Toxoplasma gondii Attachment to Host Cells Is Regulated by a Calmodulin-like Domain Protein Kinase
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m011045200
ISSN1083-351X
AutoresHeidi Kieschnick, Therese Wakefield, Carl A. Narducci, Con J. Beckers,
Tópico(s)Autophagy in Disease and Therapy
ResumoThe role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasma motility and host cell invasion. The role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasma motility and host cell invasion. Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Infection with this parasite is typically asymptomatic, although acute toxoplasmosis can be fatal in immunocompromised individuals and result in severe birth defects or abortion during the first trimester of pregnancy (1Frenkel J.K. Parasitol. Today.. 1988; 4: 273-278Google Scholar). Three developmental stages of Toxoplasma have been described. The highly infective sporozoite stage is shed in the feces of infected felines and can infect all warm-blooded animals, where it differentiates into the rapidly replicating tachyzoite stage. This form of the parasite rapidly spreads throughout the infected animal and eventually differentiates into the slow growing, encysted bradyzoite stage. The latter can infect another animal upon ingestion of infected tissues. The three stages are immunologically and biochemically distinct, due to the expression of many stage-specific proteins (2Bohne W. Holpert M. Gross U. Immunobiology.. 1999; 201: 54-248Google Scholar). All three developmental stages are obligate intracellular parasites, suggesting that infected host cells supply nutrients that supplementT. gondii biosynthetic deficiencies, such as its inability to make purines de novo (3Pfefferkorn E.R. Wyler D.J. Modern Parasite Biology: Cellular, Immunological and Molecular Aspects.W. H. Freeman and Co. 1990; : 26-50Google Scholar). As a result of these biosynthetic requirements, invasion of a host cell is critical for the growth and reproduction of T. gondii. The biochemical pathways involved in host cell invasion have not yet been identified, although it has been shown that cytoplasmic calcium in the parasite is essential for this process (4Carruthers V.B. Sibley L.D. Mol. Microbiol... 1999; 31: 421-428Google Scholar, 5Pezzella N. Bouchot A. Bonhomme A. Pingret L. Klein C. Burlet H. Balossier G. Bonhomme P. Pinon J.M. Eur. J. Cell Biol... 1997; 74: 92-101Google Scholar). The involvement of calcium in regulating parasite interaction with host cells was further strengthened by the observation of Sibley et al. that both the attachment of Toxoplasma tachyzoites to its host cells as well as parasite motility are sensitive to an inhibitor of calcium-dependent protein kinases, KT5926 (6Hashimoto Y. Nakayama T. Teramoto T. Kato H. Watanabe T. Kinoshita M. Tsukamoto K. Tokunaga K. Kurokawa K. Nakanishi S. Biochem. Biophys. Res. Commun... 1991; 181: 423-429Google Scholar, 7Dobrowolski J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar). In eukaryotic cells, protein kinases often mediate the cellular responses to external stimuli. Three major classes of protein kinases are often involved in this: cyclic nucleotide-dependent protein kinases, calcium/calmodulin-dependent protein kinases, and calcium/phospholipid-dependent protein kinases. In a number of organisms, an unusual class of calcium-dependent protein kinases has been described. These enzymes, the calmodulin-like domain protein kinases (CDPKs),1 are activated by calcium in the absence of calmodulin or phospholipids. Initially identified, characterized, and cloned in plants (8Harmon A.C. Putnam-Evans C. Cormier M.J. Plant Physiol... 1987; 83: 830-837Google Scholar, 9Putnam-Evans C.L. Harmon A.C. Cormier M.J. Biochemistry.. 1990; 29: 2488-2495Google Scholar, 10Harper J.F. Sussman M.R. Schaller G.E. Putnam-Evans C. Charbonneau H. Harmon A.C. Science.. 1991; 252: 951-954Google Scholar), CDPKs have also been identified in algae (11McCurdy D.W. Harmon A.C. Planta ( Heidelberg ).. 1992; 188: 54-61Google Scholar, 12Yuasa T. Muto S. Arch. Biochem. Biophys... 1992; 296: 175-182Google Scholar), Paramecium tetraurelia (13Gundersen R.E. Nelson D.L. J. Biol. Chem... 1987; 262: 4602-4609Google Scholar, 14Son M. Gundersen R.E. Nelson D.L. J. Biol. Chem... 1993; 268: 5940-5948Google Scholar, 15Kim K. Messinger L.A. Nelson D.L. Eur. J. Biochem... 1998; 251: 605-612Google Scholar), and the apicomplexan parasitesPlasmodium falciparum (16Zhao Y. Kappes B. Franklin R.M. J. Biol. Chem... 1993; 268: 4347-4354Google Scholar, 17Li J.L. Baker D.A. Cox L.S. Biochim. Biophys. Acta.. 2000; 1491: 341-349Google Scholar), Eimeria maxima, and Eimeria tenella (18Dunn P.P. Bumstead J.M. Tomley F.M. Parasitology.. 1996; 113: 439-448Google Scholar). The typical CDPK domain structure consists of an N-terminal serine/threonine kinase domain homologous to that of calcium/calmodulin-dependent protein kinases, followed by a highly conserved junction domain, which joins the kinase region to a C-terminal calmodulin-like domain (10Harper J.F. Sussman M.R. Schaller G.E. Putnam-Evans C. Charbonneau H. Harmon A.C. Science.. 1991; 252: 951-954Google Scholar). This calmodulin-like domain, which is 30–40% homologous to calmodulin, imparts calcium sensitivity to the CDPKs (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar, 20Zhao Y. Pokutta S. Maurer P. Lindt M. Franklin R.M. Kappes B. Biochemistry.. 1994; 33: 3714-3721Google Scholar). Activation of CDPKs has been shown to be dependent on calcium and independent of calmodulin (8Harmon A.C. Putnam-Evans C. Cormier M.J. Plant Physiol... 1987; 83: 830-837Google Scholar). The elucidation of the function of specific CDPKs has been complicated, in part, by the fact that often multiple isoforms are expressed at the same time (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar). Three soybean isoforms were found to differ in sensitivity to calcium and substrate specificity (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar), suggesting that they are involved in the regulation of different phenomena. In plants, CDPKs have been found to regulate Ca2+-pumps (21Hwang I. Sze H. Harper J.F. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 6224-6229Google Scholar), K+-transport (22Li J. Lee Y.R. Assmann S.M. Plant Physiol... 1998; 116: 785-795Google Scholar), and a Cl− channel (23Pei Z.M. Ward J.M. Harper J.F. Schroeder J.I. EMBO J... 1996; 15: 6564-6574Google Scholar) and have also been implicated in the response to environmental stress and infections (24Romeis T. Piedras P. Jones J.D. Plant Cell.. 2000; 12: 803-816Google Scholar, 25Saijo Y. Hata S. Kyozuka J. Shimamoto K. Izui K. Plant J... 2000; 23: 319-327Google Scholar). The role of CDPKs in protists and apicomplexan parasites is unclear at this time. In light of the importance of calcium-dependent regulation in host cell invasion by Toxoplasma tachyzoites and the suggestion that this might be affected in the presence of KT5926, we performed an analysis of calcium-dependent protein kinase activities in tachyzoites and their sensitivity to KT5926. We describe here the identification of two CDPKs, TgCDPK1 and TgCDPK2, in T. gondii. Of these, only TgCDPK1 is expressed in the tachyzoite stage of the parasite. This enzyme appears to be the target of KT5926 in tachyzoites and is therefore likely to play a role in their invasion of host cells. T. gondiistrain RH(EP) was obtained from D. S. Roos (University of Pennsylvania) and was maintained by serial passage in human foreskin fibroblasts or in the peritoneal cavity of Swiss/Webster mice as has been described (26Beckers C.J. Dubremetz J.F. Mercereau P.O. Joiner K.A. J. Cell Biol... 1994; 127: 947-961Google Scholar). Parasites were harvested from the culture supernatant or the peritoneal fluid, washed by centrifugation, and filtered through a 3-μm filter before use. These were essentially performed as described by Dobrowolski et al. (7Dobrowolski J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar). For attachment and invasion assays, parasites were added to confluent coverslips of human foreskin fibroblast cells at a concentration of 106/ml in Hanks' balanced salt solution containing 0.1% bovine serum albumin in the presence of increasing concentrations of KT5926 (Calbiochem) or Me2SO. Attachment and invasion were allowed to occur for 15 min at 37 °C. All subsequent operations were performed on ice. The coverslips were gently washed three times in cold phosphate-buffered saline to remove any unattached parasites. Extracellular parasites were detected by incubation with an anti-Toxoplasma rabbit antiserum in phosphate-buffered saline containing 3% bovine serum albumin. After a 30-min incubation, the coverslips were washed as before. Cells were fixed in 3% paraformaldehyde in phosphate-buffered saline, washed, and permeabilized for 5 min in ice-cold methanol. Both extracellular and intracellular parasites were detected using the mouse monoclonal antibody T41E5 (obtained from Dr. J. F. Dubremetz (Institut Pasteur, Lille, France)) to the parasite surface marker SAG1. Cells were washed and incubated in the secondary antibody solution containing fluorescein-conjugated goat anti-rabbit antibody and rhodamine-conjugated goat anti-mouse antibody and 5 μg/ml 4′,6-diamidino-2-phenylindole to label nuclei. After washing and mounting, the number of attached parasites, total parasites, and host cell nuclei were counted in eight individual fields at × 1000 magnification. All incubations were performed in triplicate. Attachment and invasion were expressed as the number of events observed per host cell nucleus. Parasites were washed and filtered as described above but using phosphate-free Dulbecco's minimal essential medium (Life Technologies, Inc.), containing 0.1% (w/v) bovine serum albumin. In a final volume of 450 μl, 4.5 × 107 parasites were mixed with 0.5 mCi [32P]sodium phosphate (Amersham Pharmacia Biotech). To one 200-μl aliquot 4 μl of Me2SO was added, and to a second aliquot 4 μl of 500 μm KT5926 was added. After a 30-min incubation at 37 °C, the parasites were recovered by centrifugation and solubilized in 40 μl of isoelectric focusing sample buffer (5% 2-mercaptoethanol, 9.2 m urea, 2% IGEPAL CA-630, 3% ampholytes 5–8, 2% ampholytes 3–10, and 2% ampholytes 3–5). Samples of 10 μl were analyzed by two-dimensional gel electrophoresis. All experiments were performed in duplicate. All manipulations were performed on ice or at 4 °C. Parasites, prepared as described above, were resuspended to a final concentration of 109 parasites/ml in 25 mmHEPES-KOH, 50 mm KCl, 1 mm phenylmethylsulfonyl fluoride, 50 μg/ml antipain, 10 μg/ml leupeptin, and 10 μg/ml aprotinin at pH 7.5. Parasites were disrupted either by two rapid freeze-thaw cycles, by brief sonication, or by the addition of Triton X-100 to a final concentration of 1%. Lysates were clarified by centrifugation for 10 min at 13,000 × g. Heat-treated extracts were prepared by incubation at 70 °C for 10 min, and denatured proteins were removed by centrifugation. A 2-ml aliquot of a parasite lysate prepared by sonication was loaded onto a 2 × 1-cm DEAE-Sepharose (Amersham Pharmacia Biotech) column equilibrated in 10 mm Tris-HCl, pH 8, 50 mm KCl. The column was washed with 10 ml of equilibration buffer, and bound protein was eluted with a 20-ml gradient of 50–500 mm KCl in 10 mm Tris-HCl, pH 8. Each 1-ml fraction was analyzed for the presence of protein kinase activity as described below. The three fractions containing the peak of calcium-dependent, KT5926-sensitive protein kinase activity were pooled, and KCl was added to a final concentration of 2m. This was loaded onto a 2 × 1-cm phenyl-Sepharose (Amersham Pharmacia Biotech) column equilibrated in 10 mmTris-HCl, pH 8, 2 m KCl. The column was washed with 10 ml of equilibration buffer, and bound proteins were eluted with a 20-ml gradient of 2 to 0 m KCl in 10 mm Tris-HCl, pH 8, and protein kinase activity in each fraction was determined as described below. Kinase assays were performed in a total volume of 20 μl containing 20 mm HEPES-KOH (pH 7.5), 10 mm MgCl2, 2 mm EGTA, 1.93 mm CaCl2 (2.5 μm free calcium), 1 mg/ml bovine serum albumin, and 0.3 μg/ml (∼5 nm) purified TgCDPK1. The purified enzyme was diluted in buffer containing 20 mm HEPES-KOH (pH 7.5), 10 mmMgCl2, and 1 mg/ml bovine serum albumin. For assays during enzyme purification, 0.2 mg/ml histones III-S and 90 μm [γ-32P]ATP (∼200 Ci/mol) (Amersham Pharmacia Biotech) were used. To assess calcium-dependent activity of the enzyme, the reactions were performed in the presence of 1 mm EGTA, or the concentration of free calcium reactions was controlled by a series of buffers containing a ratio of EGTA/CaCl2 according to Fabiato (27Fabiato A. Methods Enzymol... 1988; 157: 378-417Google Scholar). Where indicated, KT5926, calphostin C, and fluphenazine-N-2-chloroethane (all from Calbiochem) were included in the kinase reaction at the indicated concentrations. For kinetic analysis, the concentrations of peptide/protein substrates and [γ-32P]ATP were varied according to Table I. Control reactions for ATP titrations contained 25 mm EGTA, while controls for peptide titrations lacked peptide substrate. Reactions were incubated at 30 °C for 10 min, transferred immediately to an ice-water bath, and sampled by spotting 10 μl on a 2.5-cm P-81 filter paper (Whatman). Filters were air-dried and washed with constant shaking for 30 min in five changes of 75 mm o-phosphoric acid followed by 5 min in 100% acetone. Filters were air-dried, and Cerenkov radiation was quantitated using a liquid scintillation counter.Table IKinetic parameters of TgCDPK1SubstrateK m(app)V max(app)ATP155 μm5.2 μmol min−1 mg−1Glycogen synthase peptide107 μm5.3 μmol min−1 mg−1Syntide-2 peptide84 μm8.9 μmol min−1 mg−1Histones III-S3 mg/ml1.0 μmol min−1mg−1Protamine sulfate0.2 mg/ml6.5 × 10−3 μmol min−1 mg−1ATP titration was performed with 400 μm GS peptide and a dilution series of 400–50 μm [γ-32P]ATP (20–800 Ci/mol). GS peptide titration was carried out in 400 μm ATP. All other peptide titrations were carried out in 200 μm ATP. Kinetic values are derived from Eadie-Hofstee plots of data obtained. Open table in a new tab ATP titration was performed with 400 μm GS peptide and a dilution series of 400–50 μm [γ-32P]ATP (20–800 Ci/mol). GS peptide titration was carried out in 400 μm ATP. All other peptide titrations were carried out in 200 μm ATP. Kinetic values are derived from Eadie-Hofstee plots of data obtained. Values for K m and V max were obtained by analyzing data according to Eadie-Hofstee and Lineweaver-Burk. Each reaction contained 10 μl of parasite extract, 20 mm HEPES-KOH, 10 mm MgCl2, 75 μm [γ-32P]ATP (950 Ci/mol) in a final volume of 20 μl and was incubated for 15 min at 30 °C. Where indicated, reactions contained either 2 mm EGTA or 2.5 μm free Ca2+ and 200 ng of active recombinant TgCDPK1. Reactions were stopped by the addition of 60 μl of isoelectric focusing sample buffer. For the first dimension, 10-μl samples were loaded onto 7-cm isoelectric focusing gels containing 9.2 m urea, 3% acrylamide, 2% IGEPAL CA-630, 3% ampholytes 5–8, 2% ampholytes 3–10, and 2% ampholytes 3–5. Isoelectric focusing was performed for 14 h at 400 V, followed by 1 h at 800 V. After extrusion and a 15-min equilibration in SDS sample buffer, the tube gels were layered onto 15% SDS-PAGE gels. Following electrophoresis, the SDS-PAGE gels were dried, and autoradiography was used to detect 32P-labeled proteins. Total RNA was isolated from parasites with the Trizol reagent (Life Technologies) using the conditions suggested by the manufacturer. First strand cDNA was synthesized using SuperScript reverse transcriptase (Stratagene, La Jolla, CA) and either random hexamers or oligo(dT) according to the manufacturer's directions. The resulting cDNA was analyzed for the presence of protein kinase-related sequences using PCR and two primers derived from conserved domains present in a wide variety of serine/threonine protein kinases: TgPK1 (5′-CGGATCCAYMGIGAYYT-3′) and TgPK2 (5′-GGAATTCCRWARGACCAIACRTC-3′). A typical 100-μl PCR contained 2.5 units of Taq polymerase, 100 pmol of primers TgPK1 and -2, 50 ng of cDNA, 200 μm dNTPs, 1.5 mmMgCl2, 50 mm KCl, 10 mm Tris-HCl, pH 8.3, and 0.001% gelatin. The following PCR conditions were used: 30 cycles of 1 min at 95 °C, 1 min at 37 °C, and 1 min at 72 °C, followed by a 10-min incubation at 72 °C. PCR fragments of the expected size were isolated from agarose gels and cloned directly into pGEM-T vector (Promega, Madison, WI) according to the manufacturer's instructions. Clones containing inserts were identified, and the inserts were sequenced manually with Sequenase (Amersham Pharmacia Biotech). TgCDPK1- and TgCDPK2-specific probes were prepared by PCR in 50-μl reactions containing 1.25 units of Taq DNA polymerase, 100 pmol of primers TgPK1 and TgPK2, 50 ng of plasmid template, 200 μm dGTP, dTTP, dATP, and 50 μCi of [32P]dCTP (>3000 Ci/mmol; Amersham Pharmacia Biotech), 1.5 mm MgCl2, 50 mm KCl, 10 mm Tris-HCl, pH 8.3, and 0.001% gelatin. For the TgCDPK1 probe, plasmid pTgPK54 was used; for the TgCDPK2 probe, the plasmid pTgPK21 was used. The following PCR conditions were used: 10 cycles of 1 min at 95 °C, 1 min at 37 °C, and 1 min at 72 °C. A Toxoplasma cDNA library in λZAPII (AIDS Research and Reference Reagent Program, McKesson Biosciences, Rockville, MD) was screened with TgCDPK1- or TgCDPK2-specific probes. Positive phage clones were converted to plasmids using in vivo excision according to the manufacturer's instructions (Stratagene). The resulting clones were analyzed by restriction enzyme digestion and automated DNA sequence analysis (Keck Biotechnology Resource Laboratory, Yale University, New Haven, CT). Sequences were aligned employing the ClustalW algorithm, available through the Baylor University site on the World Wide Web. The phylogeny was developed from the aligned sequence data employing the branch and bound algorithm available in the PAUP program package (28Swofford D.L. PAUP: Phylogenetic Analysis Using Parsimony.Sinauer Associates Sunderland, MA. 1998; Google Scholar). In undertaking the analysis, gaps introduced during the alignment process were not considered as character states, all characters were equally weighted, and the phylogenies were not rooted. A single most parsimonious tree was identified in the branch and bound algorithm, which is shown here. Of the 632 characters analyzed, 363 were informative. The statistics for the tree are as follows: length, 2015 (minimum 1752, maximum 2672); consistency index, 0.869; rescaled consistency index, 0.621. Numbers at the nodes indicate the percentage the grouping distal to the labeled node was identified in a bootstrap analysis of 1000 replicate data sets. The entire open reading frames of TgCDPK1 and -2 were amplified using the following primers: 5′-GAAGATCTGATGGGGCAGCAGGAAAGCAC-3′ and 5′-CCAAGCTTTAGTTTCCGCAGAGCTTC-3′ for TgCDPK1; 5′-CGGGATCCCGCATCACCAGTGCAGCACC-3′ and 5′-CCAAGCTTTTACCCCGTAGCGCGAGG-3′ for TgCDPK2 with PfuTurbo DNA polymerase (Stratagene) as described by the manufacturer. Amplified products were digested withBglII (TgCDPK1) or BamHI (TgCDPK2) andHindIII and cloned into the bacterial expression vector pRSETB digested with BamHI and HindIII, resulting in the plasmids pRSETB-CDPK1 and pRESTB-CDPK2. These plasmids encode the open reading frame of the protein kinases fused at their N terminus to a polyhistidine tag and Xpress tag. For the expression of recombinant protein, these plasmids were transfected into JM109 (DE3). Overnight cultures were diluted 1:100 in 200 ml of fresh LB medium containing 50 μg/ml ampicillin and grown at 37 °C untilA 600 was 0.4–0.6. Isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and growth was continued for an additional 4 h at 37 °C. Bacteria were harvested, lysed in 6m guanidine HCl in 10 mm Tris-HCl, pH 8.0, and loaded onto 2 ml of Ni2+-nitrilotriacetic acid resin (Qiagen). The resin was washed with 20 ml of lysis buffer and 20 ml of 8 m urea, 10 mm Tris-HCl, pH 8.0. The polyhistidine-tagged fusion proteins were eluted using a linear 0–250 mm imidazole gradient in 8 m urea, 10 mm Tris-HCl, pH 8.0. Fractions containing pure fusion proteins were dialyzed overnight against phosphate-buffered saline and used as antigen. The entire open reading frame of TgCDPK1 was amplified using the primers 5′-GGAATTCCATATGGGGCAGCAGGAAAGCAC-3′ and 5′-CCAAGCTTTAGTTTCCGCAGAGCTTC-3′ and PfuTurbo as described above. The amplified product was purified by agarose electrophoresis, digested with the restriction enzymes NdeI andHindIII, and repurified by agarose gel electrophoresis. The expression plasmid pRSET-B (Invitrogen, Carlsbad, CA) was digested with the restriction enzymes NdeI and HindIII and purified by agarose gel electrophoresis. The amplification product was ligated into pRSETB, resulting in a construct (pRSETB-TgCDPK1) that encoded only TgCDPK1 without extraneous sequences. A 20-ml overnight culture of Escherichia coli JM109(DE3) containing the plasmid pRSETB-TgCDPK1 was used to inoculate 2 liters of LB medium containing 50 μg/ml ampicillin and grown at 37 °C until an A 600 of 0.8 was reached. Isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and the culture was incubated for an additional 5 h at 37 °C. All subsequent manipulations were performed at 0–4 °C. The pellet (8.1 g) was resuspended in 40 ml of ice-cold lysis buffer (50 mm Tris-HCl (pH 7.6), 25 mm NaCl, 1 mm EDTA, and 1 mmphenylmethylsulfonyl fluoride). Lysozyme was added to a final concentration of 1 mg/ml, and after a 10-min incubation the lysate was sonicated on ice. After centrifugation, the clarified supernatant was frozen in liquid N2 and stored at −80 °C. One-fourth of the frozen supernatant was thawed, and (NH4)2SO4 was added to 40% saturation. After 1 h, the precipitate was removed by centrifugation at 10,000 × g for 15 min. Additional (NH4)2SO4 was added to the supernatant to 60% saturation. After 1 h, the precipitate was collected by centrifugation at 10,000 × g for 15 min. The pellet was resuspended in 3 ml of lysis buffer and dialyzed overnight against two changes of 2 liters of lysis buffer. The dialyzed material was clarified by centrifugation and applied to a DEAE-Sepharose anion exchange column (3 × 2 cm) equilibrated with 10 mm Tris-HCl, pH 7.6, 50 mm NaCl. The column was washed with 30 ml of this buffer, and bound proteins were eluted with a 150-ml linear gradient of NaCl (50–500 mm) in 10 mm Tris-HCl, pH 7.6. Fractions (5 ml) were analyzed for kinase activity as described. The fractions containing the peak of kinase activity were pooled and frozen in liquid nitrogen. Calcium-dependent hydrophobic interaction chromatography was used as the final purification step. The peak fractions of kinase activity were pooled, and CaCl2 was added to a final concentration of 2 mm. This was applied to a (2.5 × 2 cm) phenyl-Sepharose column equilibrated with loading buffer containing 10 mm Tris-HCl pH 7.6, 50 mm NaCl, and 2 mm CaCl2. The column was washed with 45 ml of loading buffer followed by 45 ml of wash buffer (10 mmTris-HCl, pH 7.6, 50 mm NaCl, and 0.1 mmCaCl2). The enzyme was eluted with buffer containing 10 mm Tris-HCl, pH 7.6, 50 mm NaCl, and 2 mm EGTA. Fractions (2.5 ml) were assayed for kinase activity as described. Purity of the recombinant protein was assessed by SDS-PAGE. Proteins were separated by SDS-PAGE on 10 or 12% polyacrylamide gels. Following electrophoresis, proteins were transferred to nitrocellulose membrane for 60 min at 100 V. The nitrocellulose membrane was blocked in Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20. Rabbit or mouse antisera raised against purified TgCDPK1 and TgCDPK2 was used at a 1:2000 dilution in the blocking buffer. Bound antibodies were detected using horseradish peroxide-conjugated secondary antisera and the SuperSignal detection system (Pierce). Protein concentrations were measured by the method of Bradford (29Bradford M.M. Anal. Biochem... 1976; 72: 248-254Google Scholar) using bovine serum albumin as a standard. It was reported by Dobrowolski et al. (7Dobrowolski J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar) that the protein kinase inhibitor KT5926 blocked the ability of T. gondii to attach to their host cell and to move over substrates. We confirmed these findings, as can be seen in Fig. 1, and established that the IC50 of KT5926 forToxoplasma attachment is ∼100 nm. KT5926 is a selective inhibitor of calmodulin-dependent and myosin light chain kinases in animal cells. To determine the extent to which KT5926 blocks protein phosphorylation in T. gondii, tachyzoites were incubated with inorganic radioactive phosphate in the presence or absence of KT5926. As a control, we also performed an in vivo phosphorylation experiment in the presence of staurosporine, a general inhibitor of serine/threonine protein kinases. To obtain the highest possible sensitivity, the effect of the inhibitor on incorporation of [32P]PO 43− in proteins was assessed by two-dimensional gel electrophoresis. As expected, the addition of staurosporine blocks incorporation of 32P into most proteins (data not shown). The addition of KT5926 to parasites, as seen in Fig. 2, selectively blocks the incorporation of phosphate in three major proteins: PP1 (M r(app) = 67,000, pI = 6.7), PP2 (M r(app) = 31,000, pI = 5.6), and PP3 (M r(app) = 6500, pI = 4.7). These data suggest that the effect of KT5926 on protein phosphorylation inT. gondii is quite selective and might be due to an inhibition of one or only a few protein kinases. Although the IC50 of KT5926 for parasite attachment is only 100 nm, the in vivo phosphorylation experiment in Fig. 2 was performed in the presence of 10 μm inhibitor to exacerbate its effects on protein phosphorylation. A similar effect of KT5926 on protein phosphorylation was observed at a concentration of 1 μm. To characterize the effects of KT5926 on protein kinase activity in Toxoplasma tachyzoites further, we analyzed parasite extracts for the presence of protein kinase activities. As can be seen in Fig. 3, protein kinase activity is readily detected in Toxoplasmaextracts using histones as substrates. Since KT5926 is thought to be specific for calcium-dependent protein kinase (6Hashimoto Y. Nakayama T. Teramoto T. Kato H. Watanabe T. Kinoshita M. Tsukamoto K. Tokunaga K. Kurokawa K. Nakanishi S. Biochem. Biophys. Res. Commun... 1991; 181: 423-429Google Scholar), we determined whether the presence or absence of calcium affects protein kinase activity in Toxoplasma extracts. In the presence of calcium, overall protein kinase activity in parasite extracts was stimulated 2-fold when compared with reactions performed in the presence of EGTA. These data suggest that Toxoplasmaextracts do indeed contain one or more potent calcium-dependent protein kinases. The calcium-dependent increase in protein kinase activity was completely blocked in the presence of 500 nm KT5926, confirming the hypothesis that this inhibitor targets one or more calcium-dependent protein kinases in Toxoplasma. The addition of recombinant Toxoplasma calmodulin, a calmodulin antagonist, or calphostin C had no effect on the KT5926-sensitive protein kinase activity. These observa
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