Dissection of Mechanisms Involved in the Regulation of Plasmodium falciparum Calcium-dependent Protein Kinase 4
2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês
10.1074/jbc.m900656200
ISSN1083-351X
AutoresRavikant Ranjan, Anwar Ahmed, Samudrala Gourinath, Pushkar Sharma,
Tópico(s)Computational Drug Discovery Methods
ResumoRecent studies have demonstrated that calcium-dependent protein kinases (CDPKs) are used by calcium to regulate a variety of biological processes in the malaria parasite Plasmodium. CDPK4 has emerged as an important enzyme for parasite development, because its gene disruption in rodent parasite Plasmodium berghei causes major defects in sexual differentiation of the parasite (Billker, O., Dechamps, S., Tewari, R., Wenig, G., Franke-Fayard, B., and Brinkmann, V. (2004) Cell 117 503-514). Despite these findings, it is not very clear how PfCDPK4 or any other PfCDPK is regulated by calcium at the molecular level. We report the biochemical characterization and elucidation of molecular mechanisms involved in the regulation of PfCDPK4. PfCDPK4 was detected on gametocyte periphery, and its activity in the parasite was regulated by phospholipase C. Even though the Junction Domain (JD) of PfCDPK4 shares moderate sequence homology with that of the plant CDPKs, it plays a pivotal role in PfCDPK4 regulation as previously reported for some plant CDPKs. The regions of the J-domain involved in interaction with both the kinase domain and the calmodulin-like domain were mapped. We propose a model for PfCDPK regulation by calcium, which may also prove useful for design of inhibitors against PfCDPK4 and other members of the PfCDPK family. Recent studies have demonstrated that calcium-dependent protein kinases (CDPKs) are used by calcium to regulate a variety of biological processes in the malaria parasite Plasmodium. CDPK4 has emerged as an important enzyme for parasite development, because its gene disruption in rodent parasite Plasmodium berghei causes major defects in sexual differentiation of the parasite (Billker, O., Dechamps, S., Tewari, R., Wenig, G., Franke-Fayard, B., and Brinkmann, V. (2004) Cell 117 503-514). Despite these findings, it is not very clear how PfCDPK4 or any other PfCDPK is regulated by calcium at the molecular level. We report the biochemical characterization and elucidation of molecular mechanisms involved in the regulation of PfCDPK4. PfCDPK4 was detected on gametocyte periphery, and its activity in the parasite was regulated by phospholipase C. Even though the Junction Domain (JD) of PfCDPK4 shares moderate sequence homology with that of the plant CDPKs, it plays a pivotal role in PfCDPK4 regulation as previously reported for some plant CDPKs. The regions of the J-domain involved in interaction with both the kinase domain and the calmodulin-like domain were mapped. We propose a model for PfCDPK regulation by calcium, which may also prove useful for design of inhibitors against PfCDPK4 and other members of the PfCDPK family. Plasmodium falciparum is an intracellular protozoan parasite, which propagates in human erythrocyte and hepatocytes. It causes millions of deaths annually, which occur mostly in third world countries. It is very important to understand the molecular events involved in parasite development and growth in detail to develop effective drugs against this disease. In the human host, the parasite from an infected mosquito bite enters the bloodstream as a sporozoite. The sporozoite migrates and resides in the hepatocytes and replicates to yield merozoites. Upon rupture of the host cells, released merozoites invade red blood cells (RBCs), 3The abbreviations used are: RBC, red blood corpuscles; aa, amino acid(s); CDPK, calcium-dependent protein kinase; CLD, calmodulin-like domain; GST, glutathione S-transferase; JD, junctional domain; MBP, myelin basic protein; PLC, phospholipase C; RT, reverse transcription; TFE, trifluoroethanol; PfCDPK1, P. falciparum calcium-dependent protein kinase 1; Pep, peptide.3The abbreviations used are: RBC, red blood corpuscles; aa, amino acid(s); CDPK, calcium-dependent protein kinase; CLD, calmodulin-like domain; GST, glutathione S-transferase; JD, junctional domain; MBP, myelin basic protein; PLC, phospholipase C; RT, reverse transcription; TFE, trifluoroethanol; PfCDPK1, P. falciparum calcium-dependent protein kinase 1; Pep, peptide. and the asexual development takes place inside the RBC resulting in the formation of ring and trophozoite stages. During the schizont stage, the parasite undergoes nuclear division resulting in the formation of ∼24 merozoites, which infect fresh RBCs upon release. Some parasites commit to sexual differentiation resulting in the formation of male and female gametocytes. Upon ingestion into the mosquito gut, gametocytes are activated in response to environmental cues like lower temperature and xantheurenic acid (2Arai M. Billker O. Morris H.R. Panico M. Delcroix M. Dixon D. Ley S.V. Sinden R.E. Mol. Biochem. Parasitol. 2001; 116: 17-24Crossref PubMed Scopus (65) Google Scholar, 3Billker O. Lindo V. Panico M. Etienne A.E. Paxton T. Dell A. Rogers M. Sinden R.E. Morris H.R. Nature. 1998; 392: 289-292Crossref PubMed Scopus (454) Google Scholar). The male gametocyte undergoes endomitotic division followed by exflagellation. After fertilization, the zygote gives rise to a motile ookinete. Subsequently, an oocyst is formed that attaches to the outside wall of the gut.It is clear that calcium controls a wide variety of processes in the parasite (4Garcia C.R. Parasitol. Today. 1999; 15: 488-491Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Gazarini M.L. Garcia C.R. Biochem. Biophys. Res. Commun. 2004; 321: 138-144Crossref PubMed Scopus (62) Google Scholar), like invasion (6McCallum-Deighton N. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 317-323Crossref PubMed Scopus (45) Google Scholar, 7Kato N. Sakata T. Breton G. Le Roch K.G. Nagle A. Andersen C. Bursulaya B. Henson K. Johnson J. Kumar K.A. Marr F. Mason D. McNamara C. Plouffe D. Ramachandran V. Spooner M. Tuntland T. Zhou Y. Peters E.C. Chatterjee A. Schultz P.G. Ward G.E. Gray N. Harper J. Winzeler E.A. Nat. Chem. Biol. 2008; 4: 347-356Crossref PubMed Scopus (188) Google Scholar, 8Vaid A. Thomas D.C. Sharma P. J. Biol. Chem. 2008; 283: 5589-5597Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), migration (9Ishino T. Orito Y. Chinzei Y. Yuda M. Mol. Microbiol. 2006; 59: 1175-1184Crossref PubMed Scopus (183) Google Scholar), gametogenesis (1Billker O. Dechamps S. Tewari R. Wenig G. Franke-Fayard B. Brinkmann V. Cell. 2004; 117: 503-514Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 10Kawamoto F. Alejo-Blanco R. Fleck S.L. Kawamoto Y. Sinden R.E. Mol. Biochem. Parasitol. 1990; 42: 101-108Crossref PubMed Scopus (93) Google Scholar), and circadian rhythms (11Hotta C.T. Gazarini M.L. Beraldo F.H. Varotti F.P. Lopes C. Markus R.P. Pozzan T. Garcia C.R. Nat. Cell Biol. 2000; 2: 466-468Crossref PubMed Scopus (158) Google Scholar). Detailed understanding of calcium-mediated signaling pathways could provide insights into novel molecular mechanisms involved in parasite development. For instance, calcium regulation of a protein kinase B-like enzyme (12Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) via calmodulin seems to be important for erythrocyte invasion (8Vaid A. Thomas D.C. Sharma P. J. Biol. Chem. 2008; 283: 5589-5597Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Plasmodium contains calcium-dependent protein kinases (CDPKs) (13Ward P. Equinet L. Packer J. Doerig C. BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (401) Google Scholar), which have been found only in plants and some protists but are absent from almost all metazoans (14Harper J.F. Harmon A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 555-566Crossref PubMed Scopus (309) Google Scholar). These enzymes have a catalytic domain at their N terminus and a C-terminal calmodulin-like domain (CLD) composed of four calcium-binding EF-hand motifs (14Harper J.F. Harmon A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 555-566Crossref PubMed Scopus (309) Google Scholar), a junction domain (JD) separates the catalytic domain from the CLD. Biochemical studies done on plant CDPKs have revealed that the JD regulates CDPK activity by interacting with both the kinase domain and the CLD (14Harper J.F. Harmon A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 555-566Crossref PubMed Scopus (309) Google Scholar, 15Harmon A.C. Yoo B.C. McCaffery C. Biochemistry. 1994; 33: 7278-7287Crossref PubMed Scopus (147) Google Scholar). Work reported here reveals that, despite modest homology between the amino acid sequences of JD of plant CDPKs (like AtCPK1 and Soybean CDPKα) and PfCDPK4, there are some similarities between the mechanism of regulation between these kinases.Plasmodium has at least five CDPKs (13Ward P. Equinet L. Packer J. Doerig C. BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (401) Google Scholar), and the importance of CDPKs in Plasmodium signaling has been highlighted by recent gene disruption studies. PfCDPK1 seems to be an essential gene as its disruption has not been possible in P. falciparum (7Kato N. Sakata T. Breton G. Le Roch K.G. Nagle A. Andersen C. Bursulaya B. Henson K. Johnson J. Kumar K.A. Marr F. Mason D. McNamara C. Plouffe D. Ramachandran V. Spooner M. Tuntland T. Zhou Y. Peters E.C. Chatterjee A. Schultz P.G. Ward G.E. Gray N. Harper J. Winzeler E.A. Nat. Chem. Biol. 2008; 4: 347-356Crossref PubMed Scopus (188) Google Scholar). Studies performed using an inhibitor against this kinase suggest that it may be important for RBC invasion and egress (7Kato N. Sakata T. Breton G. Le Roch K.G. Nagle A. Andersen C. Bursulaya B. Henson K. Johnson J. Kumar K.A. Marr F. Mason D. McNamara C. Plouffe D. Ramachandran V. Spooner M. Tuntland T. Zhou Y. Peters E.C. Chatterjee A. Schultz P.G. Ward G.E. Gray N. Harper J. Winzeler E.A. Nat. Chem. Biol. 2008; 4: 347-356Crossref PubMed Scopus (188) Google Scholar). Reverse genetic studies done in P. berghei suggest that CDPK3 may play an important role in ookinete gliding motility and invasion (9Ishino T. Orito Y. Chinzei Y. Yuda M. Mol. Microbiol. 2006; 59: 1175-1184Crossref PubMed Scopus (183) Google Scholar, 16Siden-Kiamos I. Ecker A. Nyback S. Louis C. Sinden R.E. Billker O. Mol. Microbiol. 2006; 60: 1355-1363Crossref PubMed Scopus (135) Google Scholar). The gene disruption of CDPK4 in P. berghei caused severe defects in sexual reproduction and mosquito transmission (1Billker O. Dechamps S. Tewari R. Wenig G. Franke-Fayard B. Brinkmann V. Cell. 2004; 117: 503-514Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). These studies highlighted the importance of CDPK4 in Plasmodium biology and suggested that it may serve as a target for transmission-blocking drugs. The biochemical mechanisms involved in the regulation of this enzyme have remained unknown. In this study, we have explored the molecular and cellular mechanisms involved in the regulation of PfCDPK4.EXPERIMENTAL PROCEDURESP. falciparum Cultures—P. falciparum 3D7 strain was cultured at 37 °C in RPMI 1640 medium, supplemented either with 0.5% Albumax II (Invitrogen) or 10% AB+ human serum as described earlier (12Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 17Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6077) Google Scholar). For sexual stage studies, 3D7A, a variant of P. falciparum strain 3D7, was used to obtain gametocyte-enriched culture as described previously (18Williams J.L. Am. J. Trop. Med. Hyg. 1999; 60: 7-13Crossref PubMed Scopus (62) Google Scholar).Molecular Cloning and Site-directed Mutagenesis of PfCDPK4—Initially, PfCDPK4 gene sequence was obtained by using either TgCDPK1 or the published sequence of other CDPKs to BLAST search the P. falciparum genome sequence. Subsequently, PlasmoDb annotation (19Bahl A. Brunk B. Crabtree J. Fraunholz M.J. Gajria B. Grant G.R. Ginsburg H. Gupta D. Kissinger J.C. Labo P. Li L. Mailman M.D. Milgram A.J. Pearson D.S. Roos D.S. Schug J. Stoeckert Jr., C.J. Whetzel P. Nucleic Acids Res. 2003; 31: 212-215Crossref PubMed Scopus (287) Google Scholar) appeared in the public domain, and the gene sequence PF07_0072, which matched the PfCDPK4 sequence obtained by our studies. For PCR amplification, primers based on the nucleotide sequence of the PfCDPK4 gene were used. Total RNA from asynchronous P. falciparum cultures was used for reverse transcription (RT) along with random hexamers provided with the RT-PCR kit (Invitrogen). Both cDNA or genomic DNA were used as template for PCR. The reaction was carried out using Hi-fi Platinum Taq polymerase (Invitrogen) with the following cycling parameters: 94 °C for 2 min initial denaturation followed by 30 cycles at 94 °C for 30 s, 45 °C for 30 s, 68 °C for 2 min, and final extension at 72 °C for 10 min. Following sets of primers were used for amplification of the full-length gene PfCDPK4 forward (CDPK4F): 5′-ATGGGACAAGAGGTATCGAGTGTTAACAA-3′ and PfCDPK4 reverse (CDPK4R): 5′-TTAATAATTACAAAGTTTGACTAGCATAT-3′. PCR products were cloned in pGEM-T easy vector (Promega, Madison, WI), and the sequence for the cloned PfCDPK4 gene was obtained by automated DNA sequencing. For cloning in the expression vector pGEX4T1, PfCDPK4 or its variants were amplified using primers with overhangs containing restriction sites for SmaI and XhoI. All site-directed mutagenesis studies were performed using the QuikChange kit (Stratagene) following the standard protocol described by the manufacturer. Primer sets used for making mutants are provided in the supplemental materials.Expression and Purification of Recombinant Proteins—Plasmid DNA was transformed in Escherichia coli BL21-RIL (Stratagene) strain for the expression of GST-PfCDPK4 and its mutants. Protein expression was induced by overnight incubation of cells with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside at 18-20 °C. Subsequently, cell pellets were suspended in ice-cold lysis buffer, containing 50 mm Tris, pH 7.4, 2 mm EDTA, 1 mm dithiothreitol, 1% Triton X-100, and proteases inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin), and sonication was performed for 6 cycles of 1 min each. The resulting cell debris was removed by centrifugation at 20,000 × g for 40 min at 4 °C. Fusion proteins from the cell lysates were affinity-purified using glutathione-Sepharose resin as described previously (20Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Briefly, the resin was washed with lysis buffer, and bound proteins were eluted with 50 mm Tris, pH 8.0, with 10 mm glutathione. Finally, purified proteins were dialyzed against 50 mm Tris, pH 7.4, 1 mm dithiothreitol, and 10% glycerol. Protein concentration was determined by densitometry analysis of Coomassie-stained gels.For expression of PfCDPK1, PfCDPK1 (PFB0815w) was amplified from the asexual stage cDNA of P. falciparum and cloned in BamHI and XhoI pET-28a vector (see Table S1 in supplemental material for primer sequences) to facilitate its expression as a His6-tagged protein. Briefly, plasmid transformed BL21(DE3)RIL strain of Escherichia coli was grown in LB media containing 50 μg/ml kanamycin and 35 μg/ml chloramphenicol, and protein expression was induced using 1 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C for 16 h. Cells were harvested by centrifugation at 6000 rpm for 10 min at 4 °C, resuspended in lysis buffer (50 mm potassium phosphate, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, and 1 mm dithiothreitol), sonicated for 8 cycles of 1 min each. The soluble protein was incubated with nickel-nitrilotriacetic acid-agarose (Qiagen) with end-to-end shaking for 6 h at 4 °C, and the protein was eluted with 50 mm potassium phosphate, pH 8.0, 500 mm NaCl, 0.1% Nonidet P-40, and 1 mm dithiothreitol containing 300 mm imidazole and dialyzed against 50 mm potassium phosphate, pH 7.4, 1 mm dithiothreitol, and 10% glycerol.Assay of PfCDPK4 Activity—The catalytic activity of PfCDPK4, PfCDPK1, or their variants was assayed in a buffer containing 50 mm Tris, pH 7.5, 10 mm magnesium chloride, 1 mm dithiothreitol, and 100 μm [γ-32P]ATP (6000 Ci/mmol). Either 6 μg of myelin basic protein (MBP) or 150 μm Syntide-2 (PLARTLSVAGLPGKK, custom synthesized by Peptron Inc., South Korea) was used as phosphate-acceptor substrate. Reactions were performed in the presence of 2 mm calcium chloride or 2 mm EGTA (0 mm Ca2+) for 40 min at 30 °C. When MBP was used as the substrate, reactions were stopped by boiling the assay mix for 5 min followed by SDS-PAGE. Phosphate incorporation was adjudged by autoradiography of SDS-PAGE gels. When Syntide-2 was used as substrate, reactions were stopped by spotting the reaction mix on P81 phosphocellulose paper (Millipore), followed by washing of the paper strips with 75 mm ortho-phosphoric acid. Phosphate incorporation was assessed by scintillation counting of P81 paper. In PfCDPK4 inhibition assays, peptide inhibitors were preincubated with proteins in a kinase assay buffer at 25 °C for 30-60 min prior to the addition of substrate and ATP.Generation of Anti-PfCDPK4 Serum, Immunoblotting, Immunoprecipitation, and Immunofluorescence—A synthetic peptide (KMMTSKDNLNIDIPS) based on PfCDPK4 sequence was custom synthesized (Peptron Inc.), conjugated to keyhole limpet hemocyanin via an additional N terminus cysteine residue, and was used to raise antisera against PfCDPK4. Cell-free protein extracts were prepared from specific parasite stages as described previously (8Vaid A. Thomas D.C. Sharma P. J. Biol. Chem. 2008; 283: 5589-5597Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). After separation on 10% SDS-PAGE gel, lysate proteins were transferred to a nitrocellulose membrane. Immunoblotting was performed using anti-PfCDPK4 antisera, and blots were developed using West-pico chemiluminescence (Pierce) reagent following the manufacturer's instructions. For immunofluorescence assays, thin blood smears of parasite cultures were fixed with cold methanol, and a previously published protocol was followed (20Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The microscopy was performed on a Zeiss Axio Imager fluorescence microscope, and images were processed using the AxioVision software.Inhibitor Treatment and Immunoprecipitation—Gametocytes were distributed in six-well culture dishes and treated with various inhibitors for 30 min. Subsequently, protein lysates were prepared in a buffer containing 10 mm Tris, pH 7.4, 100 mm NaCl, 5 mm EDTA, 1% Triton X-100, phosphatase inhibitors (20 μm NaF, 20 μm β-glycerophosphate, and 100 μm sodium vanadate) and protease inhibitor mixture (Roche Applied Science). 100 μg of soluble lysate protein was incubated with PfCDPK4 anti-sera for 6 h at 4 °C. Subsequently, Protein A+G-Sepharose (Amersham Biosciences) was added to the antibody-protein complex and incubated on a end-to-end shaker for 2 h. After washing with phosphate-buffered saline-Sepharose, beads were suspended in the lysis buffer. PfCDPK4-IP was used for kinase assays as described above for the recombinant protein.Homology Modeling—The CLD-J domain shares ∼51% similarity with the CDPK from Arabidopsis thaliana AtCPK-1. The homology model of CLD-JD was determined using Swiss Model from EMBL. The template model used was CLD-JD of AtCPK-1, which was crystallized as a dimer. The J-domain helices from the two monomers were swapped with each other in this structure (21Chandran V. Stollar E.J. Lindorff-Larsen K. Harper J.F. Chazin W.J. Dobson C.M. Luisi B.F. Christodoulou J. J. Mol. Biol. 2006; 357: 400-410Crossref PubMed Scopus (53) Google Scholar). Therefore, the initial homology model generated for the complementary CLD-J domain for PfCDPK4 was also a dimer. To understand the interaction of this helix (Gln358-Lys371) with CLD of the monomer, this helix was rotated and translated keeping residues 372-375 as the flexible linker region and superimposed on to the helix from the other monomer, which resulted in the initial model for the CLD-J domain monomer. Initially, these flexible linker residues (372-375) were locally minimized using COOT (22Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22799) Google Scholar), and the overall structure was refined with slow cooling using annealing of CNS (23Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar) to remove all the short contacts. Finally, the model quality was checked with the Procheck software (24Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4329) Google Scholar).CD—CD spectra were obtained on a Jasco J-710 spectropolarimeter with a constant dispersion of 1 nm. Spectra were measured with a time constant of 1s, scan speed of 100 nm/min. Signals were averaged 10 times before measurement. The peptide concentration was between 185 and 350 μm, and a 0.1-cm path length cell was used.RESULTSMolecular Cloning of PfCDPK4 Gene—Gene-specific primers were used to amplify PfCDPK4 from genomic DNA and cDNA. The larger size of the PCR product obtained from genomic PCR suggested the presence of introns (data not shown) in PfCDPK4. Sequencing of RT-PCR product confirmed that PfCDPK4 contains an intron of 347 nucleotides (Fig. 1A), which was similar to the predictions made by PlasmoDb. The deduced amino acid sequence suggested that PfCDPK4 possesses a calmodulin-like domain at the C terminus and an N-terminal serine/threonine kinase domain, which are characteristic of CDPKs (14Harper J.F. Harmon A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 555-566Crossref PubMed Scopus (309) Google Scholar). PfCDPK4 catalytic domain possesses all 11 sub-domains that are representative of most protein kinases (supplemental Fig. S1). A glycine residue at the second position suggests that it has a putative myristoylation signal, a similar myristoylation signal in PfCDPK1 is important for its membrane targeting (25Moskes C. Burghaus P.A. Wernli B. Sauder U. Durrenberger M. Kappes B. Mol. Microbiol. 2004; 54: 676-691Crossref PubMed Scopus (92) Google Scholar). The CLD of PfCDPK4 consists of an N and C lobe and each lobe comprises of two EF-hand motifs (Fig. 1B). A ∼34 amino acid (aa) junction domain links the CLD and the kinase domain.PfCDPK4 Is Expressed in Sexual Stages of the Parasite Life Cycle—Even though previously published transcriptome in PlasmoDb and proteome (26Khan S.M. Franke-Fayard B. Mair G.R. Lasonder E. Janse C.J. Mann M. Waters A.P. Cell. 2005; 121: 675-687Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) analyses suggested gametocyte-specific expression of PfCDPK4, localization of CDPK4 in the parasite had remained unknown. PfCDPK4-specific antisera were raised against a synthetic peptide corresponding to a unique motif in the J-domain, and Western blots were performed using protein lysates from different asexual stages as well as the gametocytes. A band corresponding to ∼60 kDa, which was consistent with the predicted molecular mass of PfCDPK4, was observed only in the gametocyte lysates (Fig. 2A). These data confirmed that PfCDPK4 is expressed mainly in the sexual stages. To further confirm this, immunofluorescence studies were performed, which revealed PfCDPK4 staining mainly on gametocyte periphery (Fig. 2B). Control assays performed with pre-immune sera did not show any staining (Fig. 2A, left panel). It is likely that the presence of a myristoylation signal in this kinase (Fig. 1B) is responsible for targeting this kinase to the parasite periphery.FIGURE 2Stage-specific expression and localization of PfCDPK4. A, Western blot was performed using PfCDPK4 antisera and protein lysates from different parasitic stages: R, ring; T, trophozoite; S, schizont; and G, gametocyte. A ∼60-kDa band corresponding to the predicted size of PfCDPK4 was observed mainly in the gametocyte stages. Antiserum prepared from preimmune bleeds was used as a control (left panel). B, immunofluorescence assays were performed on gametocyte smears using anti-PfCDPK4 antisera and Alexa-594-anti rabbit IgG. Parasite nucleus was stained with Hoechst 33224 (blue).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Calcium Stimulates Autophosphorylation and Catalytic Activity of Recombinant PfCDPK4—To characterize PfCDPK4, it was expressed as a GST fusion protein in E. coli. The recombinant PfCDPK4 was active only in the presence of calcium as adjudged by its ability to phosphorylate MBP (Fig. 3A). Recombinant PfCDPK4 also exhibited calcium-dependent autophosphorylation (Fig. 3A). A small peptide, syntide-2, which has been used as an in vitro substrate for several CDPKs (15Harmon A.C. Yoo B.C. McCaffery C. Biochemistry. 1994; 33: 7278-7287Crossref PubMed Scopus (147) Google Scholar, 27Hashimoto Y. Soderling T.R. Arch. Biochem. Biophys. 1987; 252: 418-425Crossref PubMed Scopus (149) Google Scholar, 28Yoo B.C. Harmon A.C. Biochemistry. 1996; 35: 12029-12037Crossref PubMed Scopus (55) Google Scholar) was effectively phosphorylated by PfCDPK4 in a calcium-dependent manner (Fig. 3B).FIGURE 3Recombinant PfCDPK4 is autophosphorylated and activated by calcium. A, PfCDPK4 was expressed as a GST fusion protein and purified by affinity chromatography. Protein kinase assays were performed using recombinant GST-PfCDPK4 in the presence of 2 mm CaCl2 or 2 mm EGTA (absence of calcium) with MBP as phosphor-acceptor substrate. Subsequently, the kinase assay mix was electrophoresed on a SDS-PAGE gel, and phosphorimaging was performed. PfCDPK4 phosphorylates MBP only in the presence of calcium (lane 1). PfCDPK4 also exhibited calcium-dependent autophosphorylation. B, kinase assay was performed as described in panel A except syntide was used as a substrate instead of MBP. PfCDPK4 effectively phosphorylated syntide in the presence of calcium. The activity is measured in nanomoles/mg/min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phospholipase C Acts as an Upstream Regulator of PfCDPK4 in the Parasite—It has been demonstrated that the release of calcium from intracellular stores in Plasmodium is controlled by PLC (4Garcia C.R. Parasitol. Today. 1999; 15: 488-491Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29Gazarini M.L. Thomas A.P. Pozzan T. Garcia C.R. J. Cell Biol. 2003; 161: 103-110Crossref PubMed Scopus (119) Google Scholar), which is used by the parasite for various purposes. A strong correlation between increase in the levels of PLC hydrolysis product, inositol 1,4,5-trisphosphate, and gametogenesis has also been reported (30Martin S.K. Jett M. Schneider I. J. Parasitol. 1994; 80: 371-378Crossref PubMed Scopus (50) Google Scholar), which is suggestive of a role for PLC in this important parasitic process. Given the dependence of recombinant PfCDPK4 on calcium and its role in gamete formation, it was worth exploring whether PLC regulates its activity in the parasite. Gametocytes were treated with PLC inhibitor, which have been used successfully in Plasmodium (8Vaid A. Thomas D.C. Sharma P. J. Biol. Chem. 2008; 283: 5589-5597Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 12Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 29Gazarini M.L. Thomas A.P. Pozzan T. Garcia C.R. J. Cell Biol. 2003; 161: 103-110Crossref PubMed Scopus (119) Google Scholar), and the activity of immunoprecipitated PfCDPK4 was assayed. In one of the experiments, an intracellular chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), was included (Fig. 4A). This inhibitor caused a significant decrease in PfCDPK4 activity, which emphasized the importance of intracellular calcium on PfCDPK4 activation. Treatment with the PLC inhibitor, U73122, caused a significant decrease in PfCDPK4 kinase activity. In contrast, U73343, the inactive analogue of this inhibitor, failed to alter the activity of this kinase. The levels of PfCDPK4 did not change significantly upon inhibitor treatment (Fig. 4B). These data suggest that PLC acts as a regulator of PfCDPK4, which is most likely a result of its ability to control levels of free calcium in the parasite.FIGURE 4Phospholipase C acts as an upstream regulator of PfCDPK4. A, gametocytes were treated with DMSO, 30 μm U73122 or U73343, 100 μm 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM). Subsequently, PfCDPK4 was immunoprecipitated from protein lysates (see "Experimental Procedures") and PfCDPK4-IP associated kinase activity was assayed using syntide as substrate. B, immunoblotting was performed on protein lysates from the experiments described in panel A using anti-PfCDPK4 antisera.View Large Image Figure ViewerDownload Hi-res image Download (PPT)J-domain Is Responsible for PfCDPK4 Activation—Deletion and truncation mutants of PfCDPK4 domains were created to understand its regulation (Fig. 5A), and the activity of recombinant mutant proteins was determined by performing in vitro kinase assays. To evaluate the role of the J-domain in the acti
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