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Phosphorylation of Protein 4.1 in Plasmodium fakiparum-Infected Human
Red Blood Cells
By Athar H. Chishti, George J. Maalouf, Shirin Marfatia, Jiri Palek, W. Wang, Derek Fisher, and Shih-Chun Liu
The composition of the erythrocyte plasma membrane is
extensively modified during theintracellular growth of the
malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated
with the plasma membrane of human red bloodcells (RBCs)
infected with trophozoite/schizont stage malaria parasites.
However, the identity of this 80-kD phosphoprotein is controversial. One line ofevidence suggests that this protein is
a phosphorylated form of RBC protein 4.1 and that it forms a
tight complex with the matureparasite-infected erythrocyte
surface antigen. In contrast, evidence from another group
indicates that the80-kD protein isderived from theintracellular malaria parasite. To resolve whether the 80-kD protein
is indeed RBC protein 4.1, we made use of RBCs obtained
from a patient with homozygous 4.1(-1 negative hereditary
elliptocytosis. RBCs from this patient
are completely devoid
of protein 4.1. We report here that thislack of protein4.1 is
correlated with the absence of phosphorylation of the 80kD protein in parasite-infected RBCs, a finding that provides
conclusive evidence that the
80-kD phosphoprotein is indeed
protein 4.1. In addition, we also identify and partiallycharacterize a casein kinase that phosphorylates protein 4.1 in P
falciparuminfected humanRBCs. Based on these results, we
suggest that the maturation of malaria parasites in human
RBCs is accompanied by thephosphorylation of protein 4.1.
This phosphorylation of RBC protein 4.1 mayprovide a
mechanism by which the
intracellular malaria parasite alters
the mechanical properties of the hostplasma membrane and
modulates parasite growth and survival in vivo.
0 1994 by The American Society of Hematology.
T
protein as host protein 4.1. This evidence is based on the
absence of phosphorylation of this protein in RBCs obtained
from a patient with homozygous hereditary elliptocytosis
and a complete deficiency of protein 4.1. In addition, we
report the partial characterization of a casein kinase that
specifically phosphorylates protein 4.1 in infected RBCs.
Based on these results, we propose that the selective phosphorylation of protein 4.1 may provide a mechanism by
which the intracellular malaria parasite modifies the host
RBC membrane in vivo.
HE MALARIA PARASITE Plasmodium falciparum
undergoes three distinct morphologic changes during
its 48-hour life cycle inside human red blood cells (RBCs).’
After invasion into RBCs, the parasites mature to the ring
stage.2 The subsequent growth to the trophozoite stage is
associated with marked morphologic and biochemical
changes in the host plasma membrane.”3 Further maturation
of trophozoite-stage parasites to the schizont stage leads to
the loss of RBC biconcave shape and a dramatic reduction
in cell def~rmability.~
Although the molecular basis for these
changes is not yet known, it has been proposed that specific
parasite-induced alterations in the host plasma membrane
may be necessary for the growth and maturation of the intracellular malaria parasite^.^'^
In normal human RBCs, all major membrane proteins
including spectrin, ankyrin, adducin, band 3, protein 4.1,
protein 4.2, and dematin serve as substrates for a multitude
of protein kinases.‘ More importantly, the phosphorylation
of protein 4.1, dematin, and band 3 has been shown to downregulate their binary and ternary interactions in vitro.’ Therefore, it has been proposed that one of the mechanisms by
which the intracellular malaria parasite can modify the host
plasma membrane may involve the selective phosphorylation
of RBC membrane
Indeed, several RBC membrane-associated proteins are known to undergo changes in
phosphorylation on maturation of the malaria parasite.”
More specifically, a major phosphoprotein of approximately
80 kD was selectively phosphorylated at the trophozoite/
schizont
Based on the lack of reactivity of the 80kD phosphoprotein to various antibodies raised against RBC
membrane proteins including protein 4.1, it has been suggested that the 80-kD protein is derived from the intracellular
parasite.8 In contrast, the 80-kD phosphoprotein that coimmunoprecipitated with mature parasite-infected erythrocyte
surface antigen (MESA) produced one-dimensional peptide
maps that were similar to those from protein 4.1 raising the
possibility that this 80-kD protein is a phosphorylated form
of protein 4.1.9
In this study we examine the phosphorylation of the 80kD protein in RBCs that were infected with the malaria
parasite P falciparum and we identify the 80-kD phosphoBlood, Vol 83, No 11 (June l), 1994 pp 3339-3345
MATERIALS AND METHODS
Protein 4.1-deficient RBCs. The patient (DW), of African-American origin, suffersfrom homozygous 4.1(-) hereditary elliptocytosis. After splenectomy as a child, the patientis asymptomatic and
not receiving any transfusions. The patient’s RBCs are
completely
deficient of all isoforms of protein 4.1 as determinedbysodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blot analysis.
Parasite culture. A “knobby” P falciparum line 7901 (Palo
Alto, Ugandastrain)was used inthemajority of experiments. In
some experiments, the A-2(KC)strain was used. The A-2(K’) strain
is a clonal derivative of the nonclonal GambianFcR-3 line (provided
by Dr J. Ravetch, Sloan-Kettering Cancer Center, NY).The in vitro
parasiteculturemediumcontainedfresh
A+ humanerythrocytes,
RPM1 1640, glucose, gentamycin sulfate, HEPES, NaHCO,, L-glu-
From theDepartment of Biomedical Research, St Elizabeth’s Hospital, Tufts University School of Medicine, Boston; the Department
of Biochemistry and Molecular Biology and The Rowland Institute
for Science, Harvard University, Cambridge, MA; and St Jude Children’s Research Hospital, Memphis, TN.
Submitted June 24, 1993; accepted January IO, 1994.
Supported by the National Institutes of Health Grant No.
HL37462.
Address reprint requests to A.H. Chishti, PhD, ACH4, St Elizabeth’s Hospital, 736 Cambridge St. Boston, MA 02135.
The publication costs ofthis article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/831I-0011$3.~/0
3339
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3340
tamine, pyruvic acid, and15% A’ human serum as described by
Trager and Jensen.” The parasites were grown up to 6% parasitemia
at 37°C under 5% COZ, 1% Oz. and 94% NZ.Ring-stage parasites
were synchronized in 5% sorbitol.I2Trophozoite and schizont-stageinfected RBCs were then selected for the “knobby” phenotype
using the gelatin flotation method.”
Isolation of parasite-infected RBC membranes. Trophozoite/
schizont-infected RBCs at 5% parasitemia were enriched to greater
than 95% by centrifugation through sorbitol-Percoll (Sigma, St
Louis, MO) gradient^.'^ Infected RBCs were hypotonically lysed in
5 mrnol/L sodium phosphate pH 8.0,0.5 mmol/L EDTA, 0.5 mmol/
L phenylmethylsulfonyl fluoride (PMSF), and 1.0 p g h L leupeptin
and the lysates were centrifuged at 15,OOOg. The pelleted material
had two distinct layers: a tight gray zone of parasite material at the
bottom of the tube, and a less dense white layer of RBC membranes
above thegray zone. The membrane fraction was removed and
recentrifuged to remove any residual parasite material. This membrane fraction, which is referred to as “infected ghosts,” was used
in the phosphorylation assays.
Metabolic labeling of infected RBCs. For the ”P-labeling of
trophozoitelschizont-infected RBCs, intact parasite infected RBCs
were washed in phosphate- and glutamine-free Minimum Essential
Medium (Flow Laboratories, McLean, VA). The culture medium
was further supplemented with 25 mmol/L HEPES, 10 pglmL gentamycin sulfate, and15%human A+ serum. The human A+ serum
was dialyzed against sterile normal saline. Cells were labeled for 4
hours at37°Cin a culture medium towhich2.0 mCi/mL of‘*Porthophosphate was added. After incubation, the cells were washed
with the culture medium and the RBC membranes were prepared
as described above. Metabolic labeling ofthe infected RBC was
performed in methionine-free RPM1 l640 (Select-amine kit, GIBCO
Laboratories, Grand Island, NY) containing 100 pCi/mL of ” S methionine (1,140 Ci/mmol; ICN, Irvine, CA). Radiolabeled RBCs
were then washed once with the methionine-free culture medium,
and the RBC membranes were prepared as described above.
Phosphorylation assay. Phosphorylation of infected ghosts was
performed in a reaction mixture containing 50 mmoVL Tris-HCI pH
7.5,8.0 mmol/L MgCl,, 1.O mmoUL dithiothreitol (DTT), 50 pmoUL
adenosine triphosphate (ATP), and 200 pCi camer-free [y-”P]ATP.
After incubation at 30°C for 10 minutes, the radiolabeled proteins
were separated by SDS-PAGE (10% gels). For the analysis of RBC
membrane proteins that were isolated from metabolically ”P-labeled
RBCs, 50 pmolR. sodium orthovanadate and 50 pmoW sodium
fluoride were included in the lysis buffer to prevent dephosphorylation of radiolabeled proteins during the isolation procedure.
Dephosphorylation assay. Infected ghosts were phosphorylated
in vitro inthe presence of [y-”P]ATP as described above. After
the labeling reaction was complete, radiolabeled membranes were
sedimented by centrifugation at 30,000 rpm (SW 50.1 rotor) for I O
minutes to remove unreacted [y-”P]ATP, andthen washed twice
with 5 mmoW Tris-HC1,pH 8.0. Radiolabeled membranes were
incubated with either the cytosol of the uninfected RBCs, or the
cytosol of the trophozoite/schizont-infectedRBCs. The dephosphorylation assay reaction mixtures contained 40 mmoVL Tris-HCI, pH
7.5, 100 mmoln KCl, 1.0 mmom DIT, and 5 mmol/LMgC12.
After incubation at 37°C for 30 minutes, dephosphorylation of ”Plabeled 80-kD protein was evaluated by SDS-PAGE and autoradiography.
Peptide-mapping analysis. One-dimensional peptide-mapping
analysis was performed as described previously.’s The ”P-labeled
80-kD protein was excised from Coomassie-blue-stained acrylamide gels. These gel pieces were sequentially washed with water
and methanol, and then digested with 30 mg/mL of cyanogen bromide in 0.1N HCland 0.2% betamercaptoethanol. Peptides that were
produced by cleavage at methionine residues were separated using
CHlSHTl ET AL
10% to 18% gradient acrylamide gels and visualized by silver staining and autoradiography. Two-dimensional peptide mapping of the
I2’I-80-kD protein and the ”P-80-kD protein was performed as described.“
Purijication of parasite casein kinase. Trophozoite/schizont-infected RBCs were metabolically labeled with 35S-methionineas described above. The labeled parasite-infected RBCs were incubated
with 0.1% saponin for 10 minutes at 37°C to make the RBC membranes permeable. Saponin-treated parasite lysates were centrifuged
at 3,000g, and the supernatant containing RBC membrane fragments
was removed. Intact parasites were washed twice with the saponin
solution and finally withphosphate-buffered saline. 35S-labeledintact
parasites that were essentially free of RBC membranes were hypotonically lysed in 10 v01of cold distilled water. Parasite lysates
were then centrifuged at 50,000g for 30 minutes and the supernatant
collected. This supernatant, referred to as the “cytosolic extract,”
was then loaded on a casein-sepharose column (0.5 X 5 cm) that
was equilibrated with 20 mmoVL Tris-HCL, pH 7.5,and 1.0 mmoU
L DTT. After extensively washing the column, the bound proteins
were eluted using a 0 to 0.5 mmol/L KC1 gradient ( I O 0 mL). Column
fractions were assayed for both radioactivity and casein kinase activity. Affinity-purifiedcasein kinase fractions were pooled and further
purified on a diethyl aminoethyl (DEAE)-Sephacel column (Pharmacia, Piscataway, NJ). The hound enzyme was eluted with a 0 to
0.5 mom KC1 gradient. In both purification steps, the peak of casein
kinase activity overlapped that of the [%l radioactivity.
RESULTS
Selective phosphorylation of the 80-kDprotein. To compare the phosphorylationof membrane proteins inuninfected
and parasite-infected human RBCs, phosphorylation assays
were performed using both intact RBCs and isolated RBC
membranes (Fig 1). Wheninvitrophosphorylation
assays
were performed on the RBC membranes isolated from uninfected RBCs, no appreciable phosphorylation signal was detected in the region where protein4. l migrates on SDS-gels
(Fig I , lane 8). Incontrast,phosphorylation
of infected
ghosts producedselective phosphorylation of the 80-kD protein (Fig 1, lane 10, arrowhead). A comparison of invitro
protein phosphorylationsat similar spectrincontent also indicated an enhancement in the phosphorylation
of band 3 in
trophozoite-infectedRBCs (60% increase compared with uninfected cells, Fig 1: lanes 8 and lo).
The 80-M) protein was again selectively phosphorylated
when intact RBCs containing trophozoitelschizont-stageparasites were metabolically phosphorylated in the presence of
12
PO, (Fig l , lane 6, arrowhead).However,theenhanced
phosphorylation of band 3, asdetectedininfected ghosts,
was not observed in intact trophozoite-infectedRBCs. Similarly, when intact RBCs containing ring-stagemalaria parasites were metabolically phosphorylated,no phosphorylation
of the 80-kD protein was observed (Fig 1, lane 4). In fact,
ring-infected RBCs reproduciblyshowa 90% decrease in
the radiolabelingof P-spectrin compared with the same number of uninfected cells (Fig 1, lanes 2 and 4). This result is
consistent with a previous report demonstrating dephosphorylation of the major erythrocyte membrane proteins
on merozoite invasion.8
Phosphorylation of the 80-kD protein in normal and
4.1(-) RBCs. To obtainfurther insight into the origins of
the 80-kD protein and its relationship tothe phosphorylated
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PHOSPHORYLATION OF PROTEIN4.1
3341
IN MALARIA
Fig l. Membrane protein phosphorylationin uninfected and P fakiparuminfected human RBCs.C,
Coomassie blue;A, autoradiograph. Membrane proteins were phosphorylated and resolvedon 10%
SDS-PAGE gels. The position of protein 4.1 is indicated by an arrowhead. Lanes 1 and 2, membrane
fraction derived from uninfected RBCs metabolically
phosphorylated in the presence of 32P04.Note that
no phosphorylation of protein 4.1 was detected in
intact, uninfected RBCs.Lanes 3 and 4, membrane
fraction derived from ring-infected RBCs metabolically phosphorylatedin the presence of32P0,. Again
no phosphorylationof 80-kD protein was detected.
Lanes 5 and 6, membrane fraction derived from
trophozoitelschizont-infected RBCs metabolically
phosphorylated in the presence of 32P04.Note that
a prominent phosphoproteinof 80 kDwas observed
in intact RBCs. Lanes 7 and 8, in vitro phosphorylation of membrane fraction derived from uninfected
RBCs.Lanes 9 and 10, in vitro phosphorylation of
membrane fraction derived from trophozoitelschizont-infected RBCs. Notethatthe enhancedphosphorylation of band 3 was not observed (lane 101
when intact, trophozoite-infected RBCs were metabolically phosphorylated (lane 6).
Intact Red Cells "
l
-p
Unlnlscted
Unlnfected
' C
+
R
e
d
Cell Membranes 4
A '
' A
rsp
I1' -:
A '
h
4 4.1
I
2
form of RBC protein 4.I , metabolic phosphorylations were
4.l(-) RBCs that
performed in bothnormalandprotein
were infected withmalaria parasites at the trophozoite/schizont stage (Fig 2). These RBCs do not contain protein 4.I or
any of its isoforms as determined by the Western blot analysis." As shown in Fig 2, no phosphorylation of the 80-kD
phosphoprotein was observed in RBC membranes isolated
from protein 4.l(-) RBCs that were infected with malaria
parasites. It should be noted thatthis absence of phosphorylation of the 80-kD protein is not simply caused by the reduced
invasion of 4.1(-) RBCs by malaria parasites. The evidence
in support of this conclusion isprovided by thefactthat
although 4.l(-) RBCs have reduced parasitemia. both normal and 4.I (-) RBCs that contained mature parasites were
concentrated by centrifugation through Percoll and an equal
number of cells were used in the phosphorylation assays.
Moreover. the parasites in 4.l(-) RBCs appeared morphologically normaland could be propagated when cultured
with normal RBCs (not shown). These results strongly suggest thatthe 80-kD phosphoprotein in mature parasite-infected RBCs is host membrane protein 4.1 and that it is not
derived from the intracellular parasite.
Structural similarity between the 80-kD phosphoprotein
and protein 4.1. Because the mobility of the 80-kD phosphoprotein on SDS-polyacrylamide gels is identical to that
of protein 4.1 (Fig I ) , we examined structural similarities
by one- and two-dimensional peptide mapping. As shown in
Fig 3, the mobilities of three major peptides derived from
the cyanogen bromide cleavage of the 80-kD phosphoprotein
were similar to those of peptides derived from purified protein 4.1 (Fig 3, compare lanes 1 and 2). The three radiolabeled peptides derived from the "P-labeled 80-kD protein
were also contained within the silver-stained peptide map of
protein 4.1 (Fig 3, compare lane 3 with lane l). In addition.
the phosphopeptide maps of the 80-kD protein phosphorylated in either isolated RBC membranes or in intact RBCs
3
4
5
6
7
8
9
IQ
were identical (Fig 3, lanes 4 and 5) indicating that similar
sites were phosphorylated under these conditions. The structural relationship between protein 4.1 and the 80-kD phosphoprotein was further examined by two-dimensional peptide mapping analysis. Although the 'ZSI-protein4.1 peptide
map shared some spots with the phosphopeptide map of
"P-labeled 80-kD protein, there were many spots that were
unique to bothpeptidemaps
(not shown). This result is
consistent with the fact that the phosphorylated peptides exhibit altered mobilities during charge-based separation.IX
Phosphotylation of the 80-kD phosphoprotein. To elucidate the basis of hyper-phosphorylation of the 80-kD protein
in mature parasite-infected RBCs, we first compared the total
protein kinase activity in both uninfected and infected RBCs
(Fig 4).The RBCs were lysed in the hypotonic buffers, and
ruptured cells were separated into cytosol andmembrane
fractions by high-speed centrifugation. The proteinkinase
activity of the cytosol and membrane fractions was measured
using casein as the substrate. The casein kinase activity was
20-fold higher in tropho;roite/schizont-infectedred cells (Fig
4, lanes S through 8) compared with uninfected RBCs (Fig
4,lanes 1 through 4).Both the cytosol and membrane fractions of the infected RBCs exhibited elevated casein kinase
activity. A similar increase in totalprotein kinase activity
was also observed using phosvitinas an exogenous substrate.
In contrast, basicprotein substrates such as histones and
protamine werenot phosphorylated by this proteinkinase
(data not shown).
In addition to the increase in protein kinase activity, the
increase in phosphorylation of the 80-kD protein maybe a
consequence of the increased phosphate turnover caused by
the activation of protein phosphatases. To examine this possibility, membranes from parasite-infected RBCs were incubated under conditions that promoted dephosphorylation in
vitro.Iy RBC membranes that contained the "P-labeled 80kD protein were incubated with either the membrane or the
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CHlSHTl ET AL
3342
a
2
E
e
aZ .I ,1
g
A
T:
E
P
e
I
"l
-Protein
i
"
.
)
4.1
A
2
' L ?
#ilr
FV3
.4
+ 48kDa
formed within the linear range of phosphorylation kinetics.
The membrane-~tssoci~ltedprotein kinase exclusively phosphorylated serine residues of the 80-kD protein. and both
ATP and guanosine triphosphate (GTP) could be used as
phosphntc donors in thc reaction ( n o t shown). Phosphorylation of thc XO-kD proteinwasstronglyinhibited
byNnCI
and KC1 and 50% inhibition was observed a t approximately
1 0 0 mmol/L salt concentration (not shown). The phosphorylation was not affected by calcium. cnlmodulin. EGTA. or
trilluoropcrazine
and
s o excluded
participation
of
the
cnlcium/cnl~nodulinprotein kinases (Table I ). In addition,
the protein kinase
activity
was
not affected by cyclic adenosine monophosphate (CAMP) andstnurosporine (up to 36
nmol/L).suggesting that neither CAMP-dependcnt protein
kinase nor protein kinase C was directly responsible for the
phosphorylation of 80-kD protein (Table I ). Staurosporine
at 36 nmol/L is known t o inhibit CAMP kinase and protein
kinase
C
in vitro.'" In contrast.
phosphorylation
of the 80-
kD protein was inhibited by N-ethylmaleimideand 2 . 3 4 phosphoglycerate (Table I ). Heparin inhibited the in vitro
-W
kDa
1
1
2
Coomassie
1
2
497
Autorad
Fig 2. Phosphorylation of protein 4.1 in normal and protein 4.11-1
RBCs. The protein 4.11-1 RBCs were obtained from a patient with
homozygous 4.1 I-) hereditary elliptocytosis. Trophozoite-infected
normal RBCs were enriched t o greater than 95% parasitemia by Percoll-sorbitol method." Infected RBCs were incubatedwith either normal or 4.11-1 RBCs t o allow parasite invasion anddevelopment. After
48 hours, trophozoitelschizont-infected RBCs were enriched t o
greater than 9546 parasitemia and metabolically phosphorylated for
4 hours in the presence of "PO4. An equal number of radiolabeled
RBCs were hypotonicallylysed, sedimented, and the membrane fraction was directlyanalyzed by SDS-PAGE without further separation
of the intracellular parasites. It should be noted that the increased
phosphorylation of 48-kD protein and relatively higher background
in lane 1 (Autorad) may be caused by the contamination of residual
material from intracellular parasites. N o phosphorylation of 80-kD
protein wasobserved in 4.1(-1 RBCs (lane 2, Autorad). In addition, a
56-kD band was also detected in lane 2. The origin of this band is
not known at present.
4
445
(29
I '
cytosolfractionsderived
from unlabeledmatureparasiteinfected RBCs. No loss of radioactivity from the "P-labeled
80-kD protein was observed in the presence of magnesium,
which is known t o promote dephosphorylation of the RRC
membrane proteins'" (data not shown). suggesting that the
observed hyper-phosphorylation of the 80-kD protein is
likely caused by the increased protein kinase activity in parasite-infected RBCs.
l3fw1 of' IIIP proIeit1 kir1mc irlhihitars. To identifyand
characterize the protein kinase activity that catalyzed phosphorylation of the 80-kD protein. we first determinedthe
kinetics of phosphorylation. All subsequent assays were per-
66
2 1
l 3
4
5
I
Silver
Autoradiograph
Stain
Fig 3. One-dimensional CNBr peptide maps ofprotein 4.1 and
the 80-kD phosphoprotein. Lane 1, purified protein 4.1 isolated from
uninfected RBCs. Lane 2, 3'P-labeled 80-kDprotein from trophozoite/
schizont-infected RBC membranes phosphorylated in vitro. Lane 3,
autoradiograph of lane 2. Lane 4, 3ZP-labeled80-kD protein isolated
frommetabolicallyphosphorylated
trophozoitelschizont-infected
RBCs. Lane 5, 3ZP-labeled80-kD protein isolated from trophozoite/
schizont-infected RBC membranes phosphorylated in vitro. The 80kD phosphoproteinin lanes 3 and 5 werederived from t w o different
parasite strains (7901 Palo Alto strain, A2 strain). The similar phosphopeptide patternsin lanes 4 and 5 suggest that the 80-kD protein
was phosphorylated on similar
sites in vitro as well as in intactRBCs.
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PHOSPHORYLATION OF PROTEIN4.1
S P
1
S
P
IN MALARIA
S P
S
3343
P
. I
+4.1
1
2
Coomassle
3
4
Autorad
7 0
5 6
Coomassle Autorad
Fig 4. Measurement of thecasein kinase activity in uninfected and
trophozoitelschizont-infected RBCs. S, supernatant; P, pellet. Protein
kinase activity was measured in the presence of carrier-free [y-'*P]ATP and exogenous casein as substrate. Lanes 1 and 2, SDS-PAGE
of uninfected humanRBCs lysed in thehypotonic buffer. Lanes 3 and
4, autoradiograms oflanes 1 and2. Note that only residual phosphorylation of casein was observed under these conditions. Lanes 5 and 6,
lysis of trophozoitelschizont-infected human RBCs in the hypotonic
buffer. Lanes 7 and 8, corresponding autoradiograms. The position
of the isoforms of casein is indicated by the dark diffuse bands at
the bottom of SDS-gels. The bands were excised from supernatant
and pellet fractions
and radioactivity wasmeasured in a liquid scintillation counter. Note that in contrast t o uninfected red cells (lanes 3
and 4). there is a prominent phosphorylation ofcasein by the fractions derived from infected
RBCs (lanes 7 and 81. The phosphorylation
is
with theresults shown
of 80-kD protein in lane 8 (arrow) consistent
in Fig 1.
phosphorylation of the 80-kD protein in a dose-dependent
manner with a 50% inhibition at I .0 pg/mL (Table 1 ). Finally, the identity of the protein kinase was establishedusing
CKI-7, a specific inhibitor of the casein kinases (Table 1 ).
CKI-7, an N-(2-aminoethyl)-S-chloro-isoquinoline-8-sulfonamide, is known to inhibit casein kinase I and casein kinase
11 with IC?,, valuesof 9.5 and 195 pmol/L, respectively."
The ICsc,value of CKI-7 for the inhibition of phosphorylation
of the 80-kD protein was 30 pmol/L (Table 1 ). These results
strongly suggest that the phosphorylation of the 80-kD protein was catalyzed by a casein kinase in trophozoite/schizontinfected RBCs.
Pur$fic~rtior7 r r r d c~hrrrrccteri~ntiorlof t h c cctsch kirlnsc.
As shown in Fig 4. thecaseinkinaseactivity
was significantly higher in trophomite/schizont-infected cells than in
uninfected RBCs. To determine theorigin of thiscasein
kinase activity, trophozoitekchizont-infected RBCs were
metabolicallylabeled in thepresenceof["S]-methionine.
The mature parasites were then isolated using selective lysis
in the prcscnceofsaponin."
The cytosoliccaseinkinase
fraction was then obtained by hypotoniclysis of purified
parasites, and further purified by affinity chromatography on
theimmobilized casein. Asshown in Fig SA. thecasein
kinase activity peak completely overlapped the ["S] radioactivity peak. strongly suggesting that the casein kinase was
metabolically labeled and therefore derived from the malaria
parasite. The affinity-purified casein kinase was further purified using anion-exchange chromatography (FigSR). Again,
the caseinkinaseactivityandthe
["S] radioactivitypeaks
overlapped. Theproperties of the purificd casein kinase were
similar to those of the membrane-bound casein kinase that
phosphorylatedthe 80-kD protein in trophozoitekhizontinfected RBCs (data not shown). Theseobservations suggest
that these two enzyme activities. which show similar biochemical properties, may be the samc and may be derived
from the intracellular parasite. However, further experiments
are necessary to prove the hypothesis that the parasite-derived casein kinase is translocated to the host membrane in
infected RBCs.
DISCUSSION
The overall aim of this study was to identify an 80-kD
phosphoprotein that isprominentlyphosphorylated
at the
trophozoite/schizont stage of parasite development, and to
characterize the protein kinase that mediates this phosphorylation.Asshown
in Fig l , phosphorylation of the 80-kD
protein was dependent on the stage of parasite development.
Such developmentally regulated phosphorylation of the 80kD protein is consistent with previously reported phosphoryhuman RBCS.~."
Howlation events in P,fnlci~nr~frn-infccted
ever, there aretwo conflictingviews with respect to the
identity of the 80-kD phosphoprotein. One group' suggested
that the 80-kD phosphoprotein was derived from the intracellular malaria parasite. based on the observation that antibodiesspecific to protein 4.1 failed to immunoprecipitate "Plabeled 80-kD protein from infected RBCs. In contrast, others" concluded that the 80-kD protein is aphosphorylated
form of RBC protein 4. I . as suggested by ( I ) the absence of
metabolic radiolabeling of 80-kD protein in infected RBCs in
the presence of [?3]-methionine or a mixture of I6 radiolabeled amino acids,and ( 2 ) thesimilarities of one-dimensional peptide maps of the 80-kD protein and protein 4. I .
Our results confirm the observation
of Lustigman et a19
Table 1. Inhibition of Phosphorylation of Erythrocyte
Membrane-Associated 80-kD Protein
Inhibition of
Compounds
Concentration
Kinase Activity (%)
CaCI2
Calmodulin
EGTA
Trifluoroperazine
CAMP
Staurosporine
N-ethylmaleimide
2.3-Diphosphoglycerate
Heparin
CKI-7*
16 pmol/L
16 pg/mL
5 mmol/L
25-160 pmol/L
60-300 pmol/L
4-36 nmol/L
8 mmol/L
5 mmol/L
None
None
None
None
None
None
75
50
50
7
1 pglmL
10 pmol/L
30 pmol/L
100 pmol/L
52
70
CKI-7, N-~2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide.
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3344
CHlSHTl ET AL
A
300-
-b
m
2x 200 -
v
z
v 100-
lrJ
m
v)
Fig 5. Purification of the caseinkinase
from trophozoite/
schizont-infected RBCs. (A) Affinity purification of the casein
kinaseon a casein-agaroseaffinity column. [B) Further purification of the casein kinase on a
DEAE-sephacelanionexchange
column. The casein kinase activity was measured by a filter kinase
assay
as
described
previously?’
The
details of the
enzyme isolation are described
in Materials and Methods.
and show that the 80-kD protein is indeed protein 4.1 based
kinases in RBCS.~~””
Boivin and Galand” have purified a
on the following observations: (1) The elliptocytic RBCs,
casein kinase from the cytosol of normal human RBCs that
which completely lack all isoforms of protein 4.1, fail to
was stimulated by monovalent salts and inhibited by calcium.
show any phosphorylation of the 80-kD protein on parasite
These properties are different from the casein kinase we
development (Fig 2). (2) Both the [”PI-labeled 80-kD prohave characterized in infected RBCs. A CAMP-kinase was
tein and RBC protein 4.1 have similar molecular weights
previously characterized from normal RBC cytosol,” which
(Fig l), one-dimensional peptide maps (Fig 3), andmemphosphorylated casein, spectrin, and protein 4. l . This casein
brane association properties (Fig 4).In contrast, the twokinase was not inhibited by 2,3-diphosphoglycerate, a propdimensional peptide maps of [‘251]-protein 4.1and the [32P]- erty different from the casein kinase reported in this manuscript. Similarly, Clari and MoretZ9have characterized a ca80-kD protein were distinctly different and shared only three
sein kinase from human RBC cytosol that phosphorylated
spots (not shown). This apparently contradictory result is
spectrin and protein 4.1. This enzyme was not inhibited by
consistent with the reported altered migration of phosphoryeither NaCl or 2,3-diphosphoglycerate.
lated peptides during charge separation.” On account of this
Our data also show that the casein kinase isolated from
fact the two-dimensional peptide maps cannot beusedto
parasite-infected RBCs can bemetabolically labeled (Fig 5 ) ,
establish the identity of these two proteins.
and this further supports the hypothesis that the enzyme is
Our second task wasto characterize the protein kinase
of parasite origin. Although the functional significance of
involved in the phosphorylation of the 80-kD protein in parathese observations is presently unknown, the phosphorylasite-infected RBCs. A type I casein kinase has been previously characterized in both P bergheiZ3and P f a l c i p a r ~ m . ~ ~tion of protein 4.1 is known to dramatically reduce both its
spectrin-actin and membrane-binding properties.’ The selecMore recently, phosphorylation of the parasite proteins was
tive phosphorylation of RBC membrane proteins by the incorrelated with parasite invasion and development in intracellular malaria parasite may influence events such as the
fected RBCS”; however, the corresponding protein kinases
loss of cell shape and deformability, reorganization of the
were not identified.2s The data presented here describe the
membrane skeleton and membrane permeability, membrane
properties of the casein kinase that phosphorylates the 80rupture during merozoite release, and the modification of
kD protein in P fulcipururn-infected human RBCs. The propRBC surface antigens. Whether the parasite induced phoserties of the casein kinase in infected RBCs resemble those
phorylation of protein 4.1 brings about any such effects in
of casein kinase I1 with respect to substrate specificity and
intact RBCs remains to be investigated.
enzyme inhibition by specific inhibitors (Table l), whereas
the elution of enzyme from the ion-exchange matrix resemACKNOWLEDGMENT
bles that of casein kinase I (Fig 5B).26Because the biochemical characteristics of the casein kinase reported here appear
We thank Linda Oak for valuable technical assistance with the
to share the features of both type I and type I1 casein kiculture of malaria parasites.
nases,26primary structure determination will be required to
unambiguously establish its classification.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
PHOSPHORYLATION OF PROTEIN4.1
IN MALARIA
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1994 83: 3339-3345
Phosphorylation of protein 4.1 in Plasmodium falciparum-infected
human red blood cells
AH Chishti, GJ Maalouf, S Marfatia, J Palek, W Wang, D Fisher and SC Liu
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