MOLECULAR

iikEMIcAL
PARAsIToLoGy
ELSEVIER Molecular and Biochemical Parasitology 82 (1996) 37-49

Plasmodium falciparum protein kinase 5 and the malarial

nuclear division cycles

Ralph Graeser, Barbara Wernli, Richard M. Franklin, Barbara Kappes*

Departmnzt of’ Structural Biology, Biozentrum, (irkwrsiiy of Basel. Klingelbergstrasse 70. CH-4056 Basel, Swit-_erland

Received 9 May 1996: revised 10 July 1996: accepted 18 July 1996

Abstract

In the course of our studies on cell cycle regulation mechanisms of P/usnzodi~r~~ fulciparum, we investigated
expression pattern, kinase activity, and localization of PfPKS, a putative malarial member of the family of
cyclin-dependent protein kinases (cdks). The kinase was immunoprecipitated from parasites of selected stages and
from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from
aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite’s S-phase.
To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors
flavopiridol and olomoucine, which act on PfPKS in vitro at similar concentrations as on other cdks. When applied
during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from
our proposition that PfFK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies
provide further evidence for this potential role of PfPK5.

Kr)~~\~ords: Malaria; Plusmodium jhkiparum; PfPK5; cdk; Nuclear division cycle

1. Introduction

Compared to the knowledge available con-

cerning surface antigens, immunogenicity, and
Abbreviations: cdk, cyclin-dependent protein kinase; IC,,, epidemiology of the human malaria parasite,
50% inhibitory concentration; NTA, nitriloacetic acid; PfPK5, Plasmodium falcipavum, not much is known
Plasmodium fakiparum protein kinase 5; RBC, red blood cell: about its cell biology. Regulation of the cell di-
TCA, trichloroacetic acid: TOT0 1, benzothiazolium-4-quino-
vision cycle, understood in detail in other or-
hum dimer.
* Corresponding author, Swiss Tropical Institute, Socin-
ganisms, is one example. During the asexual
strasse 57, 4002 Basel, Switzerland. Tel.: + 41 61 2848205; fax: intraerythrocytic life cycle of P. fulciparum, the
+ 41 61 2718654; email: kappes@ubaclu.unibas.ch syncytial division cycles take place after about

0166-6851/96/$15.00 8 1996 Elsevier Science B.V. All rights reserved

PII S166-6851(96)02716-6
38 R. Graeser et al. / Molecular
30 h of maturation in the red blood cell
(RBC), in the so-called schizont stage. This
lasts about 6-8 h. Usually 10 to 20 newly
formed nuclei are produced, which would re-
quire 4-5 rounds of mitosis. Additional knowl-
edge concerning the nuclear division cycle of P.
falciparum can be summarized as follows:
(1) DNA synthesis can be reversibly blocked by
aphidicolin and irreversibly by hydroxyurea [ 11.
Some properties of the malarial DNA poly-
merase a have been reported [2], and a partial
sequence is known [3]. The complete sequence
of DNA polymerase 6 has been published [3],
and also that of a proliferating cell nuclear
antigen (PCNA) homologue [4].
(2) Based largely on electron microscopy stud-
ies, it has been suggested that the structural
organization of mitotic nuclear division in the
asexual cycle is of a crypto-ortho-mitotic type.
In such cases the nuclear envelope remains in-
tact during separation of the chromosomes. The
spindle apparatus is generated by a single cen-
triolar plaque which gives rise to a hemispindle
and is subsequently split into two daughter
spindles. These migrate in opposite directions
to form the mitotic spindle apparatus, which is
involved in separating the chromosomes [5].
(3) During mitosis, the chromosomes are not
condensed in the M-phase [6], although Plas-
modium seems to have nucleosomes [7]. Several
histone genes, as well as a gene coding for a
HMG (high mobility group)-like protein, have
been described [8- 1I]. Furthermore, homo-
logues of ran (ras-related nuclear protein), and
a chromatin binding protein, RCC 1 (regulator
of chromatin condensation l), which can act as
a guanidine exchange factor for ran, have been
reported for P. falciparum ([12], and abstract
published in Ann. Trop. Med. Parasit. 89, 210).
In BHK21/13 cells containing a temperature-
sensitive mutant allele of RCCl, a shift to the
restrictive temperature during the Gl phase of
the cell cycle provoked premature chromosome
condensation [13]. RCCl plays a role in other
cellular processes as well (see [14]).
The cdk (cyclin-dependent kinases) family of
protein kinases is involved in the regulation of the
cell cycle of many organisms (see [15]). Originally,
and Biochemical Parasitology 82 (1996) 37-49
a single gene product, cdc2 (now cdkl), in a
complex with cyclin B was thought to be the
regulatory factor. In the meantime many more
kinases and their cyclin partners have been found
(see [16]). A gene with 60% identity to human
cdc2, PfPK5 (Plasmodium falciparum protein ki-
nase 5) has been isolated from P. falciparum [ 171.
The phosphorylation pattern and cyclin binding
of PfPK5 in vivo have not yet been investigated.
In vitro data suggest, however, the presence of a
similar site for activating phosphorylation as in
cdks from other species [18]. Another cdc2-related
gene, Pfcrkl (Plasmodium falciparum cdc2-related
kinase l), was identified and this gene may be
involved in gametogenesis [19]. In this paper we
present data on the putative function of PfPK5 in
vivo.
2. Materials and methods
2.1. Materials
Aphidicolin was purchased from ICN, olo-
moucine from Promega, [Y-~~P]ATP from Amer-
sham Life Science, and Expre35S35S Protein
Labeling Mix and [G-3H]hypoxanthine from
NEN-DuPont. Flavopiridol was a gift from Dr.
P.J. Worland [20]. TOT0 1 (benzothiazolium4-
quinolium dimer) was purchased from Molecu-
lar Probes.
2.2. Parasite cultures and in vivo labeling
P. falciparum isolate Kl (Thailand), adapted
to grow in horse serum, was kindly provided
by Dr. H. Matile (F. Hoffmann-La Roche,
Basel) and was used for all experiments. The
parasites were cultured in RPMI-1640 (Gibco-
BRL) supplemented with 25 mM HEPES, 0.37
mM hypoxanthine, 100 mg l- ’ neomycin base,
and 10% heat-inactivated horse serum. The cul-
tures were set up with a 5% hematocrit and
incubated at 37°C with 4% CO,, 3% O,, and
93% N,, at 94% relative humidity. Cultures
were synchronized by three consecutive sorbitol
treatments [21]. The second and the third treat-
ments followed 34 and 75 h after the first.
 R. Graeser et al. I Molecular and BiochemicalParasitolog))82 (1996) 37-49
Time zero was set at 48 h after the start of the last
sorbitol treatment
To label parasitic proteins with [35S]methionine,
parasites were incubated 3 h in RPM1 lacking
methionine, followed by 6 h in the presence of 200
PCi of Expre35S35S Label. To label parasitic nu-
cleic acids with [3H], parasites were preincubated
12 h in RPM1 lacking hypoxanthine. Then, 0.5
ml/well of the culture was plated on 24-well-
plates. Finally, 30 PCi [G-3H]hypoxanthine was
added per well, as described in the Results (Sec-
tion 3).
2.3. Nucleic acid extraction
RNA and DNA were separated as described in
[22], with the exception that the centrifugation
parameters were modified. All centrifugation steps
were done at 9000 x g, except for the last, which
was at 15000 x g.
2.4. Determination of in vivo IC,,
Concentrations of drugs required to inhibit par-
asite growth by 50% after 48 h (IC,,) were deter-
mined by the method of [G-3H]hypoxanthine
incorporation [23], using cultures of 1% haemat-
ocrit and 0.8% initial parasitaemia. The data were
plotted using the Cricket Graph software, and the
IC,, value was determined.
2.5. Preparation of PfPKS Sepharose and
antibody pur$cation
An overnight culture of the bacterial strain
SG13009 [24] harbouring the plasmid pDS56/
RBSII, 6 x His [25] with the complete PfPK5
open reading frame and six adjacent histidines at
the N-terminus was diluted 1:60 into 5 1 of 2TY
medium containing kanamycin (25 pg ml - ‘) and
ampicillin (100 pg ml - I). The bacteria were al-
lowed to grow for 1 h at 37°C. The temperature
was then reduced to 30°C and, after the OD,,,
reached 0.8 to 1, the culture was grown for an
additional 5 h in the presence of 2 mM IPTG
(isopropyl P-D-thiogalactoside). After collecting
the cells by centrifugation at 8300 x g for 15
min. they were resuspended in 50 ml of buffer A
39
(50 mM Tris-HCl pH 7.4, 300 mM NaCl). Af-
ter six pulses of sonication (75%, 30 s in a
Braun sonicator), the supernatant was cleared
twice by centrifugation at 18 000 x g for 20 min
and then applied to a 1 ml column of nickel-
NTA-Agarose (Qiagen). After washes with buffer
B (50 mM MES (2-(N-morpholino)ethanesulfonic
acid) pH 6, 300 mM NaCl) and buffer C (50 mM
Tris-HCl pH 7.4, 300 mM NaCl, 40 mM imida-
zole), the protein was eluted with buffer D (50
mM Tris-HCl pH 7.4, 300 mM NaCl, 200 mM
imidazole), and concentrated in Centricon- con-
centrators (Amicon) to 4--6 ml. The buffer was
changed to coupling buffer (100 mM NaHCO,,
500 mM NaCl) by gel filtration on a Fast Desalt-
ing HRlOjlO column (Pharmacia). Coupling to
CNBr activated Sepharose 6MB beads (Pharma-
cia) was done according to the instructions of the
manufacturer.
The PfPK5 antibody [17] was affinity-purified
on the PfPK5 Sepharose column as described in
P61.
2.6. Immunoprecipitation and kinase assay
Parasites were isolated as described in [27].
The parasites were then lysed in the appropriate
volume of RIPA buffer (50 mM Tris-HCl pH
8, 150 mM NaCl, 50 mM NaF, 5 mM EDTA,
0.5% sodium deoxycholate, 0.1% SDS, 1% Tri-
ton X-100) supplemented with 1 mM PMSF
(phenylmethylsulfonyl fluoride), pepstatin A (1
pg ml - ‘), and leupeptin (1 pg ml ~ ‘). The lysate
was cleared twice by centrifugation at 15 000 x g
for 15 min, and treated two times 30 min with
Pansorbin (Calbiochem). The affinity-purified
antibody was diluted in 1 ml of RIPA buffer
and rotated for 2 h at 4°C in the presence of 75
~1 of PfPKS-Sepharose or BSA (bovine serum
albumin) Sepharose. The supernatant, containing
the non-bound material, was given to the appro-
priate amount of parasite lysate, and rotated for
2 h. To collect the complexes, a volume of 40 ~1
of a 10% suspension of protein-A-Sepharose
CL4B (Pharmacia) were added to each reaction
and left rotating for 1 h at 4°C. The beads were
40 R. Graeser et al. / Molecular and Biochemical Parasitology 82 (1996) 37-49

washed three times with 1 ml of RIPA buffer and 3. Results

four times with 1 ml of kinase wash buffer (50
mM Tris-HCl pH 7.4, 15 mM MgCl,). [3sS]-la- 3.1. Stage-speciJic expression, kinase activity, and
beled immunoprecipitates were then taken up in localization of PfPKS
1 x sample buffer, those to test for kinase activity
in 20 ul of kinase assay buffer (50 mM Tris-HCl In order to establish a possible function of
pH 7.4, 15 mM MgCl,, 5 mM EGTA, 1 mM PfPK5 (Plasmodium falciparum protein kinase 5)
1,4-dithio-DL-threitol, and 2.5 yg of histone Hl during the malarial nuclear division cycle in the
(Boehringer). Reactions were started by the addi- erythrocyte, the presence and activity of the en-
tion of ATP to 25 uM and 1 uCi of [Y-~~P]ATP, zyme during this period of asexual development
allowed to proceed for 30 min at 30°C and of the parasite had to be analysed. The nuclear
stopped by the addition of sample buffer and division cycles take place in the late trophozoite/
heating to 95°C for 5 min. early schizont stage, at around 28 to 38 h after
invasion of the red blood cell (RBC). At that
2.7. Confocal immunojIuorescence analysis time, newly formed nuclear bodies are detected
within the parasite by Giemsa staining of fixed
Parasitized erythrocytes at a hematocrit of 20% parasitized RBCs. We chose four timepoints: 24,
were washed twice in RPM1 1640 medium. Ten ul 30, 36, and 42 h (see Materials and methods,
of the cell suspension was fixed and permeabilized Section 2 for definition of the age). encompassing
with 20 ul 4% paraformaldehyde containing 0.1% this period. Synchronized cultures with an age
SDS for 30 min on poly-(L-lysine)-coated (1 mg range of approximately f 3 h were used to deter-
ml ~ ‘) slides, followed by six washes in PBS (137 mine PfPK5 kinase activity, level of expression,
mM NaCl, 2.7 mM KCl, 8.1 mM Na,HPO,, 1.8 and amount of protein, as well as total protein
mM KH,PO,, pH 7.4) for 5 min each. Samples synthesis and amount of protein. All the determi-
were blocked by incubation in PBS containing 1% nations dealing with PfPK5 were done with equal
bovine serum albumin (BSA, Sigma A 7030) for amounts of parasites.
30 min. The affinity-purified PfPK5 antibody was The expression level of PfPK5, which was mea-
applied at a dilution of 1:4 for 60 min. Six washes sured by immunoprecipitation of the kinase from
in PBS containing 1% BSA were done. Then, the cultures grown for 6 h in the presence of
second antibody, which was a CySTM coupled [35S]methionine, peaks at 30 h (Fig. 1, panel b).
donkey anti-rabbit antibody (Jackson Im- The highest relative protein level, determined by
munoResearch Laboratories), together with the means of a Western blot using a PfPK5 specific
DNA stain, TOT0 1 (0.1 PM), were applied. The antiserum, was detected at 36 h (Fig. 1, panel c).
slides were rinsed six times in PBS, mounted in To test the enzymatic activity of the PfPK5 ki-
90% glycerol in 100 mM TrissHCl pH 8 contain- nase, it was immunoprecipitated from unlabeled
ing 0.2% o-phenylendiamine, and examined using cell lysates and incubated in the presence of [y-
a confocal laser scanning microscope (Noran, “P]ATP with histone Hl as substrate. The en-
Odyssey XL). zyme activity was highest at 36 h (Fig. 1, panel a).
According to these results, PfPK5 may play a role
2.8. Other techniques in the malarial nuclear division cycle.
In other eukaryotes. the cellular localization of
Western blotting was done according to [17]. cdks is strictly regulated (see [29]). We were there-
Protein concentration was determined according fore interested whether, in addition to expression
to [28]. [35S]-incorporation in parasite proteins and activity, the cellular localization of PfPK5 is
was measured by TCA precipitation of labeled also regulated during the parasite development in
lysates, filtration through glass microfiber filters the RBC. We addressed this question by using
(Whatman), and subsequent counting by liquid confocal immunofluorescence microscopy. PfPK5
scintillation spectroscopy. was detected using the affinity-purified antiserum,
 R. Grueser et al. i Molecular and Biochemical Parasitolog! 82 (1996) 37-49 41

DNA was localized with TOT0 1 (Fig. 2A). The 3.2. Effect of aphidicolin treatment on PfPK.5
upper panel shows two parasites at the tropho- kinase activity
zoite stage, before or at the beginning of DNA
synthesis (age < 30 h). PfPK5 colocalizes with the Most cdks play a role in (a) specific phase(s) of
DNA stain. In the middle panel, a parasite in the the cell cycle and thus their activity oscillates
process of nuclear division is depicted (age 30-36 during the cell cycle [16]. An investigation of
h). PfPK5 localizes as discrete spots at the edge of PfPK5 activity within the individual phases of the
the DNA. Only rarely, PfPK5 colocalizes with nuclear division cycles requires a synchronization
TOT0 1 during the process of nuclear division of the parasite nuclear division cycles. An impor-
(Fig. 2B). In the lowest panel of Fig. 2A, a tant prerequisite to achieve this is the presence of
parasite at the end of the intraerythrocytic cycle is so-called checkpoints, which control, e.g. the
shown, in which the new merozoites have already availability of certain components or the correct
been formed (age > 40 h). PfPK5 immunofluores- order of events during the cell cycle. Upon chal-
cence is decreased to minute spots close to the lenge of such a checkpoint by starvation or drug
DNA stain, suggesting that merozoites contain a inhibition, the cells are arrested at a distinct phase
small amount of PfPK5. The presence of PfPK5 during the cell cycle.
in merozoites could be confirmed by Western Difluoromethylornithine, aphidicolin, and taxol
or vinblastine block the eukaryotic cell cycle at
blotting (data not shown). Taken together the
G,, S, and M-phase, respectively, by challenging
immunofluorescence data suggest a regulation of
specific checkpoints [30-331. All of these drugs
PfPK5 localization within the developmental cycle
had been shown to act on their specific targets in
of the parasite in the RBC.
P. falciparum as well ([34,35]; R. Graeser, submit-
ted for publication). The effect of aphidicolin is
reversible [36], indicating that the parasite pos-
a. sesses a checkpoint controlling for DNA synthe-
sis. Checkpoints controlling polyamine levels or
24 30 36 42 24 30 36 42 mitotic spindle function, however, are missing
during the malarial nuclear division cycles (R.
Graeser, submitted). Therefore, difluoromethylor-
nithine, taxol and vinblastine do not block and
b.
hence cannot be used to synchronize the malarial
30 36 42
nuclear division cycles.
24 30 36 42 24
We tried to synchronize the parasite nuclear
division cycles by reverting the block obtained by
aphidicolin. Recovery from the inhibitor was slow
C. and resulted in cultures with a wide age span. We
therefore compared PfPK5 kinase activity from
24 30 36 42 lysates of aphidicolin blocked cells to untreated
cells, an approach used for the investigation of the
Fig. 1. Activity, expression and protein level of PfPK5 in
function of other cdks as well [37-391. Cultures of
relation to the age of the parasite. Parasites 24, 30, 36. and 42
h old were investigated. (a) Histone Hl phosphorylated by an average age of 30 h were incubated for 6 h in
PfPK5 immunoprecipitates. (b) PfPK5 immunoprecipitated the presence of 5 uM aphidicolin, or left untreated
from [“Slmethionine-labeled parasites. The immunoprecipita- (control). The aphidicolin concentration corre-
tions in panels (a) and (b) were done with affinity-purified sponded to about IO-fold the concentration re-
PfPK5 antibodies, which were preincubated with BSA-Sep-
quired for a 50% inhibition (IC,,) of the growth
harose (left panel). or PfPKS-Sepharose (right panel. negative
control). The same amount of parasites was taken for each of our cultures and was in the range of that
assay. (c) Western blot analysis with the PfPK5 antibody from applied in an earlier study [36]. If PfPK5 plays a
lysates of equal amounts of parasites. role in the initiation or the maintenance of the
42 R. Grueser et al. 1 Molecular and Biochemical Parasitology 82 (1996) 37-49

Fig. 2. Confocal immunofluorescence images, Left column (red): PfPK5 immunofluorescence; middle column (green): TOT0 1; right
column: composite. (A) Parasites of various ages - upper row: two parasites, less than 30 h old; middle row: parasite, 30-36 h old;
lower row: parasite, older than 40 h. The upper two size bars are 2.5 urn, the one in the bottom row 2 urn. (B) Three parasites at
the time of nuclear divisions. Note the nuclear localization of PfPK5. The size bar is 5 urn.

S-phase of the parasite nuclear division cycles, its stimulation of kinase activity when compared to
kinase activity would be expected to be elevated control cultures (Fig. 3). The drug did not change
by the drug treatment. If not, PfPK5 activity from PfPK5 protein and expression levels, nor did it
aphidicolin blocked cells should be decreased. affect total protein synthesis and amount (data
Aphidicolin treatment resulted in a 1.6 to 2 times not shown). The increased activity of PfPK5 in
 R. Graeser et al. 1 Molecular and Biochemical Parasitology 82 (1996) 37-49
aphidicolin blocked cells suggests a possible in-
volvement of the kinase in the initiation or
maintenance of the S-phase of the parasite’s nu-
clear division cycles.
3.3. Olomoucine and flavopiridol
One great obstacle in investigating a gene’s
function in P. falciparum is the inability to easily
and reproducibly transfect or knockout genes. We
searched for alternative routes to support our
proposal that PfPK5 is active in the S-phase of
the nuclear division cycle. Recently several highly
specific cdk inhibitors have been described. Olo-
moucine was tested on a wide range of protein
kinases, and shown to act very specifically on
most members of the cdk family with IC,, values
in the range of 3-50 uM [40]. Furthermore, at a
concentration of 50-100 PM, the drug arrests
plant cells at the G,/S and G,/M transitions [41],
and affects cell cycle progression in a wide range
of other eukaryotic organisms [42]. Also, 0.2 uM
control aphidicolin
Fig. 3. When compared to control cells, aphidicolin treatment
of parasites increased PfPK5 kinase activity. PfPK5 was im-
munoprecipitated from untreated or aphidicolin-treated
sites, and incubated with [y-3’P]ATP and histone
Autoradiographs from several independent experiments
scanned with a laser densitometer. Values from the control
experiments were set to loo%, and the aphidicolin values were
calculated. The standard error is indicated.
43
flavopiridol arrests the cell cycle of breast car-
cinoma cells at the G,/M transition [43]. More-
over, the drug inhibits purified sea star cdc2
kinase with an IC,, of 0.5 uM [20]. Both drugs,
olomoucine and flavopiridol, act as cdk-specific
ATP-analogues [20,40].
We tested the effect of these two inhibitors on
the kinase activity of PfPK5 immunoprecipitated
from aphidicolin-blocked cells, as well as on the
growth of parasite cultures. The IC,, values of the
drugs on kinase activity were determined by eval-
uating scans of the respective autoradiographs
(Fig. 4a), those on the growth of parasite cultures
by the method of [G-3H]hypoxanthine incorpora-
tion [23] (Fig. 4b). The ICY,,values for flavopiridol
were 0.06 uM for the immunoprecipitated PfPK5
kinase and 2 uM when tested on cultures. For
olomoucine both values were approximately 15
PM. Since these values were comparable to those
reported for other eukaryotic systems, both in-
hibitors were considered as potential tools to in-
vestigate PfPK5 function in vivo.
3.4. Drug effects on DNA synthesis
If PfPK5 acts as an activator of, or is necessary
to maintain, the S-phase of the parasite, in vivo
inhibition of PfPK5 by flavopiridol or olomoucine
should result in a block of DNA synthesis. To test
this, parasites with an average age of 30 h were
treated with these two drugs and also aphidicolin,
as a control for DNA synthesis inhibition. The
effect of the inhibitors on [G-3H]hypoxanthine
incorporation into DNA was determined. The
concentrations of flavopiridol and olomoucine
were 15 and 150 PM, corresponding to 7.5 or 10
times, respectively, the in vivo IC,, value.
In the control culture, the [3H]-incorporation of
label into DNA peaks between 32 and 33 h (Fig.
5a), whereas incorporation into RNA is con-
stantly decreasing during this time period (data
not shown). The RNA synthesis peaks before
DNA synthesis [ 1,221. Aphidicolin reduced
para- [3H]hypoxanthine incorporation into DNA very
Hi.
rapidly and efficiently to about 30% of the control
were
value (Fig. 5b). Flavopiridol decreased DNA syn-
thesis after 30 min to about 50%; olomoucine was
less effective, reducing the hypoxanthine incorpo-
44 R. Graeser et al. / Molecular and Biochemical Parasitology 82 (1996) 37-49

a. In a further experiment, we tested the effect of

* the drugs on PfPK5 kinase activity and expres-
olomoucine
I sion as well as on total parasite protein and
nucleic acid synthesis. Additionally, the amount
of total parasite protein was determined (Fig. 6).
Both drugs decreased total parasite protein syn-
flavopifidfd thesis to approximately 50% of the control val-
ues, reducing the amount of total parasite
protein by 25%. The 50% inhibition of PfPK5
100 10 1 0.1 0.01 0 -
expression followed the decrease of total parasite
w protein synthesis and resulted in a total kinase
b. activity of PfPK5 of 80% when compared to
control lysates. Since the drugs act as cdk-spe-
cific ATP-analogues, their effect is reversible
[41,43], and the decrease of PfPK5 activity will
probably be caused by the inhibition of its ex-
pression.
If the effect of the cdk inhibitors on DNA,
RNA and parasite protein synthesis is the result
of a general toxicity of the compounds on P.
falciparum, a prolonged incubation of parasite
cultures in the presence of the drugs should en-
hance their effect on DNA and RNA synthesis.
To check this, we incubated parasite cultures with
olomoucine and flavopiridol from 10 h before the
.Ol .I I 10 100 loo0
onset of nuclear divisions to the end of the nu-
w clear division cycles, at an age of 36 h. The
extended treatment did not markedly alter the
Fig. 4. Effect of olomoucine and flavopiridol on PfPK5 kinase
effect of the drugs, indicating the specificity of
activity and growth of the parasite. (a) Kinase assay. Thirty h
old cultures were grown for 6 h in the presence of aphidicolin. their action on the nuclear division cycles of the
PfPK5 was immunoprecipitated and incubated with [y- parasite (data not shown). Furthermore, the cor-
““PIATP, histone HI and increasing amounts of olomoucine relation of the onset of drug action with PfPK5
or flavopiridol (indicated in PM). 0, indicates the untreated activation at a parasite age of 30 h may indicate
positive control; -, the preincubation with PfPKS-Sepharose
that PfPK5 is indeed the target of the drugs.
(negative control). (b) Effects on the growth of parasite cul-
tures were determined using the method of [G-3H]hypoxan-
thine incorporation [23]. Drug concentrations are again
indicated in uM. Open squares are flavopiridol values, closed 4. Discussion
squares olomoucine. The means of duplicate determinations
are shown.
Due to the present unavailability of methods
used for other eukaryotes - such as overexpress-
ration to around 60-70% of control synthesis (Fig. ing or knocking out genes, microinjection tech-
5c,d). The slightly delayed and weaker inhibition of niques, etc. - it has proven very difficult to
DNA synthesis by the latter two drugs suggests an investigate the in vivo function of a given protein
indirect mechanism of action, which may be ex- in P.falciparum. A gene of P.falciparum encoding
plained by a primary inhibition of PfPK5, which a cdk-like kinase, PfPK5, had previously been
then results in inhibition of DNA synthesis. This isolated, and its product had been demonstrated
interpretation would support our hypothesis that to be functional in in vitro assays [17]. Using
PfPKS plays a role in the malarial S-phase. immunoprecipitations with an affinity purified
 R. Graeser et ul. / Molecular and Biochemical Parasitology S-1 (1996) 37-49 45

30.5 31 31.5 32 32.5 33 33.5 34 34.5 35 35.5 36 30.5 31 31.5 32 32.5 33 33.5 34 34.5 35 35.5 36
parasite age parasite age

30.5 31 31.5 32 32.5 33 33.5 34 34.5 35 35.5 36 30.5 31 31.5 32 32.5 33 33.5 34 34.5 35 35.5 36
parasite age parasite age

Fig. 5. Effect of aphidicolin. flavopiridol, and olomoucine on parasite DNA synthesis, Thirty h old cultures were aliquoted into
24-well plates, and the drugs were added. Untreated cultures served as controls. Duplicate cultures were pulsed with 30 nCi of
[G-lH]hypoxanthine for 30 min, and pulses were applied every half hour, until the parasites reached an age of 36 h. The pulses were
stopped by directly pipetting the cultures in 10% TCA 1221. The x-axis indicates the endpoint of the incubation period. (a) Untreated
(control) cultures, (b) aphidicohn-, (c) flavopiridol-, and (d) olomoucine-treated cultures. Similar results were found in several
independent experiments.

PfPK5 antiserum, we were able to isolate the play a role at a distinct phase of the cell cycle (see
active protein from parasite lysates. PfPK5 is [ 161). We therefore considered synchronization of
present and active at the time of nuclear divisions the nuclear division cycles in order to specify the
in the developmental cycle (Fig. 1). Many cdks role of PfPK5 more precisely. Since the nuclear
46 R. Graeser et al. 1 Molecular and Biochemical Parasitology 82 (1996) 37-49
division cycles of Plamodiunt are fast, i.e. 668 h
for 4-5 rounds of mitosis [44], and the recovery
from the reversible drug aphidicolin was too slow
to permit synchronization, we decided to test
lysates from arrested cells for kinase activity. The
kinase activity of PfPK5 was elevated in cells
blocked by aphidicolin, when compared to the
control culture (Fig. 3). This suggests a role of the
kinase in the parasite S-phase. The relatively weak
stimulation of PfPK5 kinase activity in the aphidi-
colin-blocked cells may be explained by the rapid
succession of the nuclear division cycles. The
replication of the genome will probably make up
a large part of one cycle. Therefore many para-
sites of the control cultures would be expected to
be in S-phase as well.
In order to get additional evidence for a role of
PfPK5 in the nuclear division cycles, we tested the
effect of two cdk inhibitors, olomoucine and
flavopiridol, on PfPK5 kinase activity and on
parasite cultures. Since the IC,, values obtained
were similar to those found in other eukaryotes,
Fig. 6. Effect of olomoucine and flavopiridol on PfPK5 ex-
pression and activity, total parasite protein synthesis as well as
amount, and nucleic acid synthesis. Thirty h old cultures were
incubated for 6 h in the presence of the individual drugs. The
values given for PfPK5 expression and kinase activity are
derived from scans of the respective autoradiographs. The
cultures used to determine the effect on nucleic acid synthesis
were aliquoted into 24-well plates and supplemented with
[3H]hypoxanthine. DNA and RNA were prepared as described
in [22]. The control values were set to 100%. and the others
were calculated.
we used the drugs to mimick the effect of knock-
ing out PfPK5 during the nuclear division cycles.
If these inhibitors do act via PfPK5 inhibition,
and PfPK5 does play a role in the nuclear division
cycle, the parasite’s DNA synthesis should be
affected by the drugs. Both drugs significantly
reduced incorporation of [3H]hypoxanthine into
DNA. The inhibition of DNA synthesis by the
two cdk inhibitors support a function of PfPK5
during the nuclear division cycle, although we
cannot exclude that other cdk-like kinases have
been inhibited as well. Together with the stimula-
tory effect of the aphidicolin treatment on PfPK5
kinase activity, however, these results strongly
suggest a role of PfPK5 in the malarial S-phase.
The cdk inhibitors, and aphidicolin (data not
shown), hence the inhibitors which block DNA
synthesis, affect total RNA synthesis as well. This
effect is restricted to the time of the nuclear
division cycles, and may be caused by the reduc-
tion of DNA synthesis. The nucleolus is not dis-
persed during the division cycles [5], and total
RNA synthesis, albeit constantly decreasing, con-
tinues throughout the nuclear division cycles
([ 1,221, unpublished results). Therefore, the ob-
served reduction of RNA synthesis may reflect the
reduced availability of template in the treated
cultures when compared to the untreated.
The effect of the cdk inhibitors olomoucine and
flavopiridol on parasite protein synthesis might be
mediated by the inhibition of PfPK5. If a feed-
back loop, as postulated for the self-amplification
of MPF during Xenopus oocyte maturation [45],
would be involved in the regulation of protein
synthesis during the nuclear division cycles, the
inhibition of PfPK5 kinase activity might cause
the decrease of protein translation. The inhibition
of (an)other cdk-like protein kinase(s) can, how-
ever, not be excluded. An involvement of PfPK5
is supported by the restriction of drug activity to
the time of the nuclear division cycles during
which PfPK5 is active. Thus, although the peak of
protein synthesis takes place before the onset of
the nuclear division cycles, an incubation of para-
site cultures in the presence of the drugs from
20-36 h did not enhance their effect on DNA or
RNA synthesis when compared to the 30-36 h
incubation.
 R. Graeser et at. / Molecular and Biochemical Parasitology 82 (1996) 37-49 31

The question about the exact timing and the tion to the DNA. Both of these tasks of PfPK5
mode of PfPK5 activation can not be fully an- would be expected to require a nuclear localiza-
swered yet. Fig. 1 indicates that PfPK5 requires tion of the kinase, since the nuclear envelope is
an activation step: compared to the similar not broken down during mitosis and the mitotic
protein amount at 24 and 42 h, PfPK5 kinase spindles are intranuclear [5]. The centrosomes
activity is significantly higher at the later time- are, however, located to pores in the nuclear
point. This suggests that PfPK5 is activated at membrane and organize both, cytosolic and nu-
the beginning of the nuclear division cycles. The clear spindles [5]. Cytosolic PfPK5 may therefore
activation will probably involve a phosphoryla- have access to the centrosomes. A nuclear local-
tion on Thr15’, as suggested by in vitro experi- ization of the kinase is not excluded by the re-
ments [18] and results from cdks, and, also by sults from our previous study. in which PfPK5
analogy to cdks [16], an association with a cyclin was found exclusively in the cytosol [17], since
subunit. From 30-36 h, PfPK5 activity increases the nuclei were probably destroyed while break-
in parallel with its expression, suggesting that the ing up the cells, and non-membrane bound nu-
portion of activated versus inactive PfPK5 re- clear proteins were released to the cytosolic
mains constant in a culture during the nuclear fraction.
division cycles. This indicates that the increasing In summary, our data strongly suggest that
number of newly formed nuclei requires a con- PfPK5 plays a role in the nuclear division cycles
stantly growing pool of PfPK5. The mechanism of the malarial parasite. The distinctness of the
how PfPK5 is regulated within the individual parasites’ nuclear division cycles when compared
rounds of nuclear divisions remains yet to be to the cell cycle of other eukaryotes renders in-
elucidated. Shortly after the end of the division vestigations on this subject exciting and demands
cycles, the level and activity of PfPK5 decrease for an effort to obtain more detailed information
abruptly, thereby resetting both to the levels be- about their regulation and organization.
fore the nuclear divisions.
The confocal immunofluorescence studies
provide some additional information about the Acknowledgements
role of PfPK5 in the nuclear division cycles. At
the time of the onset of the nuclear divisions, We would like to thank M. Gschwind and Dr.
PfPK5 colocalizes with the DNA stain, TOT0 1. A. Bell for many helpful discussions. This work
During the process of nuclear division, however, was funded in part by funds from the Canton
PfPK5 is only rarely colocalized with TOT0 1. City of Base1 and the Swiss National Foundation
Part of it is cytosolic (data not shown), but the Grant 31-29969.90.
most prominent staining is found at the edge of
the DNA stain. The localization pattern of
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