(Received for publication, August 27, 1996, and in revised form, December 30, 1996)
From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446
KFR1, a mitogen-activated protein (MAP) kinase
identified in the African trypanosome, Trypanosoma brucei,
is a serine protein kinase capable of phosphorylating the serine
residues in histone H-1, myelin basic protein, and -casein. It
phosphorylates four proteins with estimated molecular masses of 22, 34, 46, and 90 kDa from the T. brucei bloodstream-form lysate
in vitro. KFR1 bears significant sequence similarity to the
yeast MAP kinases KSS1 and FUS3 but cannot functionally complement the
kss1/fus3 yeast mutant. It is encoded by a single-copy gene
in the diploid T. brucei, and only one of the two alleles
can be successfully disrupted, suggesting an essential function of
KFR1 in T. brucei. KFR1 activity is present at
a much enhanced level in the bloodstream form of T. brucei
when compared with that in the insect (procyclic) form. This enhanced
activity can be eliminated in vitro by the treatment with
protein phosphatase HVH2 known to act specifically on MAP kinases. It
can also be decreased in the bloodstream form of T. brucei
by serum starvation but induced specifically by interferon-
. The
production of interferon-
in the mammalian host is known to be
triggered by T. brucei infection, and this cytokine, as has
been reported, promotes the proliferation of T. brucei in the mammalian blood. Since none of these phenomena can be observed in
the procyclic form of T. brucei, activation of KFR1 is most likely involved in mediating the interferon-
-induced proliferation of T. brucei in the mammalian host.
Many parasites employ unique strategies to escape the host
immunity by taking advantage of their environmental niches. For example, Trypanosoma cruzi, a protozoan parasite causing
chronic debilitating Chagas' disease in Latin America, avoids the host immune response by invading host cells. This invasion is mediated by
activation transforming growth factor-
(TGF-
)1 signaling pathway in the
mammalian cells (1). On the other hand, Trypanosoma brucei,
an extracellular parasite that causes African sleeping sickness in man
and nagana in cattle, escapes the host defense system primarily by
repeatedly changing its variant surface glycoprotein coat (2, 3). This
flagellated parasite exists primarily in two distinct forms in its life
cycle, the bloodstream form in the mammalian host and the procyclic
form in the transmitting vector, the tsetse fly. It has been reported that there is a bidirectional interaction between T. brucei
and the mammalian host immune system (4-7). Upon invasion of the host,
T. brucei releases a 44-kDa lymphocyte-triggering factor (8), which binds to host CD8+ T cells and triggers
interferon-
production in the host (7). By injecting monoclonal
antibody against the lymphocyte-triggering factor or depleting
CD8+ cells in T. brucei-infected animals,
interferon-
production was abrogated, growth of trypanosome
suppressed, and survival of the infected animals prolonged (4, 8).
Interferon-
can stimulate proliferation of trypanosome bloodstream
cells in culture (6). Administration of interferon-
antibody to the
infected animals can reduce parasitemia and prolong the survival of the animals (7). The molecular mechanism of this interferon-
action is,
however, unclear at present.
Interferons, which exhibit a wide range of biological effects on cells
and animals, play important roles in modulating the immune system
(9-11). Among the three major classes of interferons (IFN-,
IFN-
, and IFN-
), interferon-
is the most important immunomodulator. It induces major histocompatibility complex class I
and class II, regulates isotype expression of Igs, and activates monocytes and macrophages by enhancing Fc receptor expression (12, 13).
It also has antiviral, antitumor, and antiproliferation activities in
mammals (9, 10). The antiproliferation activity is partly through
blockage of the protein kinase C-dependent
mitogen-activated protein (MAP) kinase activation pathway (14, 15).
Interferon-
can specifically block platelet-derived growth factor
(PDGF)- and phorbol ester-stimulated activation of Raf-1 and MAP
kinases in fibroblast cells (14). It can also block the
colony-stimulating factor-1-stimulated MAP kinase activation via
Jak1/Jak2 (Janus kinase) in macrophages (15). This blockage of MAP
kinase activation contributes to the antiproliferation effects of
interferons in mammals (15).
We have previously cloned a gene encoding a MAP kinase homologue,
KFR1, from T. brucei that bears significant
sequence homologies to the yeast MAP kinase homologues,
KSS1/FUS3, and other Xenopus and mammalian MAP
kinases, ERK1 and ERK2 (extracellular
signal-regulated kinase) (16). The characteristic MAP kinase motif,
including the amino acid residues TEY between protein kinase subregions VII and VIII, is very well conserved in KFR1. The KFR1
messenger RNA level in the bloodstream form of T. brucei is
much higher than that in the insect (procyclic) form (16), suggesting
that KFR1 may play an important role in the former. Here we
report that KFR1 is capable of phosphorylating the serine residues in histone H-1, myelin basic protein, and -casein. This KFR1 activity is much higher in the bloodstream form of T. brucei than in
the procyclic form, and it can be inactivated by a MAP kinase-specific phosphatase HVH2 in vitro. This activity in the bloodstream
form can also be drastically reduced by serum starvation, which also arrests the cell growth. Several growth factors were tried to stimulate
the KFR1 activity. Only interferon-
strongly enhanced the KFR1
activity as well as the cell proliferation. The lower KFR1 activity in
procyclic form was, however, unaffected by interferon-
.
Trypanosoma brucei brucei
strain 427 bloodstream-form cells (antigen variant clone MITat 1.4 (117)) were cultured in HMI-10 medium (17), and the procyclic form
cells were cultured in Cunningham's medium (18) supplemented with 10%
heat-inactivated fetal bovine serum (Atlanta Biologicals, Inc.,
Atlanta, GA). Saccharomyces cerevisiae strain IH2725
(MATa trp1 leu2 ura3 his4 can1 fus3::URA3
kss1::URA3) was from Dr. I. Herskowitz and cultured in
YPD standard rich medium. Murine interferon-, recombinant human
basic fibroblast growth factor, histone H-1, and myelin basic protein
were purchased from Life Technologies, Inc.
-Casein was from Sigma.
Recombinant human PDGF B/B was purchased from Boehringer Mannheim.
Yeast plasmids pRS314,
pRS-FUS3, and pFus1-LacZ were from Dr. I. Herskowitz. pRS-FUS3 contains
FUS3 gene in pRS314 under GAL1 promoter.
pFus1-LacZ contains the Fus1-LacZ gene and the
LEU2 gene (19, 20). To clone KFR1 into pRS314
vector, BamHI and SalI sites were generated
flanking the KFR1 (16) by polymerase chain reaction (PCR)
using primer B (5-GAAGGATCCATGGTGTCGTTCAGCATC) and primer S
(5
-GGAGGTCGACTATGGCTGAGCAACATTTC). The PCR-amplified fragment was
inserted into vector pCR1000 (Invitrogen, La Jolla, CA). The nucleotide
sequence of the KFR1 fragment was confirmed by DNA
sequencing. The fragment was released by
BamHI/SalI digestion and subsequently inserted
into the BamHI/SalI site of pRS314, which is
located downstream of GAL1 promoter. The plasmid thus constructed is named pRS-KFR1.
S. cerevisiae strain IH2725 cells were transformed with
SphI-linearized pFus1-LacZ and selected on a leucine-free
plate for the stable transformants resulting in the strain SH101
(MATa trp1 leu2 ura3 his4 can1 fus3::URA3
kss1::URA3 FUS1-LacZ). SH101 cells were then transformed
with pRS314, pRS-FUS3, and pRS-KFR1, respectively. GAL1
promoter was activated by dextrose-free SC medium containing 2%
galactose, 0.5% sucrose, 2% glycerol, 1% raffinose, and
tryptophan-free amino acid mixture. After a 1-h induction by
-factor, LacZ expression was monitored as described (19).
The KFR1 cDNA insert in pBluescript
SK(), designated pB-KFR1 (16), was amplified by PCR using the 5
primer (5
-AAACCATGTGTCGTTCAGCATCGATG) and the T3 primer
(5
-ATTAACCCTCACTAAAG). The PCR-amplified fragment was treated with
Klenow fragment of DNA polymerase I and further digested with
BamHI. The digested PCR fragment was inserted into the
trypanosome overexpression vector, pTSA-HYG2 (21), which had been
treated with XhoI and Klenow and further with
BamHI. The plasmid thus constructed was designated pTKH-Hyg.
The orientation and nucleotide sequence of KFR1 in pTKH-Hyg
were confirmed by sequencing. Expression of both KFR1 and
hygr is under the control of the PARP promoter,
and there is a splicing signal site upstream of each open reading frame
(22). Purified plasmid DNA of pTKH-Hyg was linearized at the
MluI site in the trypanosome tubulin intergenic region in
the plasmid and transformed into procyclic T. brucei cells
by electroporation (22). After stable transformants were selected with
50 µg/ml hygromycin B (Calbiochem), cell lines were cloned by
limiting dilution (22). Expression of KFR1 was examined at
mRNA and protein levels by Northern hybridization and Western
blotting, respectively, as described (16).
A DNA fragment containing the PARP promoter and hygr was released from pTSA-HYG2 (21) by treating with SacI and T4 DNA polymerase and followed by BssHII. Likewise, a fragment containing the PARP promoter and neor was released from pTSA-NEO2 (22) with SacI and T4 DNA polymerase and followed by a partial digestion of BssHII. Due to the presence of BssHII site in the open reading frame of neor, only the fragment containing the complete neor open reading frame was recovered from an agarose gel. The recovered DNA fragments, containing hygr or neor, were ligated into pB-KFR1 after SnaBI and BssHII digestion, which released a 250-base pair fragment at the center of the KFR1 open reading frame. Thus constructed plasmids were designated pNKH-Hyg and pNKH-Neo, respectively. Correct inserts were confirmed by restriction mapping. Purified plasmid DNA of pNKH-Hyg was digested with SspI and SacI and transformed into procyclic trypanosome cells by electroporation. Stable transformants were selected by hygromycin B, and cell lines were cloned as described above. Cell lines were examined by Southern hybridization. One allele of KFR1 gene in the T. brucei genome was successfully disrupted in all the cell lines examined. To disrupt the second allele of KFR1, one of the above cell lines was transformed with pNKH-Neo digested with SspI and SacI. The transformants were then selected under both hygromycin B (50 µg/ml) and G418 (50 µg/ml) (geneticin, Life Technologies, Inc.). Cell lines were then cloned and examined by genomic Southern hybridization after overnight digestion with PstI or BamHI as described previously (16).
Immunoprecipitation and in Vitro Kinase AssayT.
brucei cells (5 × 106 cells) were harvested by
centrifugation at 3,000 × g for 10 min. Cells were
lysed on ice for 1 h in 1 ml of lysis buffer (50 mM
Tris-HCl, pH 6.8, 150 mM NaCl, 1% Nonidet P-40, 0.2%
sodium cholate, 2 mM EGTA, 1 mM sodium
orthovanadate, 10 mM NaF, 100 nM okadaic acid,
1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
10 µg/ml chymostatin, 10 µg/ml aprotinin, 10 µg/ml
phenanthroline, and 15 µg/ml benzamidine HCl). The lysate was
centrifuged at 16,000 × g for 15 min. The supernatant
was precleared with preimmune serum (16) and Pansorbin (Calbiochem) essentially as described (23, 24). The precleared lysate was incubated
with 40 µl of 50% protein-A beads (Zymed, Inc.) on ice for 1 h
with shaking. KFR1 protein was then precipitated with 5 µl of
protein-A beads cross-linked with rabbit antibody against the unique
carboxyl-terminal region of KFR1 (16). The antibody beads had been
presaturated with 6% bovine serum albumin in lysis buffer.
Alternatively, 5 µl of bovine serum albumin-saturated regular rabbit
IgG beads (Zymed, Inc.) was used as controls in the
immunoprecipitation. The immunocomplex was washed three times with
lysis buffer, followed by two times in kinase assay buffer (25 mM Tris-HCl, pH 6.8, 10 mM MgCl2, 2 mM MnCl2, 100 mM NaCl, 0.5 mM sodium orthovanadate, 10 µM ATP). The
immunocomplex was then suspended in 10 µl of kinase assay buffer.
Five micrograms of substrate protein and 10 µCi of
[-32P]ATP (Amersham Corp.) were added to the
immunocomplex. The mixture was incubated at room temperature for 1 h, and the reaction was stopped by adding 10 µl of SDS-PAGE loading
solution (2% SDS, 65 mM Tris-HCl, pH 6.8, 5%
-mercaptoethanol, 20% glycerol) and immediately heated in a boiling
water bath for 5 min. The phosphorylated substrates were subsequently
separated by SDS-PAGE, transferred to Immobilon membrane (Millipore),
and visualized by autoradiography. The radioactive bands were excised,
and their radioactivities were quantitated by Cerenkov counting in a
scintillation counter (Beckman).
Phosphorylated substrate proteins (32P-labeled) were separated by SDS-PAGE, and electrotransferred to polyvinylidene difluoride membrane (Millipore). The phosphorylated substrate bands were visualized by autoradiography, excised, and hydrolyzed by constant boiling of HCl at 110 °C for 1.5 h. The phosphoamino acids were separated by electrophoresis at pH 3.5 on a TLC plate essentially as described (25).
HVH2 Protein Expression and KFR1 InactivationPlasmid
pGEX-HVH2 was from Dr. K.-L. Guan. GST-HVH2 fusion protein was
expressed in Escherichia coli and purified as described (26). Proteins were quantitated by densitometry analysis of Coomassie
R-250-stained SDS-PAGE gels using bovine serum albumin as a standard.
Purified GST-HVH2 protein was mixed with immunoprecipitated KFR1 on
protein-A beads (described above) in 20 µl of phosphatase buffer (50 mM HEPES, pH 7.5, 0.1% -mercaptoethanol) with
occasional shaking at 37 °C for 60 min. After four washes with lysis
buffer and two washes in kinase buffer, the kinase activity of
GST-HVH2-treated KFR1 was determined by in vitro kinase
assay described above.
Poly(A)+ RNA was isolated and analyzed by Northern blotting as described (16). Immunostaining was performed as described previously (16). Antibody against KFR1 was diluted 3,000-fold.
The
MAP kinase homologue KFR1 from T. brucei has a
deduced amino acid sequence bearing over 40% identity to those of
KSS1/FUS3 (16). To analyze whether KFR1 is a
functional homologue of KSS1/FUS3 in S. cerevisiae, we used KFR1 to complement a yeast
kss1/fus3 double mutant. It has been shown that the
pheromone -factor can activate FUS3/KSS1, and either KSS1 or FUS3 is
sufficient to activate the expression of another gene FUS1
(20). We therefore transformed KFR1 under the control of
GAL1 promoter into a yeast strain containing a
FUS1-LacZ gene and a kss1/fus3 double mutation
(see "Experimental Procedures"). After induction of KFR1 expression
with galactose and treatment with
-factor, the yeast cells were
assayed for
-galactosidase activity. As shown in Fig.
1A, KFR1 did not complement kss1/fus3 on FUS1-LacZ gene expression, even
though KFR1 protein was successfully translated in the transformant and
detected by Western blot using antibody against a unique
carboxyl-terminal peptide of KFR1 protein, TbKH1 (16) (Fig.
1B). In contrast, an introduced FUS3 gene could
efficiently activate FUS1 expression. These results suggest
that KFR1 is functionally distinct from KSS1 and
FUS3.
The Controlled Expression Level and Indispensability of KFR1 in T. brucei
To further study the biological function of
KFR1, we overexpressed KFR1 in the procyclic form
of T. brucei. The KFR1 cDNA was inserted into
a trypanosome expression vector, pTSA-HYG2, and transfected into
procyclic T. brucei cells (21). Expression of
KFR1 and its downstream hygr gene was
controlled by the same constitutive PARP promoter and the polycistronic
transcript processed by trans-splicing (27). Stable
transfectants were selected under hygromycin B and were subsequently
cloned by limiting dilution (22). Northern hybridizations indicated
that the transfected KFR1 gene was successfully transcribed (Fig. 2A). There are two species of the
transcripts; the 1.3-kilobase species is of the same size as the wild
type mRNA (16). The 2.8-kilobase species is probably the unspliced
polycistronic transcript of KFR1-hygr, because
both KFR1 and hygr probes hybridized
with the same band (Fig. 2, A and B). The
KFR1 transfectant has approximately a 20-fold higher level
of KFR1 messenger RNA than those in the untransfected or
vector-transfected T. brucei (Fig. 2A). However,
when the cell lysates were immunostained with the antibodies against
TbKH1 (16), the KFR1 transfectant exhibited the same level
of KFR1 protein as in the controls (Fig. 2D). These results
suggest that expression of KFR1 is post-transcriptionally regulated to maintain its protein at a fixed level in the transfected procyclic cells of trypanosome.
KFR1 gene disruption experiments were also carried out. KFR1 is present as a single-copy gene in the diploid T. brucei genome (16). Driven by a PARP promoter, either hygr or neor gene was inserted into the plasmid pB-KFR1 to replace a central portion of the KFR1 coding region resulting in pNKH-Hyg and pNKH-Neo, respectively (see "Experimental Procedures"). The pNKH-Hyg construct was electroporated into the procyclic T. brucei cells, and the transfected cells were selected under hygromycin B and subsequently cloned by limiting dilutions. Genomic Southern hybridizations showed that one allele of the KFR1 gene was successfully disrupted in all the cloned cell lines (data not shown). We then proceeded to disrupt the second allele of KFR1 gene in the diploid T. brucei. A cell line, with one allele of the KFR1 gene disrupted, was transfected with the pNKH-Neo construct. The transfected cells were selected under both hygromycin B and G418 and further cloned. Genomic Southern analysis revealed that all 18 cloned double drug-resistant cell lines still contained the intact second allele of KFR1 gene (data not shown), while the neor gene was integrated into other loci in the T. brucei genome. The failure in disrupting the second allele of KFR1 gene cannot be attributed to the neor gene because two alleles of the ODC (ornithine decarboxylase) gene were successfully disrupted in T. brucei by using an identical strategy (28). These results suggest that KFR1 gene is essential for growth of T. brucei procyclic cells.
Differential Kinase Activities in KFR1To assay for its
kinase activity, KFR1 protein was immunoprecipitated from cell lysates
(5 × 106 cells) of both bloodstream and procyclic
forms of T. brucei using antibodies against the unique
carboxyl-terminal peptide of KFR1 protein (16). The immunoprecipitated
protein was then assayed for phosphorylation of histone H-1, myelin
basic protein, and -casein, respectively, in the presence of
[
-32P]ATP (29, 30). The phosphorylated substrates were
then separated by SDS-PAGE and visualized by autoradiography. The
results indicated that all three protein substrates were effectively
phosphorylated (Fig. 3A). The KFR1 from
bloodstream cells has much higher specific activities than that from
procyclic cells in these assays, even though the KFR1 protein levels in
the bloodstream and procyclic form are about equal (16) (data not
shown). Densitometry analysis of the autoradiograms indicated that the
former is approximately 4-10-fold higher than the latter.
A dual specific phosphatase HVH2 has been reported to selectively dephosphorylate and inactivate MAP kinases, ERK1 and ERK2, from mammalian cells (26). KFR1 immunoprecipitated from 5 × 106 bloodstream cells was first treated with approximately 110 ng of the purified GST-HVH2 fusion protein from transformed E. coli for 1 h at 37 °C (26) and then analyzed in the kinase assay. The KFR1 kinase activity was completely abolished by the HVH2 pretreatment, whereas the level of KFR1 protein was not changed by the treatment as demonstrated in the immunostains (Fig. 3B). Thus, like mammalian MAP kinases, the activity of KFR1 can be regulated by the status of phosphorylation with the phosphorylated KFR1 being the activated form. It is also possible that KFR1 is present in different states of phosphorylation in the bloodstream and procyclic forms.
Serum Induction of KFR1 ActivityA major distinction between
the bloodstream and procyclic forms of T. brucei is that the
former propagates in mammalian blood whereas the latter grows in the
midgut of the tsetse fly. To examine whether the high KFR1 activity in
the bloodstream form could be related to the presence of serum in its
living environment, both bloodstream and procyclic cells were first
incubated in the serum-free media for up to 12 h. Fetal calf serum
was then added to the cultures, and cell samples were harvested at
different time points thereafter. KFR1 protein was immunoprecipitated
and assayed for kinase activities. As shown in Fig.
4A, the kinase activity in KFR1 from the
bloodstream form was gradually reduced to the basal level within 6 h of serum starvation and remained at that level. Upon serum addition,
KFR1 activity was rapidly restored within 30 min to the original level prior to serum starvation, whereas the protein level of KFR1 in the
cell was constant during the entire experiment as determined by Western
analysis of the immunoprecipitated samples. On the other hand, the KFR1
activity in the procyclic form responded poorly to serum (Fig.
4B). It dropped also to the basal level within 30 min upon
serum starvation, but it took 6 h to restore the original low
level of KFR1 kinase activity after the serum was supplemented. Similar
to the bloodstream form, the KFR1 protein level also remained unchanged
during the study.
Interferon-
In trying
to identify the specific component(s) of the serum responsible for
inducing the KFR1 kinase activity in T. brucei bloodstream
cells, interferon- (200 units/ml), PDGF (100 ng/ml), and basic
fibroblast growth factor (20 ng/ml) were each added to the cell
cultures starved of serum for 12-14 h. The KFR1 protein was
immunoprecipitated from the cells harvested at different time points
and assayed for kinase activity. The kinase activity in KFR1 was
reduced approximately 3-4-fold following the serum starvation but was
rapidly activated within 30 min upon interferon-
treatment (Fig.
5, A, B, and C),
whereas bovine serum albumin (Fig. 5D), PDGF, and basic
fibroblast growth factor did not show any effect (data not shown), even
though all the samples contained approximately an equal amount of KFR1
protein by Western blot analysis (Fig. 5E). Similar
experiments revealed that the same interferon-
treatment did not
stimulate KFR1 activity in procyclic cells within the same time period
(data not shown). Thus, interferon-
is able to activate KFR1 only in
the bloodstream form of T. brucei.
It has been reported that T. brucei invasion stimulates
interferon- production in the mammalian host which in turn promotes the growth of bloodstream-form T. brucei both in
vivo and in vitro (6-8). We also observed a rather
moderate enhancement of bloodstream cell proliferation by
interferon-
(500 units/ml) in HMI-10 medium for 24 h
(approximately 15-20% increase in cell numbers over the control
without interferon-
) (data not shown). In contrast, growth of
procyclic cells was not at all promoted by the interferon-
treatment.
Phosphoamino acid analysis of
the KFR1-phosphorylated substrates, histone H-1, myelin basic protein,
and -casein, was performed. The substrates were labeled with
[
-32P]ATP by the catalysis of the immunoprecipitated
KFR1 from bloodstream cells, separated in SDS-PAGE, transferred to
polyvinylidene difluoride membrane, and visualized by autoradiography.
The radiolabeled protein substrates were then hydrolyzed in constant
boiling HCl and separated by electrophoresis (25). As shown in Fig.
6A, all three protein substrates were
phosphorylated on the serine residues by KFR1. Neither phosphothreonine
nor phosphotyrosine was detected in the hydrolysates. Therefore,
similar to other MAP kinases (31-33), KFR1 from trypanosome also
belongs to the serine/threonine protein kinase superfamily.
We also followed the time course of changes in KFR1 activity upon
interferon- treatment of the bloodstream form T. brucei by analyzing the phosphoamino acid in the substrate of KFR1.
Phosphorylated myelin basic protein as illustrated in Fig.
5A was hydrolyzed and its phosphoamino acids analyzed. The
results in Fig. 6B confirmed that KFR1 is a serine kinase,
and its activity stimulated upon interferon-
treatment did not show
change of substrate specificity, thus ruling out possible involvement
of another protein kinase co-immunoprecipitated with KFR1. The same
activity was diminished after treatment with HVH2 (Fig. 6C)
suggesting that the interferon-
-stimulated KFR1 must be in the
phosphorylated form in the bloodstream form of T. brucei.
In an attempt to
identify the endogenous substrates of KFR1 in T. brucei, we
first prepared the T. brucei bloodstream-form lysate by
sonicating the cells in the presence of protease inhibitors. The
sonicated lysate was boiled for 5 min and followed by another sonication. The bloodstream-form lysate thus prepared was then used as
a substrate for immunoprecipitated KFR1 in kinase assay as described
(see "Experimental Procedures"). Results in Fig. 7
demonstrated that KFR1 was able to phosphorylate four endogenous substrates with molecular masses of 22 kDa (p22), 34 kDa (p34), 46 kDa
(p46) and 90 kDa (p90), respectively. Among these substrates, the
phosphorylation signals from p22 and p34 were stronger than those from
the other two. However, the identities of these endogenous substrates
are yet to be determined.
In the present investigation, we have examined the function of a MAP kinase homologue, KFR1, in T. brucei. By sequence comparison, KFR1 is classified in the ERK subfamily of the MAP kinase family (34). Despite high percentages of sequence identity with the yeast MAP kinases, KSS1 and FUS3, our complementation experiment suggests that KFR1 is functionally different from KSS1 and FUS3. Functional diversity has been observed among MAP kinases (32-34). For example, ERK1/ERK2 and stress-activated protein kinases in mammalian cells are involved in cell proliferation and differentiation, and stress responses, respectively, whereas FUS3/KSS1, MPK1, and HOG1 in S. cerevisiae are involved in the mating, cell-wall biosynthesis, and osmoregulation, respectively (34). This may in part explain why there is more than one member of MAP kinase in a living organism. Our T. brucei genomic Southern blots have consistently revealed very weak hybridization signals other than the major signal from the single copy KFR1 gene when the genomic DNA was digested with several restriction enzymes and probed with KFR1 cDNA.2 This result suggests that there is probably at least one additional MAP kinase homologue in T. brucei.
Our experimental evidence indicates that KFR1 is a serine protein
kinase that specifically phosphorylates the serine residues in the
substrates tested. It has also been clearly indicated that KFR1 is most
likely activated in its phosphorylated form but inactivated through
dephosphorylation by a MAP kinase-specific phosphatase HVH2 in
vitro. We also identified four proteins in the bloodstream-form lysate that can be phosphorylated by KFR1, although the identities of
these endogenous substrates remain unknown at present. The mechanism of
a probable KFR1-mediated proliferation of T. brucei bloodstream form is also unclear for the time being. Based on what is
known on the cascade mechanisms behind MAP kinase activation in other
living organisms (32-34), however, certain speculation can be made at
the present time. It is known that activation of protein kinase C can
lead to activation of MAP kinase and further promote cell growth
(35-38). Protein kinase C is activated by diacylglycerol released from
phosphatidylinositol, which is hydrolyzed by phospholipase C upon
stimulation by many extracellular signals including growth factors and
mitogens in mammals (39-41). T. brucei contains a
glycosylphosphatidylinositol-specific phospholipase C that cleaves the
carboxyl-terminal glycosylphosphatidylinositol anchor of the variant
surface glycoproteins forming diacylglycerol and 1,2-cyclic phosphate
on the inositol ring (42, 43). The enzyme is present in the bloodstream
forms but not in the procyclic forms (44, 45). Although it is unclear
at present whether diacylglycerol generated from the
glycosylphosphatidylinositol anchor is capable of activating protein
kinase C in T. brucei, protein kinase C activities have been
characterized in T. brucei. There are apparently two
diacylglycerol-dependent protein kinase C activities in the
bloodstream form, but no protein kinase C activity was detected in the
procyclic form (46). This distinction between the two forms could
constitute the basis of their different responses to interferon-. It
remains to be examined, however, what the link is between
interferon-
, the activation of protein kinase C, and the activation
of KFR1 in T. brucei bloodstream form if such a link does
exist. Molecular cloning of the bloodstream form-specific protein
kinase C genes from T. brucei would supply powerful tools
for the study.
Our results suggest that KFR1 is probably involved in the proliferation of bloodstream-form cells of T. brucei. However, the role of KFR1 in the insect form needs to be further investigated. In view of the fact that the second allele of KFR1 gene could not be disrupted by gene replacement in the procyclic cells, it would be reasonable to propose that KFR1 plays also an essential role in the insect form of T. brucei. The procyclic cells of trypanosome expressed lectin-binding sites, and lectins secreted in the gut of tsetse fly not only interfere with the establishment of the parasites in the midgut but also provide a signal for maturation of the parasites and induce hypopharyngeal infection (47-49). In other systems, lectins, such as concanavalin A, phytohemagglutinin, and wheat germ agglutinin, can stimulate protein kinase activities through G-protein-coupled receptor, growth factor receptor, and protein kinase C pathways and further promote cell proliferation and differentiation (50-52). Studying the potential role of KFR1 in the lectin-induced maturation of T. brucei procyclic cells would thus be a very interesting subject in the future.
T. brucei invasion of mammalian host stimulates production
of TGF- and interferon-
by the host T cells (6, 7). This parasite-induced host cytokine production suggests that they could play
important pathophysiological roles during parasite infection. T. brucei may benefit from host-produced TGF-
and interferon-
in two different ways. First, it has been reported that production of
TGF-
from T cells is associated with immunosuppression by its
down-regulatory role of autoimmune responses in certain diseases such
as encephalomyelitis (53, 54). On the other hand, interferon-
induces immunosuppression in trypanosome-infected animals, and this
immunosuppression, at least in part, is mediated by nitric oxide
released from suppressor macrophages (55, 56). Therefore, immunosuppression induced by TGF-
and interferon-
may allow the
parasite to evade host immune responses. Second, as we found in the
current studies, interferon-
stimulates the kinase activity in KFR1,
which is correlated with the moderate promotion of proliferation of the
T. brucei bloodstream cells. Thus, the parasite apparently takes advantage of the host immune system to promote its own
propagation in the mammalian host. However, in view of the relatively
modest in vitro growth promotion, a note of caution should
be added to the correlation between induction of KFR1 activity and cell
proliferation. On the other hand, the growth-promoting role of
interferon-
for the bloodstream form of T. brucei
contrasts with that for another trypanosome parasite, T. cruzi, in which interferon-
-mediated macrophage activation
inhibits intracellular parasite replication in the host cells (57, 58).
It is most likely that the mechanisms of these two trypanosomes in
response to interferon-
are different. It is also intriguing to
notice that interferon-
exerts opposite effects on the bloodstream
forms of T. brucei and certain mammalian cells. In
fibroblasts and macrophages, interferon-
induces activation of
Jak1/Jak2, which inhibits protein kinase C, and thereby blocks MAP
kinase activation (14, 15). Studying the mechanism of KFR1 activation
in T. brucei by interferon-
would thus be an interesting
endeavor.
We are grateful to Dr. M. Peters for advice on yeast complementation, to Dr. I. Herskowitz for yeast strains and plasmids pRS314 and pRS-FUS3, and to Dr. K.-L. Guan for plasmid pGEX-HVH2. We thank Drs. M. Mutomba, J. Sommer, and A. L. Wang for critical reading of the manuscript.