Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum. J Biol Chem 278:39839–39850PubMedCrossRefGoogle Scholar
  33. 33.
    Ubersa JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541CrossRefGoogle Scholar
  34. 34.
    Chen C, Turk BE (2010) Analysis of serine-threonine kinase specificity using arrayed positional scanning peptide libraries. Curr Protoc Mol Biol. Chapter 18, Unit 18.14Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Dominique Dorin-Semblat
    • 1
  • Andrew R. Bottrill
    • 2
  • Lev Solyakov
    • 2
  • Andrew Tobin
    • 2
  • Christian Doerig
    • 3
    Email author
  1. 1.Centre National de Transfusion SanguineUniversité ParisPARIS CEDEXFrance
  2. 2.MRC Toxicology UnitUniversity of LeicesterLeicesterUK
  3. 3.Department of MicrobiologyMonash UniversityClaytonAustralia

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