From the Division of Geographic Medicine and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, December 7, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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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 Toxoplasma
tachyzoites 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 regulating
Toxoplasma 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 (1). 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 (2).
All three developmental stages are obligate intracellular parasites, suggesting that infected host cells supply nutrients that supplement T. gondii biosynthetic deficiencies, such as its inability
to make purines de novo (3). 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 (4, 5). 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 (6, 7).
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 (8-10), CDPKs have
also been identified in algae (11, 12), Paramecium tetraurelia (13-15), and the apicomplexan parasites
Plasmodium falciparum (16, 17), Eimeria maxima,
and Eimeria tenella (18).
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 (10). This calmodulin-like
domain, which is 30-40% homologous to calmodulin, imparts calcium
sensitivity to the CDPKs (19, 20). Activation of CDPKs has been shown
to be dependent on calcium and independent of calmodulin (8). 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 (19). Three soybean isoforms were found to differ in
sensitivity to calcium and substrate specificity (19), suggesting that
they are involved in the regulation of different phenomena. In plants, CDPKs have been found to regulate Ca2+-pumps (21),
K+-transport (22), and a Cl 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.
Growth and Isolation of Parasites--
T. gondii
strain 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 (26). Parasites were harvested from the culture
supernatant or the peritoneal fluid, washed by centrifugation, and
filtered through a 3-µm filter before use.
Attachment and Motility Assays--
These were essentially
performed as described by Dobrowolski et al. (7). 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.
In Vivo Phosphorylation Reactions--
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.
Preparation and Fractionation of Toxoplasma
Extracts--
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 mM
HEPES-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 2 M. This was loaded onto a 2 × 1-cm phenyl-Sepharose
(Amersham Pharmacia Biotech) column equilibrated in 10 mM
Tris-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.
Protein Kinase Assay--
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 mM
MgCl2, and 1 mg/ml bovine serum albumin. For assays during
enzyme purification, 0.2 mg/ml histones III-S and 90 µM [
Values for Km and Vmax were
obtained by analyzing data according to Eadie-Hofstee and
Lineweaver-Burk.
In Vitro Phosphorylation of Toxoplasma Proteins by
TgCDPK1--
Each reaction contained 10 µl of parasite extract, 20 mM HEPES-KOH, 10 mM MgCl2, 75 µM [ Two-dimensional Analysis of Phosphorylation Reactions--
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.
Amplification of Protein Kinase-related Sequences from Toxoplasma
cDNA--
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 mM
MgCl2, 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).
cDNA Library Screening--
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 Phylogenetic Analysis of TgCDPK Sequences--
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 (28). 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.
Production of Antisera to TgCDPK1 and TgCDPK2--
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 with
BglII (TgCDPK1) or BamHI (TgCDPK2) and
HindIII 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 until A600 was 0.4-0.6.
Isopropyl-1-thio- Expression and Purification of Enzymatically Active
TgCDPK1--
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 and
HindIII, 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 A600 of 0.8 was reached.
Isopropyl-1-thio- SDS-PAGE and Immunoblotting--
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 Quantitation--
Protein concentrations were measured
by the method of Bradford (29) using bovine serum albumin as a standard.
KT5926 Blocks Parasite Motility and Cell Attachment--
It was
reported by Dobrowolski et al. (7) 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 for
Toxoplasma attachment is ~100 nM. KT5926 is a
selective inhibitor of calmodulin-dependent and myosin
light chain kinases in animal cells.
KT5926 Is a Selective Inhibitor of Toxoplasma Protein
Phosphorylation in Vivo--
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 A Calcium-dependent Protein Kinase Activity Is
Inhibited by KT5926--
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 Toxoplasma extracts using histones as substrates. Since KT5926 is thought to be
specific for calcium-dependent protein kinase (6), 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 Toxoplasma
extracts 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 observations suggest
that Toxoplasma does not possess major
calmodulin-dependent protein kinase or protein kinase C
activities and that therefore the target of KT5926 may be a different
class of calcium-dependent protein kinase.
To characterize the calcium-dependent protein kinase
activity in Toxoplasma extracts further, we fractionated
parasite extracts by ion exchange chromatography and analyzed each
fraction for calcium-dependent and KT5926-sensitive protein
kinase activity and the effect of KT5926 on that activity. As can be
seen in Fig. 4A, soluble
extracts of Toxoplasma contain a number of protein kinase
activities that can phosphorylate histones, but only a single peak of
calcium-dependent protein kinase activity was detected, and
this activity was effectively inhibited in the presence of 500 nM KT5926. Further fractionation of this activity by
hydrophobic interaction chromatography revealed only a single sharp
peak of calcium-dependent, KT5926-sensitive protein kinase
activity (Fig. 4B). As before, the addition of neither
recombinant Toxoplasma calmodulin nor calmodulin antagonists
affected any of the protein kinase activities detected in Fig. 4 (data
not shown). Taken together, these observations suggest that
Toxoplasma tachyzoites contain a single KT5926-sensitive
protein kinase activity that is calcium-dependent but does
not require calmodulin.
Identification of Two CDPKs in Toxoplasma--
To identify
possible candidates for the calcium-dependent,
KT5926-sensitive protein kinase activity in Fig. 4, we performed PCR on
Toxoplasma cDNA using primers that were known to allow amplification of the catalytic domain of a wide variety of
serine/threonine protein kinases. The only candidates for
calcium-dependent protein kinases identified in an
exhaustive analysis of protein kinase sequences were two members of the
family of CDPKs. No candidates for calmodulin-dependent
protein kinases or protein kinase C were identified. Full-length clones
were isolated from a cDNA library using both PCR products and
characterized by DNA sequencing. Since the predicted amino acid
sequences of both clones are highly homologous to known CDPKs, these
are henceforth referred to a TgCDPK1 and TgCDPK2. Low stringency
Southern blot analysis of Toxoplasma genomic DNA did not
reveal any additional CDPK-related sequences (data not shown). The open
reading frame of TgCDPK1 encodes a 507-amino acid protein with a
predicted molecular mass of 57.3 kDa. TgCDPK2 is predicted to be a
520-amino acid protein with a predicted molecular mass of 58.3 kDa.
The predicted sequences of both proteins contain the domains typically
encountered in CDPKs: an N-terminal catalytic domain, a short
autoinhibitory domain, and a C-terminal calcium-binding domain (Fig.
5A). The two CDPK isoforms of
Toxoplasma are 36% identical to each other. TgCDPK1 is most
homologous to the CDPKs described in the related apicomplexan
parasites E. maxima and E. tenella and CDPK1 of
P. falciparum. TgCDPK2 is more homologous to CDPK2 and -3 of
P. falciparum and Arabidopsis thaliana CDPK4 and
soybean CDPK-
Motifs commonly found in protein kinases (30) are present in both
enzymes. Both TgCDPK1 and -2 contain a glycine loop motif (G58XG60XXG63
in TgCDPK1 and
G56XG58XXG61
in TgCDPK2) as well as an invariant lysine (Lys80 in
TgCDPK1 and Lys78 in TgCDPK2) presumably involved in ATP
binding. Both enzymes also have a number of motifs commonly found in
serine/threonine protein kinases: Val65, Ala78,
195DFG197, 174DLKPEN179
in TgCDPK1 and Val63, Ala76,
Asp190-Phe191-Gly192,
169DLKPEN174 in TgCDPK2. The junction domains
of both enzymes (residues 299-352 in TgCDPK1, residues 294-345 in
TgCDPK2) are ~35% homologous to the junction domains of
Arabidopsis CDPK4 and soybean CDPK-
The evolutionary relationship of the Toxoplasma CDPKs to
known CDPKs is shown in Fig. 5B. The CDPKs from higher
plants and Paramecium form two clades that are distinct from
each other and from the CDPKs from apicomplexans. The latter form two
distinct clades. One contains TgCDPK1, the Eimeria CDPKs, and P. falciparum CDPK1; the second clade comprises TgCDPK2 and P. falciparum CDPK2 and CDPK3.
Expression of TgCDPK1 and -2 in Toxoplasma
Tachyzoites--
Monospecific antisera to both TgCDPK1 and -2 were
prepared using recombinant proteins and were used to determine their
expression in the parasite. The resulting antisera are highly specific
for each of the TgCDPKs, and no cross-reactivity is observed (Fig. 6A). As can be seen in Fig.
6A, TgCDPK1 can be readily detected in parasite extracts as
a protein with an apparent molecular mass of 58 kDa, close to the
predicted value of 57.3 kDa. The antiserum to TgCDPK2 failed to detect
any protein in tachyzoite extracts, although it reacted very strongly
with recombinant protein (Fig. 6A). To address the
possibility that the TgCDPK2 open reading frame might be part of a
pseudogene, we analyzed TgCDPK2 expression in tachyzoites using reverse
transcription-PCR. As can be seen in Fig. 6B, the TgCDPK2
transcript can be readily detected by reverse transcription-PCR,
suggesting that the TgCDPK2 gene is indeed transcribed but that
expression of the encoded protein may be regulated at the
post-transcriptional level. Since the TgCDPK2 protein does not appear
to be expressed in Toxoplasma tachyzoites, it was not
analyzed further.
TgCDPK1 Is a KT5926-sensitive, Calcium-dependent
Protein Kinase--
Purified recombinant TgCDPK1 demonstrated
calcium-dependent protein kinase activity. To analyze
activation of TgCDPK1 by calcium, in vitro protein kinase
reactions were performed at different concentrations of free calcium
(Fig. 7). Half-maximal activation of
TgCDPK1 occurred at ~600 nM free calcium, and enzyme
activity was maximal at calcium concentrations between 1 and 5 µM. A slight inhibition of activity was observed at
higher calcium concentrations. For all subsequent experiments,
reactions were performed in the presence of 2.5 µM free
calcium. Activation of TgCDPK1 was solely dependent on the presence of
calcium. The addition of 200 µg/ml phosphatidyl serine to in
vitro protein kinase reactions failed to stimulate the activity of
TgCDPK1 beyond that seen with calcium alone (data not shown). The
addition of recombinant Toxoplasma calmodulin also failed to
enhance calcium-stimulated TgCDPK1 activity (data not shown).
The results of kinetic analysis of TgCDPK1 are shown in Table
I. Several protein and peptide substrates
commonly used in the characterization of protein kinases were
investigated as possible TgCDPK1 substrates. Protamine sulfate and
neurogranin peptide were very poor substrates. Histone III-S, which has
been used as a substrate for plant CDPKs, was a poor substrate for
TgCDPK1 (Km = 3 mg/ml, Vmax = 1 µmol min
To characterize the enzymatic activity of TgCDPK1 further, we
investigated the effects of a variety of inhibitors on the enzyme. Several inhibitors of serine/threonine kinases had no effect on the
activity of TgCDPK1. Calphostin, an inhibitor of protein kinase C had
an IC50 greater than 10 µM. A potent
inhibitor of CaMKII, KN93, had an IC50 in excess of 30 µM for TgCDPK1. The IC50 value of
fluphenazine-N-2-chloroethane and calmidazolium, classic
calmodulin antagonists, was greater than 25 µM (data not
shown). The most potent inhibitor of TgCDPK1 was KT5926, a compound
commonly used as an inhibitor of calmodulin-dependent
protein kinase II and myosin light chain kinase that are inhibited with
Ki values of 4 and 18 nM, respectively
(6). As can be seen in Fig. 1, TgCDPK1 is less sensitive to KT5926 than
either enzyme with half-maximal inhibition of protein kinase activity
occurring at about 100 nM and complete inhibition at 1 µM. Strikingly, however, TgCDPK1 protein kinase activity
and Toxoplasma attachment to host cells display a virtually
identical sensitivity to KT5926 (Fig. 1), suggesting that TgCDPK1 is
the main target of KT5926 in T. gondii and is required for
the attachment of the parasite host cells.
To determine the relationship between TgCDPK1 and the major
KT5926-sensitive protein kinase activity detected during fractionation of Toxoplasma tachyzoite extracts, we analyzed all
chromatographic fractions by immunoblot analysis. As can be seen in
Fig. 4, TgCDPK1 cofractionates exactly with the
calcium-dependent, KT5926-sensitive protein kinase,
suggesting that they are identical.
Proteins Phosphorylated in Vitro by TgCDPK1 Are Similar to
KT5926-sensitive Phosphoproteins Identified in Vivo--
To identify
soluble endogenous substrates for TgCDPK1, in vitro protein
phosphorylation reactions were performed and analyzed by
two-dimensional gel electrophoresis and autoradiography. We first
identified proteins within T. gondii cytosol that were
phosphorylated as a result of endogenous calcium-dependent
protein kinase activities. To this end, protein kinase reactions
containing parasite cytosol were performed in the presence and absence
of calcium, and the corresponding autoradiographs were compared (Fig.
8, A and B). Three
major substrates of endogenous calcium-dependent protein kinase activity were identified as well as several minor ones.
To identify specifically the substrates for TgCDPK1,
T. gondii cytosol was heated at 70 °C for 10 min to
inactivate endogenous protein kinases. As is evident from Fig.
8C, this treatment is very effective at inactivating the
endogenous protein kinases. The addition of recombinant TgCDPK1 to
in vitro protein kinase reactions containing heat-treated
cytosol results in the calcium-dependent phosphorylation of
a number of proteins. The three major TgCDPK1 substrates appear
identical to the major substrates of calcium-dependent protein kinase activity in parasite extracts. More importantly, with
respect to molecular weight and isoelectric point, they appear to be
identical to the three KT5926-sensitive phosphoproteins identified in
Toxoplasma tachyzoites in vivo, PP1-3 (Fig. 2). In addition to the major substrates, heat-treated cytosol also contains
a number of minor substrates for TgCDPK1. Since these are absent in
untreated cytosol, they most likely represent proteins that were
denatured during the heat treatment of cytosol, exposing cryptic
TgCDPK1 phosphorylation sites in the process.
Although TgCDPK1 is a major protein kinase activity in
parasite extracts (Fig. 4), comparison of Figs. 2 and 8 suggests that this enzyme actually represent only a small fraction of the total protein kinase activity found in Toxoplasma tachyzoites.
This discrepancy suggests that other protein kinase activities are lost
during preparation and fractionation of extracts or that they are
inactive in the incubation conditions used to detect TgCDPK1. In
addition, the removal or inactivation of one or more TgCDPK1-specific
protein phosphatase activities during the preparation and subsequent
fractionation of the extracts would also result in an apparent increase
in its relative activity.
Cytoplasmic calcium fluctuations have been implicated
in the regulation of host cell invasion by the apicomplexan parasites Theileria parva (31), P. falciparum (32), and
T. gondii tachyzoites (4, 7, 33), suggesting that this
process is regulated by a common mechanism in apicomplexan parasites.
Although the specific calcium-dependent regulatory
mechanism involved has not been identified directly, inhibitor studies
have implicated calmodulin and a myosin light chain kinase-like
activity (5, 7).
Here, we describe the identification of one
calcium-dependent regulatory system required for
Toxoplasma attachment to host cells as a member of the CDPK
family. Two members of this family, TgCDPK1 and TgCDPK2, were
identified in T. gondii. Whereas TgCDPK1 protein is
expressed in tachyzoites, TgCDPK2 protein is not detectable in this
stage of the Toxoplasma life cycle. The predicted amino acid
sequences of these protein kinases are highly homologous to those of
CDPKs previously described in plants (8-10) and in the apicomplexan
parasites P. falciparum (16, 17) and E. maxima and E. tenella (18). Like these enzymes, the
Toxoplasma CDPKs are composed of three domains: an
N-terminal catalytic domain, a short junction domain, and a C-terminal
calmodulin-like domain with four EF-hand calcium-binding sites. Further
characterization of TgCDPK1 revealed that, in addition to structural
homology, the Toxoplasma enzyme also shares functional
properties with other CDPKs in that its activity is absolutely
dependent on the presence of calcium yet is insensitive to calmodulin
antagonists. Despite the high degree of homology, the catalytic
properties of TgCDPK1 are different from other CDPKs with respect to
the observed Km(app) values for peptide substrates
and ATP (9, 19). These differences most likely reflect the different
substrate specificities of these enzymes in vivo. Another
indication of diverse cellular activities among CDPKs lies in their
activation by calcium (19). While the activity of TgCDPK1 is
half-maximal at 600 nM free calcium, a concentration often
encountered in activated cells, the activity of the P. falciparum CDPK1 is half-maximal at 15 µM free
calcium (20). This difference could reflect the need for different
CDPKs to be activated at different levels of cellular stimulation,
although it could also reflect the absence of essential cofactors in
the kinase assays for P. falciparum CDPK1.
A possible function for TgCDPK1 in T. gondii is
suggested by its sensitivity to KT5926, an inhibitor of myosin light
chain kinases in animal cells. This compound was previously shown to be
an effective inhibitor of Toxoplasma motility as well as its attachment to host cells. The IC50 of KT5926 for in
vitro kinase activity of TgCDPK1 and parasite attachment to host
cells is almost identical. In addition, in vivo
phosphorylation experiments reveal that the effect of KT5926 on protein
phosphorylation in Toxoplasma is quite specific, since it
blocks the phosphorylation of only three proteins. These three proteins
are, with respect to their apparent molecular weights and isoelectric
points, indistinguishable from the three major substrates of TgCDPK1
identified in Toxoplasma extracts in vitro.
Finally, during fractionation of Toxoplasma cytosol, a
single peak of KT5926-sensitive protein kinase activity was detected,
which cofractionated consistently with TgCDPK1. Taken together, these
data demonstrate that TgCDPK1 is the most likely target for KT5926 in
Toxoplasma and is therefore likely to be involved in
bringing about parasite motility and attachment to host cells.
The physiological effects of TgCDPK1 activation are most
likely brought about by the phosphorylation of the three substrates we
detected in Toxoplasma extracts. The identity of these
substrates and the manner in which their phosphorylation could
contribute to parasite motility and host attachment is unclear at this
time. One possible mechanism is suggested by the observation that
treatment of Toxoplasma with KT5926 inhibits the secretion
of the adhesin MIC2 (and probably other proteins as well) from the
parasites' micronemes (7). It is therefore possible that
phosphorylation of one or more of the TgCDPK1 substrates is required to
bring about an aspect of microneme secretion, such as movement of
micronemes to their site of secretion or the actual fusion of
micronemes with the parasite plasma membrane. Since the secretion of
MIC2 and other adhesins by Toxoplasma appears to be an
essential prerequisite for parasite motility, it is possible that all
three TgCDPK1 substrates are involved in microneme secretion. It is
equally likely, however, that microneme secretion and parasite motility
are regulated independently by TgCDPK1 phosphorylation of different
substrates. Identification of the TgCDPK1 substrates will probably shed
light on these issues.
The possible role of TgCDPK2 is unclear at this time.
Although its transcript is readily detected in the tachyzoite stage of
the Toxoplasma life cycle, the protein product is not. A
number of examples have been described where the transcripts of
bradyzoite-specific antigens are actually detectable in both
tachyzoites and bradyzoites, but the proteins themselves are only
detectable in either the tachyzoites (34) or the bradyzoites (35).
Therefore, the possibility remains that TgCDPK2 protein will be
expressed in bradyzoites or sporozoites. The recent observation that
P. falciparum CDPK3 is only expressed in the sexual
erythrocytic stage of P. falciparum (17) strengthens this hypothesis.
The role of CDPKs in other apicomplexan parasites is unclear at this
time. But considering that all apicomplexan parasites are obligate
intracellular parasites and that cytoplasmic calcium appears to be
essential for host cell invasion, they are likely to possess similar
calcium-dependent regulatory mechanisms to bring about host
cell attachment and invasion. It is therefore likely that one or more
of the CDPKs identified thus far in other apicomplexan parasites will
perform a role similar to that of TgCDPK1 in Toxoplasma
gondii. If true, this would put apicomplexan CDPKs in control of
one of the most essential processes in parasite survival, the ability
to invade animal host cells. Since no members of the CDPK family have
been described in the animal hosts of apicomplexan parasites, these
enzymes may prove to be suitable targets for the development of novel
antiparasitic chemotherapeutics.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel (23) and
have also been implicated in the response to environmental stress and
infections (24, 25). The role of CDPKs in protists and apicomplexan
parasites is unclear at this time.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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 (27). 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 [
-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.
-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.
ZAPII (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).
-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 6 M 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.
-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 mM
phenylmethylsulfonyl 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 mM
Tris-HCl, pH 7.6, 50 mM NaCl, and 0.1 mM
CaCl2). 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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Toxoplasma attachment to host cells and
TgCDPK1 activity are equally sensitive to KT5926. The sensitivity
to KT5926 of Toxoplasma attachment to human foreskin
fibroblast cells (closed symbols) was determined
as described under "Experimental Procedures." Half-maximal
inhibition was observed at ~90 nM KT5926. The in
vitro sensitivity of TgCDPK1 to increasing concentrations of
KT5925 was determined as described under "Experimental Procedures"
in the presence of 2.5 µM free calcium. Half-maximal
inhibition of protein kinase activity was observed at ~100
nM. Data represent the means ± S.E.
(n = 3).
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[in a new window]
Fig. 2.
KT5926 is a selective inhibitor of protein
phosphorylation in T. gondii. Extracellular
T. gondii were incubated 30 min at 37 °C with
[32P]sodium phosphate in the presence of
Me2SO (DMSO) (A and
C) or 10 µM KT5926 (B and
D) and analyzed by two-dimensional gel electrophoresis as
described under "Experimental Procedures." Autoradiography for each
gel was performed for 24 h (A and B) and
96 h (C and D). Those phosphoproteins that
were reproducibly sensitive to KT5926 are boxed.
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Fig. 3.
Toxoplasma extracts contain
calcium-dependent and KT5926-sensitive protein kinase
activities but no calmodulin-dependent protein kinase
activity. Parasite extracts were prepared, and the endogenous
protein kinase activities were analyzed using histones III-S as
substrate as described under "Experimental Procedures." Each
20-µl reaction contained an amount of parasite extract equivalent to
5 × 106 organisms and was performed in the presence
of 2.5 µM free calcium, 1 mM EGTA, or 2.5 µM free calcium in combination with 500 nM
KT5926, 50 ng of recombinant Toxoplasma calmodulin, 30 µM of the calmodulin antagonist
fluphenazine-N-2-chloroethane (FCE), or 1 µM calphostin C (CP).
View larger version (23K):
[in a new window]
Fig. 4.
Cofractionation of KT5926-sensitive protein
kinase activity and TgCDPK1 in Toxoplasma
extracts. Soluble proteins were prepared from 2 × 109 parasites and fractionated by chromatography on
DEAE-Sepharose (A) and phenyl-Sepharose (B) and
analyzed for protein kinase activity as described under "Experimental
Procedures." Protein kinase activity was determined in the presence
of 2.5 µM free calcium (closed
circles), 2 mM EGTA (open
circles), or 2.5 µM free calcium in
combination with 500 nM KT5926 (open
squares). After chromatography on DEAE-Sepharose
(A), fractions containing the majority of
calcium-dependent, KT5926-sensitive protein kinase activity
were pooled as indicated and, after the addition of KCl to 2 M, were further fractionated by chromatography on
phenyl-Sepharose (B). This resulted in the detection of a
single peak of calcium-dependent, KT5926-sensitive protein
kinase activity. Using immunoblot analysis, TgCDPK1 was shown to
cofractionate precisely with the KT5926-sensitive protein kinase
activity during chromatography on DEAE-Sepharose (C) and
phenyl-Sepharose (D).
. Homologies extend along the entire length of the
predicted protein sequences but are most pronounced in the catalytic
domains (residues 1-298 in TgCDPK1 and 1-293 in TgCDPK2), where they
range from 44 to 93%.
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Fig. 5.
Alignment of phylogenetic analysis of TgCDPK
sequences. A, the predicted protein sequences of
TgCDPK1 and TgCDPK2 were aligned with the sequence of Glycine
max CDPK and human calmodulin. Boxes I
and II mark two conserved sequence elements conserved
between protein kinases and thought to be required for binding of ATP.
Box III indicates a sequence element conserved
between serine/threonine protein kinases and thought to be involved in
catalysis. Box IV indicates the four EF-hand
calcium-binding domains found in all CDPKs and calmodulin. The
sequences of TgCDPK1 and TgCDPK2 have been deposited at
GenBankTM under accession numbers AF333958 and
AF333959. Sequences of Toxoplasma cDNA clones that are homologous
to TgCDPK1 and TgCDPK2 were identified in the GenBankTM
data base (accession numbers AAC02532 and AAD17247, respectively).
B, TgCDPK1 and TgCDPK2 are members of two distinct classes
of apicomplexan CDPKs. CDPK sequences most homologous to TgCDPK1 and -2 were identified using BLAST and FASTA, and a phylogenetic tree was
generated as described under "Experimental Procedures."
AtCDPK2, A. thaliana CDPK2 (BAA04830);
AtCDPK4, A. thaliana CDPK4 (CAB82124);
EmCDPK, E. maxima CDPK (CAA96438);
EtCDPK, E. tenella CDPK (CAA96439);
GmCDPK
, G. max CDPK-
(AAB80693);
PfCDPK1, P. falciparum CDPK1 (CAA47704);
PfCDPK2, P. falciparum CDPK2 (CAA68090);
PfCDPK3, P. falciparum CDPK3 (AAF63154);
PtCDPKA, P. tetraurelia CDPK-A (AAC13356);
PtCDPKB, P. tetraurelia CDPK-B (AAC13355).
. The
calmodulin-like C-terminal domains of TgCDPK1 and -2, like all
known CDPKs, contain four EF-hand calcium-binding sites. The overall
homology with the calmodulin-like domains of other characterized CDPKs
ranges from 34 to 74%. The homology of this domain to
Toxoplasma, plant, and animal calmodulin is confined to the
EF-hands and does not exceed 34%.
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Fig. 6.
Expression of TgCDPK1 and TgCDPK2 in T. gondii tachyzoites. A, immunoblot analysis
of TgCDPK expression in tachyzoites. Total lysates of
Toxoplasma tachyzoites were prepared in SDS-PAGE sample
buffer, and aliquots corresponding to 5 × 105
(TgCDPK1) or 107 (TgCDPK2) were analyzed by SDS-PAGE and
immunoblotting with specific antisera to TgCDPK1 and TgCDPK2. As
positive controls, 20 ng of purified recombinant TgCDPK1 and TgCDPK2
were analyzed in parallel. B, reverse transcription-PCR
analysis of TgCDPK2 mRNA expression in tachyzoites. Total RNA was
isolated and analyzed by reverse transcription-PCR as described under
"Experimental Procedures."
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Fig. 7.
Calcium dependence of TgCDPK1 activity.
The protein kinase activity of TgCDPK1 was determined in the presence
of different concentrations of free calcium as described under
"Experimental Procedures" (n = 3).
1 mg
1).
While histone III-S initially proved useful in the identification and
purification of a number of CDPKs, it has been noted that potential
variation in peptide composition among different preparations of the
substrate limits the reliability of kinetic comparisons (36). We
found that the synthetic peptides, syntide-2 and the glycogen synthase
peptide, provided more consistent kinetic measurements than histone
III-S. We obtained values of 107 µM and 5.3 µmol min
1 mg
1 for
Km and Vmax for the glycogen
synthase peptide and found the Km to be 155 µM and the Vmax to be 5.2 µmol min
1 mg
1 with
respect to ATP.
Kinetic parameters of TgCDPK1
-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.
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Fig. 8.
In vitro phosphorylation of
Toxoplasma proteins by TgCDPK1. Soluble parasite
extracts were prepared as described under "Experiments Procedures."
To detect endogenous calcium-dependent protein kinase
activity in parasite extracts, aliquots corresponding to
107 parasites were incubated directly with
-[32P]ATP in the presence of 2 mM EGTA
(A) or 2.5 µM free calcium (B). The
presence of specific TgCDPK1 substrates in parasite extracts was
determined using heat-treated extracts (10 min at 75 °C)
corresponding to 107 parasites that were incubated with
[
-32P]ATP in the absence (C) or presence of
200 ng of purified recombinant TgCDPK1 (D). Phosphorylation
reactions were analyzed by two-dimensional chromatography and
autoradiography. Phosphoproteins that are phosphorylated specifically
in the presence of TgCDPK1 are boxed. The
asterisk indicates a minor contaminant of recombinant
TgCDPK1 that is phosphorylated effectively by this enzyme.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Julie Hopkins for excellent technical assistance and thank Tara Mann and Drs. Gary Ward, David Sibley, and Vern Carruthers for helpful discussions. We also thank Dr. Jean Francois Dubremetz for generously supplying the monoclonal antibody to SAG1. We are especially grateful to Dr. Tom Unnasch for performing the phylogenetic analysis of the CDPK sequences.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI41765 and a New Investigator Award from the Burroughs Wellcome Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF333958 and AF333959.
To whom correspondence should be addressed: Division of Geographic
Medicine, 845 19th St. S., BBRB 206, Birmingham, AL 35294-2170. Tel.:
205-934-1633; Fax: 205-933-5671; E-mail: cbeckers@uab.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M011045200
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ABBREVIATIONS |
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The abbreviations used are: CDPK, calmodulin-like domain protein kinase; TgCDPK1 and -2, T. gondii CDPK1 and -2; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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