An Essential Role for the Plasmodium Nek-2 Nima-related Protein Kinase in the Sexual Development of Malaria Parasites
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.017988
ISSN1083-351X
AutoresLuc Reininger, Rita Tewari, Clare Fennell, Zoe Holland, J. P. Dean Goldring, Lisa Ranford‐Cartwright, Oliver Billker, Christian Doerig,
Tópico(s)Research on Leishmaniasis Studies
ResumoThe molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission. The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission. Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is a major public health problem in the developing world (1Breman J.G. Alilio M.S. Mills A. Am. J. Trop. Med. Hyg. 2004; 71: 1-15Crossref PubMed Scopus (482) Google Scholar). The species responsible for the vast majority of lethal cases is Plasmodium falciparum. The life cycle of malaria parasites consists of a succession of developmental stages: asexual multiplication occurs in the human host (first in a single round of schizogony in a hepatocyte infected by a sporozoite injected by the mosquito vector, and then multiple rounds of schizogony in erythrocytes), whereas the sexual cycle is initiated by the formation of cell cycle-arrested gametocytes in infected erythrocytes and proceeds, in the midgut of the mosquito vector, to gametogenesis, fertilization, and formation of a motile ookinete. The ookinete crosses the midgut epithelium and establishes an oocyst at the basal lamina, in which sporogony occurs, generating sporozoites that render the vector infectious once they reach its salivary glands. The alternation of proliferative and non-proliferative phases implies that the control of cell cycle progression is of prime importance for completion of the life cycle of the parasite. The NIMA-related protein kinases (Neks) 5The abbreviations used are: NekNIMA-related protein kinasesRTreverse transcriptaseMAPKmitogen-activated protein kinaseGFPgreen fluorescent proteinRTreverse transcriptaseDHFRdihydrofolate reductaseTRITCtetramethylrhodamine isothiocyanateUTRuntranslated regionGSTglutathione S-transferaseMBPmyelin basic protein. 5The abbreviations used are: NekNIMA-related protein kinasesRTreverse transcriptaseMAPKmitogen-activated protein kinaseGFPgreen fluorescent proteinRTreverse transcriptaseDHFRdihydrofolate reductaseTRITCtetramethylrhodamine isothiocyanateUTRuntranslated regionGSTglutathione S-transferaseMBPmyelin basic protein. constitute an extended family of eukaryotic mitotic serine/threonine kinases. The best characterized members of the Nek family include NIMA (never in mitosis/Aspergillus), the founding member from the fungus Aspergillus nidulans (2Oakley B.R. Morris N.R. J. Cell Biol. 1983; 96: 1155-1158Crossref PubMed Scopus (93) Google Scholar), and its closest homologue in mammals, Nek2 (3Schultz S.J. Fry A.M. Sutterlin C. Ried T. Nigg E.A. Cell Growth & Differ. 1994; 5: 625-635PubMed Google Scholar, 4Fry A.M. Meraldi P. Nigg E.A. EMBO J. 1998; 17: 470-481Crossref PubMed Scopus (343) Google Scholar). Initially identified as a kinase essential for mitotic entry in Aspergillus, NIMA has been also shown to participate in nuclear membrane fission (5Davies J.R. Osmani A.H. De Souza C.P. Bachewich C. Osmani S.A. Eukaryot. Cell. 2004; 3: 1433-1444Crossref PubMed Scopus (21) Google Scholar). Eleven members of the NIMA kinase family (Nek1–11) have now been identified in various human tissues, and together fulfill a number of cell cycle-related functions in centrosome separation, mitosis, meiosis, and checkpoint control (reviewed in Ref. 6O'Regan L. Blot J. Fry A.M. Cell Div. 2007; 2: 25Crossref PubMed Google Scholar). It has been proposed that expansion of the Nek family accompanied the evolution of a complex system for the coordination of progression through the cell cycle with the replication of cellular components such as cilia, basal bodies, and centrioles. Several human Neks have C-terminal extensions to their catalytic domain, which contain regulatory elements (e.g. PEST sequences that function as target for cell cycle-dependent proteolytic degradation, or coiled-coil domains mediating dimerization). Nek6 and Nek7 have no large extensions, but bind to the C-terminal non-catalytic tail of Nek9, an enzyme that becomes activated during mitosis and is likely to be responsible for the activation of Nek6 (7Belham C. Roig J. Caldwell J.A. Aoyama Y. Kemp B.E. Comb M. Avruch J. J. Biol. Chem. 2003; 278: 34897-34909Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). This may represent a novel cascade of mitotic NIMA family protein kinases whose combined function is important for mitotic progression. NIMA-related protein kinases reverse transcriptase mitogen-activated protein kinase green fluorescent protein reverse transcriptase dihydrofolate reductase tetramethylrhodamine isothiocyanate untranslated region glutathione S-transferase myelin basic protein. NIMA-related protein kinases reverse transcriptase mitogen-activated protein kinase green fluorescent protein reverse transcriptase dihydrofolate reductase tetramethylrhodamine isothiocyanate untranslated region glutathione S-transferase myelin basic protein. The P. falciparum kinome includes four NIMA-related serine/threonine kinases (8Ward P. Equinet L. Packer J. Doerig C. BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (403) Google Scholar). Pfnek-1 (PlasmoDB identifier PFL1370w) clusters within the Aspergillus NIMA/human Nek2 branch in phylogenetic trees, whereas clear orthology to mammalian or yeast Neks could not be assigned for the three other P. falciparum sequences (Pfnek-2, -3, and -4, PlasmoDB identifiers PFE1290w, PFL0080c, and MAL7P1.100, respectively) (9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Microarray data (10Le Roch K.G. Zhou Y. Blair P.L. Grainger M. Moch J.K. Haynes J.D. De La Vega P. Holder A.A. Batalov S. Carucci D.J. Winzeler E.A. Science. 2003; 301: 1503-1508Crossref PubMed Scopus (1019) Google Scholar) available in the PlasmoDB data base (11Bahl 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 (288) Google Scholar) indicate that Pfnek-1 is expressed in asexual and sexual stages, whereas mRNA encoding the other three enzymes is predominantly or exclusively expressed in gametocytes, suggesting a possible role in the sexual development of the parasite. Consistent with this hypothesis, we previously showed that rodent malaria parasites Plasmodium berghei lacking the Nek-4 enzyme are unable to complete DNA replication to 4C in the zygote prior to meiosis (9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Pfnek-1, -3, and -4 have been characterized at the biochemical level and are active as recombinant enzymes (9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 12Dorin D. Le Roch K. Sallicandro P. Alano P. Parzy D. Poullet P. Meijer L. Doerig C. Eur. J. Biochem. 2001; 268: 2600-2608Crossref PubMed Scopus (106) Google Scholar, 13Lye Y.M. Chan M. Sim T.S. FEBS Lett. 2006; 580: 6083-6092Crossref PubMed Scopus (39) Google Scholar). Pfnek-1 and Pfnek-3 have surprisingly been implicated as possible regulators of an atypical mitogen-activated protein kinase (MAPK), as both enzymes synergize with the Pfmap-2 MAPK in vitro (12Dorin D. Le Roch K. Sallicandro P. Alano P. Parzy D. Poullet P. Meijer L. Doerig C. Eur. J. Biochem. 2001; 268: 2600-2608Crossref PubMed Scopus (106) Google Scholar, 13Lye Y.M. Chan M. Sim T.S. FEBS Lett. 2006; 580: 6083-6092Crossref PubMed Scopus (39) Google Scholar); the physiological relevance of these observations remains to be demonstrated. Here, we demonstrate that Pfnek-2, like the other three members of the P. falciparum Nek family, is a bona fide protein kinase. Analysis of the expression pattern demonstrates that low levels of Pfnek-2 mRNA are actually detectable in asexual parasites, even though transgenic parasites expressing a green fluorescent protein (GFP)-tagged Pfnek-2 under the control of its cognate promoter display female gametocyte-specific expression. To investigate the function of this kinase, parasite clones with a disrupted nek-2 gene were generated in P. falciparum and P. berghei; transmission experiments identified an important role for nek-2 in sexual development: nek-2− parasites are able to differentiate into mature gametocytes and to undergo gametogenesis, but do not develop into ookinetes. Further investigations on the pbnek-2− parasites showed that pre-meiotic DNA replication is dysregulated in the mutant clones. Oligonucleotides (forward, OL10 GGGGGATCCATGTCTAAACCCAAAATG; reverse, OL9 GGGGGTCGACTCAAATTTGGCTATTCCT) were designed to contain the start and stop codons of the full-length, 8-exon Pfnek-2 open reading frame combining the prediction by the Glimmer algorithm and Pf annotation on PlasmoDB (gene identifier PFE1290w), as well as BamHI and SalI restriction sites (underlined), respectively. The open reading frame was amplified from a gametocyte cDNA library (a gift from Pietro Alano) and the 883-bp amplified product was inserted between the BamHI and SalI sites of the pGEX-4T3 vector yielding the plasmid pGEX-Pfnek-2. Catalytically inactive recombinant pGEX-K38M-Pfnek-2 was obtained by site-directed mutagenesis using the overlap extension PCR technique. A similar strategy was used to generate pGEX-T169A-Pfnek-2 mutant. All inserts were verified by DNA sequencing prior to expression of the recombinant proteins in Escherichia coli (strain BL21-CodonPlus). Briefly, cells were grown at 37 °C until an A600 of 0.6 was reached, at which time expression of Pfnek-2 was induced by the addition of 0.2 mm isopropyl β-d-galactoside. Expression was induced for 3 h and then harvested by centrifugation. Purification of wild-type and mutated GST-Pfnek-2 was performed following published procedures (9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). SDS-PAGE analysis of purified GST-Pfnek-2 revealed a band corresponding to 59 kDa, the predicted molecular mass of the GST-Pfnek-2 fusion protein. Kinase assays were performed in a standard reaction (30 μl) containing 25 mm Tris-HCl, pH 7.5, 15 mm MgCl2, 2 mm MnCl2, 15 mm ATP, 5 μCi of [γ-32P]ATP (3000 Ci/mmol, Amersham Biosciences), and 5 μg of substrate (β-casein, myelin basic protein (MBP), or histone H1 (purchased from Sigma)). Reactions were initiated by the addition of 1 μg of the recombinant wild-type or mutated Pfnek-2. The reaction proceeded for 30 min at 30 °C and was stopped by the addition of Laemmli buffer, boiled for 3 min, and analyzed by electrophoresis on 12% SDS-polyacrylamide gel. The gels were dried and submitted to autoradiography. Asexual stages of the 3D7 clone of P. falciparum, its F12 subclone, and the 3D7 transfectants described in this paper were grown in human erythrocytes as described previously using Albumax I instead of human serum (14Ringwald P. Meche F.S. Bickii J. Basco L.K. J. Clin. Microbiol. 1999; 37: 700-705Crossref PubMed Google Scholar). Gametocytes were prepared according to the protocol of Carter et al. (15Carter R. Ranford-Cartwright L. Alano P. Methods Mol. Biol. 1993; 21: 67-88PubMed Google Scholar). Parasites were released from infected erythrocytes by saponin (0.1% w/v) lysis, washed in phosphate-buffered saline, pH 7.5, and kept frozen at −80 °C until use. Total RNA samples were extracted from parasite pellets using TRIzol lysis solution (Invitrogen). DNase treatment of RNA samples prior to RT-PCR was performed by incubation at 37 °C for 30 min using the RQ1 RNase-free DNase purchased from Promega. The DNase was inactivated by incubation at 65 °C for 10 min. RT-PCRs were performed with 500 ng of total RNA/reaction using the ImPromII reverse transcription system purchased from Promega. In control reactions reverse transcriptase was omitted, and only Taq polymerase (TaKaRa) was present. The RT reactions were incubated at 42 °C for 1 h. For the PCR (30 cycles at 94 °C for 45 s, 55 °C for 45 s, and 68 °C for 2 min 30 s), Pfnek-2-specific primers were the forward OL10 and reverse OL9 oligonucleotides described above used for the cloning and expression of the full-length Pfnek-2 open reading frame. For nested PCR (25 cycles at 94 °C for 45 s, 55 °C for 45 s, and 68 °C for 2 min), 1 μl (1/25) of each PCR product was reamplified using the Pfnek-2-specific primers forward OL42 and reverse OL43 oligonucleotides used for construction of the knock-out plasmid described below. Products of both series of reactions were resolved on a 1% agarose gel. The Pfnek-2 disruption plasmid (pCAM-Pfnek-2) was generated by inserting a PCR product corresponding to a central portion of the catalytic domain of the enzyme into the pCAM-BSD vector (a gift from David Fidock), which contains a cassette conferring resistance to blasticidin. The insert was obtained using 3D7 genomic DNA as template and the following oligonucleotides: forward, OL42 GGGGGGATCCTCGTTTGGAATTGTAACTGC; reverse, OL43 GGGGCGGCCGCTGGTGCCATATATCCTA, which contain BamHI and NotI sites (underlined), respectively. The Pfnek-2-GFP plasmid (pCHD-Pfnek-2) was generated by using the pHGB and pCHD-1/2 transfection vectors based on GatewayTM recombinational cloning established by Tonkin et al. (16Tonkin C.J. van Dooren G.G. Spurck T.P. Struck N.S. Good R.T. Handman E. Cowman A.F. McFadden G.I. Mol. Biochem. Parasitol. 2004; 137: 13-21Crossref PubMed Scopus (346) Google Scholar). The ∼1-kb 5′-flanking region of Pfnek-2 was amplified from 3D7 genomic DNA using the forward (GGGTCGACCTATTAGGAAATATGAAG) and reverse (CCAGATCTACTAATATGATTATTCATAC) oligonucleotides containing SalI and BglII sites (underlined), respectively, and inserted into the SalI/BglII sites of pHGB to produce the plasmid pHGB-Pfnek-2 5′. We next amplified the Pfnek-2 open reading frame from the gametocyte cDNA library indicated above and the oligonucleotides forward, CCCAGATCTATGTCTAAACCCAAAATGATAG and reverse, CCCCCTAGGAATTTGGCTATTCCTTTCTTGC, containing BglII and AvrII sites (underlined), respectively. The digested product was ligated into the BglII/AvrII sites of plasmid pHGB-Pfnek-2 5′ to produce the pHGB-Pfnek-2 entry clone. This plasmid was used in a recombination reaction with the pCHD-1/2 destination vector containing the cassette responsible for expression of hDHFR, the gene mediating resistance to WR99210 treatment, to produce the final transfection vector pCHD-Pfnek-2. pCHD-Pfnek-2 contains the full-length Pfnek-2 coding sequence in-frame with the downstream fluorescent reporter GFP gene, driven by its own 5′ promoter region and terminated by the P. berghei DHFR-TS 3′ terminator. Transfections were carried out by electroporation of ring stage 3D7 parasites with 50–100 μg of plasmid DNA, according to Sidhu et al. (17Sidhu A.B. Valderramos S.G. Fidock D.A. Mol. Microbiol. 2005; 57: 913-926Crossref PubMed Scopus (281) Google Scholar). Blasticidin (Calbiochem) or WR99210 (Jacobus Pharmaceutical Co., Inc., Princeton, NJ) were added to a final concentration of 2.5 μg/ml and 5 nm, respectively, 48 h after transfection to select for transformed parasites. Resistant parasites appeared after 3–4 weeks and were maintained under drug selection. Subsequent to genotyping indicating that integration occurred at the target locus, Pfnek-2 knock-out parasites were cloned by limiting dilution in 96-well plates for further genotypic and phenotypic analyses. Genotypes of Pfnek-2 knock-out parasites were analyzed by PCR and Southern blotting, using standard procedures. Genomic DNA from transfectants and 3D7 control parasites were extracted from frozen saponin lysis pellets by standard proteinase K digestion in the presence of SDS, phenol/chloroform/isoamyl alcohol (24:24:1) extraction, and ethanol precipitation. Genomic DNA pellets were resuspended in 10 mm Tris-HCl, pH 8.0, 1 mm EDTA (TE) buffer prior to analysis. Disruption of the Pfnek-2 locus was analyzed by diagnostic PCR using various primer pairs. The primer pair OL10/OL9 produced a 1.8-kb fragment corresponding to the undisrupted Pfnek-2 locus only with template from wild-type 3D7. The primer pair OL167 (TATTCCTAATCATGTAAATCTTAAA) and OL168 (CAATTAACCCTCACTAAAG) specific for the pCam-BSD vector, produced a 1.4-kb fragment corresponding to the episome only in pCAM-Pfnek-2 transfectants. Primer pairs OL10/OL168 and OL167/OL9 amplified across the 5′ and 3′ ends of the integration site, giving rise to 1.3- and 1.9-kb products only in the disrupted locus, respectively. For Southern blot analysis, 5 μg of genomic DNA was digested with EcoRI, separated on a 0.7% agarose gel and transferred to Hybond N+ membrane according to the manufacturer's procedures (Amersham Biosciences). The blot was probed with the fluorescein isothiocyanate-labeled Pfnek-2 sequence amplified from 3D7 genomic DNA with the primer pair OL42/OL43 (used to produce the Pfnek-2 knock-out construct), and incubated with an anti-fluorescein isothiocyanate monoclonal antibody conjugated to horseradish peroxidase purchased from Amersham Biosciences. Chemoluminescence detection was performed using the Western Lightning Chemoluminescence Reagent Plus detection kit (PerkinElmer) and exposure to imaging Hyperfilm MP (Amersham Biosciences). Parasites expressing GFP were fixed with methanol. Images were captured using a Delta vision deconvolution fluorescence microscope (×100 objective Olympus IX-70) after counterstaining (i) with a rat anti-Pfg377 antibody (a kind gift from Pietro Alano), using a red Alexa Fluor 594-conjugated rabbit anti-rat IgG (H+L) secondary antibody and (ii) with 4′,6-diamidino-2-phenylindole. The pattern of microtubules in gametocytes was determined on methanol-fixed parasites stained with a mouse monoclonal antibody specific for chick brain α-tubulin (clone DM1A, purchased from Sigma) and a secondary TRITC-labeled goat anti-mouse IgG (Southern Biotech, Birmingham, AL). Western blot analysis was performed on cell-free extracts prepared by resuspending parasite pellets in phosphate-buffered saline containing 0.1% SDS, 0.05% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, and ComplexTM mixture protease inhibitor tablet from Roche Applied Science. 10 μg of each extract were boiled in Laemmli sample buffer, separated on 12% SDS-polyacrylamide gels, and subsequently electrotransferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% skim milk and Tris-buffered saline containing 0.05% Tween 20 overnight at 4 °C. Immunoblotting was performed using mouse monoclonal anti-GFP antibody (1:5000 dilution) (Roche) and horseradish peroxidase-conjugated sheep anti-mouse antiserum (1/10 000 dilution) (Sigma). Bound antibodies were detected using Western Lightning Chemiluminescence Reagent Plus detection kit (PerkinElmer) and exposure to imaging hyperfilm MP (Amersham Biosciences). Gametocytes of each parasite clone were grown in vitro and fed to Anopheles gambiae mosquitoes through membrane feeders as described previously (15Carter R. Ranford-Cartwright L. Alano P. Methods Mol. Biol. 1993; 21: 67-88PubMed Google Scholar), using medium containing 10% human serum instead of Albumax. Mosquitoes were dissected at 48 h or 10 days post-infection for microscopic analysis of ookinete formation and oocyst infection of the midgut, respectively. pbnek-2−P. berghei (ANKA) parasites were generated by double homologous recombination using the targeting vector pBSDHFR, in which the Toxoplasma gondii dihydrofolate reductase/thymidylate synthase gene (DHFR/TS) is flanked by the upstream and downstream control elements from P. berghei DHFR/TS. A 776-bp fragment of the 5′ UTR of the pbnek-2 gene was amplified from genomic DNA using the following primers (restriction sites are underlined): forward (K0071), GGGGGGTACCTTGGTTCAAAATCATACATAATG; reverse (K0072), GGGGGGGCCCTGCCATTCTTCAATGACTTAT. The amplicon was inserted into pBSDHFR as a KpnI/ApaI fragment upstream of the DHFR/TS cassette. 550 bp of the pbnek-2 3′ UTR were then amplified from genomic DNA using the following primers: forward (K0073), GGGGGGATCCGCCTGATCCACTTCCTAGTA; reverse (K0074), GGGGCCGCGGATTCAATGGACGGACGC. The amplicon was inserted as a BamHI/SacII fragment downstream of the DHFR/TS cassette. The final construct was digested with KpnI and SacII to excise the fragment prior to transfection into P. berghei. A second independent pbnek-2− clone was generated in P. berghei ANKA clone 507 expressing GFP (18Mair G.R. Braks J.A. Garver L.S. Wiegant J.C. Hall N. Dirks R.W. Khan S.M. Dimopoulos G. Janse C.J. Waters A.P. Science. 2006; 313: 667-669Crossref PubMed Scopus (334) Google Scholar). The pyrimethamine-resistant parasites were then cloned by limiting dilution and two independent clones (one from each transfection) were genotyped. For pulsed field gel electrophoresis, chromosomes of wild-type and pbnek-2− clones were separated on an LKB 2015 Pulsaphor system using a linear ramp of 60–500 s for 72 h at 4 V/cm. The gel was blotted and hybridized with a probe that binds to the 3′ UTR of DHFR/TS detecting both the endogenous dhfr locus (chromosome 7) and the modified pbnek-2 locus (chromosome 10). For Southern analysis, genomic DNA from wild-type mutant parasites was digested with EcoRI. The fragments were separated on a 0.8% agarose gel, blotted onto a nylon membrane, and probed with the fragment of the pbnek-2 3′ UTR used in the knock-out vector (see above). We followed procedures similar to those described earlier (9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 19Liu J. Gluzman I.Y. Drew M.E. Goldberg D.E. J. Biol. Chem. 2005; 280: 1432-1437Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In short, gamete activation was triggered by treating the parasite-infected blood with 50 μm xanthurenic acid. Zygote formation and ookinete conversion rates were monitored in in vitro cultures by immunolabeling the macrogamete/zygote/ookinete marker P28 as reported earlier (9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 19Liu J. Gluzman I.Y. Drew M.E. Goldberg D.E. J. Biol. Chem. 2005; 280: 1432-1437Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar); Hoechst 33342 was used for nuclear staining. The stained cells were analyzed on a Leica DMR microscope fitted with an Axiovision digital camera. For mosquito transmission triplicate sets of 50–70 Anopheles stephensi mosquitoes were allowed to feed on anesthetized infected mice on days 3–4 of blood infection for 30 min at 20 °C. Gene prediction algorithms on PlasmoDB proposed conflicting gene structures for Pfnek-2. To resolve this issue, we amplified, cloned, and sequenced the Pfnek-2 open reading frame from a 3D7 gametocyte cDNA library, using primers hybridizing to the most distal of the predicted START and STOP codons. Sequences obtained from 12 clones concurred to show that the 864-bp Pfnek-2 open reading frame comprises eight exons, a gene structure that differs from all gene predictions proposed in PlasmoDB: exons 1 and 2 follow the “Glimmer” prediction, whereas exons 3–8 are as proposed by the Pf annotation. Translation of the Pfnek-2 open reading frame would generate a 286-amino acid, 33.3-kDa protein with a pI of 7.8 (Fig. 1). The 11 subdomains characteristic of serine/threonine protein kinases, as well as most of the key residues that are largely invariant in this family, are conserved in Pfnek-2. In contrast to most Nek family members, Pfnek-2 has a very short (19 amino acids) C-terminal extension with no identified motifs or domains. However, a possible PEST motif (PESTfind score +7.63), targeting proteins to proteolytic degradation, is found within the catalytic domain. Although some Neks have been reported to oligomerize via a coiled-coil domain present in their C-terminal extension, no such sequence is found within Pfnek-2. The Pfnek-2 protein was expressed in E. coli with an N-terminal GST tag. Purified recombinant GST-Pfnek-2 possessed kinase activity, as demonstrated by its ability to autophosphorylate (Fig. 2A) and to phosphorylate exogenous substrates such as myelin basic protein (MBP), histone H1 (Fig. 2B), and β-casein (data not shown). To verify whether the activity was indeed due to GST-Pfnek-2 rather than to a co-purifying contaminant, we showed that a catalytically inactive mutant enzyme (Lys38 → Met) did not yield any signal in the phosphorylation assay. Thus, Pfnek-2 is a genuine protein kinase like Pfnek-4, but displays a different substrate preference (Pfnek-4 is unable to phosphorylate MBP or histone H1 (Fig. 2C and Ref. 9Reininger L. Billker O. Tewari R. Mukhopadhyay A. Fennell C. Dorin-Semblat D. Doerig C. Goldring D. Harmse L. Ranford-Cartwright L. Packer J. Doerig C. J. Biol. Chem. 2005; 280: 31957-31964Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Accordingly, kinase assays using heat-inactivated parasite extracts as substrates consistently display different patterns of phosphorylated proteins depending on whether GST-Pfnek-2 or GST-Pfnek-4 is used in the reaction (data not shown). Thr169 is conserved in many protein kinases (including Neks) as the site for activating phosphorylation, and mutant human NEK2 lacking this residue display altered kinase activity (20Rellos P. Ivins F.J. Baxter J.E. Pike A. Nott T.J. Parkinson D.M. Das S. Howell S. Fedorov O. Shen Q.Y. Fry A.M. Knapp S. Smerdon S.J. J. Biol. Chem. 2007; 282: 6833-6842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Likewise, replacement of this threonine with an alanine ablated Pfnek-2 kinase activity, indicating that the amino acid at this position is crucial to enzyme function and suggesting possible regulation by autophosphorylation; the Thr169 residue may also be a target for other upstream kinase(s) in vivo; the corresponding residue in Nek6 (Ser206) has been shown to be the target of phosphorylation by Nek9 (7Belham C. Roig J. Caldwell J.A. Aoyama Y. Kemp B.E. Comb M. Avruch J. J. Biol. Chem. 2003; 278: 34897-34909Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). We did not observe any synergy between the activities of GST-Pfnek-2 and GST-Pfmap-2, as had been observed for Pfnek-1 (12Dorin D. Le Roch K. Sallicandro P. Alano P. Parzy D. Poullet P. Meijer L. Doerig C. Eur. J. Biochem. 2001; 268: 2600-2608Crossref PubMed Scopus (106) Google Scholar) and Pfnek-3 (13Lye Y.M. Chan M. Sim T.S. FEBS Lett. 2006; 580: 6083-6092Crossref PubMed Scopus (39) Google Scholar) (data not shown). Kinase assays using wild-type and kinase-dead enzymes performed to detect a possible phosphorylation/activation of recombinant Pfnek-2 by Pfnek-4 (or vice versa) did not provide any evidence that such cross-activation occurs, at least in vitro (Fig. 2C). Microarray analyses indicate that the Pfnek-2 mRNA is predominantly expressed in gametocytes (10Le Roch K.G. Zhou Y. Blair P.L. Grainger M. Moch J.K. Haynes J.D. De La Vega P. Holder A.A. Batalov S. Carucci D.J. Winzeler E.A. Science. 2003; 301: 1503-1508Crossref PubMed
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