Interferon-gamma Activation of a Mitogen-activated Protein Kinase, KFR1, in the Bloodstream Form of Trypanosoma brucei*

(Received for publication, August 27, 1996, and in revised form, December 30, 1996)

Shao-Bing Hua Dagger and Ching C. Wang

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 beta -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-gamma . The production of interferon-gamma 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-gamma -induced proliferation of T. brucei in the mammalian host.


INTRODUCTION

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-beta (TGF-beta )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-gamma production in the host (7). By injecting monoclonal antibody against the lymphocyte-triggering factor or depleting CD8+ cells in T. brucei-infected animals, interferon-gamma production was abrogated, growth of trypanosome suppressed, and survival of the infected animals prolonged (4, 8). Interferon-gamma can stimulate proliferation of trypanosome bloodstream cells in culture (6). Administration of interferon-gamma antibody to the infected animals can reduce parasitemia and prolong the survival of the animals (7). The molecular mechanism of this interferon-gamma 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-alpha , IFN-beta , and IFN-gamma ), interferon-gamma 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-gamma 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 beta -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-gamma strongly enhanced the KFR1 activity as well as the cell proliferation. The lower KFR1 activity in procyclic form was, however, unaffected by interferon-gamma .


EXPERIMENTAL PROCEDURES

Cells and Materials

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-gamma , recombinant human basic fibroblast growth factor, histone H-1, and myelin basic protein were purchased from Life Technologies, Inc. beta -Casein was from Sigma. Recombinant human PDGF B/B was purchased from Boehringer Mannheim.

S. cerevisiae Complementation

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 alpha -factor, LacZ expression was monitored as described (19).

KFR1 Overexpression Vector Construction and Transformation

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).

Gene Disruption Constructs

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 Assay

T. 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 [gamma -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% beta -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).

Phosphoamino Acid Analysis

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 Inactivation

Plasmid 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% beta -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.

Northern Blot and Immunostaining Analyses

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.


RESULTS

KFR1 Does Not Complement Function of KSS1/FUS3 in Yeast

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 alpha -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 alpha -factor, the yeast cells were assayed for beta -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.


Fig. 1. Experiments of complementation of KFR1 to yeast FUS3/KSS1. KFR1 cDNA was inserted into yeast vector pRS314 under GAL1 promoter. This construct, pRS-KFR1, and pRS-FUS3 (positive control) were then, respectively, transformed into a yeast strain SH101 that contains double deletion mutation fus3/kss1 and a lacZ gene under FUS1 promoter. Yeast cells were treated with galactose and followed by alpha -factor. Yeast cells were then lysed and assayed for beta -galactosidase activity (A) or subjected to SDS-PAGE and further immunostaining with antibody against KFR1 (B). Lane 1, SH101; lane 2, SH101/pRS314; lane 3, SH101/pRS-FUS3; and lane 4, SH101/pRS-KFR1.
[View Larger Version of this Image (22K GIF file)]


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.


Fig. 2. KFR1 overexpression in procyclic cells of T. brucei. KFR1 cDNA was inserted into the trypanosome overexpression vector pTSA-HYG2 (21) and transfected into procyclic cells of T. brucei. Poly(A)+ RNA was isolated from the cloned transfectants and analyzed with Northern blotting using KFR1 probe (A). The same blot was reprobed with hygr DNA (B) and beta -tubulin DNA (C) sequentially. Cell lysates were also subjected to SDS-PAGE and immunostaining using antibody against KFR1 (D). Lanes 1, untransfected T. brucei cells; lanes 2, T. brucei transfected with vector pTSA-HYG2 alone; lanes 3, T. brucei transfected with pTKH-Hyg containing KFR1 (see "Experimental Procedures"). The upper band (2.9 kilobases (kb)in size) in panels A and B is presumably derived from the unspliced polycistronic transcript of KFR1-hygr. The weak band around 75 kDa in panel D was also detected when T. brucei cell lysates were immunostained with preimmune serum (16).
[View Larger Version of this Image (35K GIF file)]


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 KFR1

To 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 beta -casein, respectively, in the presence of [gamma -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.


Fig. 3. In vitro kinase activity of KFR1 from bloodstream and procyclic cells of T. brucei. A, KFR1 protein was immunoprecipitated from 5 × 106 cells of either bloodstream form (BSF) or procyclic form (PCF) by using antibody against the unique carboxyl-terminal domain of KFR1 (see "Experimental Procedures"). The immunoprecipitated KFR1 was then assayed for kinase activity using histone H-1 (H-1), myelin basic protein (MBP), and beta -casein (beta -C) as substrates in the presence of [gamma -32P]ATP. The phosphorylated products were subjected to SDS-PAGE and visualized by autoradiography. Regular rabbit IgG (IgG ctrl) was also used as control in the experiments. B, immunoprecipitated KFR1 from bloodstream cells was assayed for kinase activity (lane 1) or after treatment of GST protein (lane 2) or GST-HVH2 fusion protein (lane 3) using myelin basic protein as substrate. The same blot was further immunostained with antibody against KFR1 and is shown in the bottom panel (rabbit IgG bands have been cut off).
[View Larger Version of this Image (26K GIF file)]


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 Activity

A 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.


Fig. 4. KFR1 activation by serum. Both bloodstream (A) and procyclic cells (B) were serum starved for up to 12 h and followed by addition of serum to the starved cells. At the indicated time points, KFR1 protein was immunoprecipitated from 5 × 106 cells and assayed for kinase activity using histone H-1 as substrate. Rabbit IgG (IgG ctrl) was included as control. Immunoprecipitated samples were also detected with antibody against KFR1 as shown in the bottom panels of A and B.
[View Larger Version of this Image (43K GIF file)]


Interferon-gamma Activates KFR1 in Bloodstream Form

In trying to identify the specific component(s) of the serum responsible for inducing the KFR1 kinase activity in T. brucei bloodstream cells, interferon-gamma (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-gamma 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-gamma treatment did not stimulate KFR1 activity in procyclic cells within the same time period (data not shown). Thus, interferon-gamma is able to activate KFR1 only in the bloodstream form of T. brucei.


Fig. 5. KFR1 activation by interferon-gamma in bloodstream cells. Bloodstream cells were starved of serum for 14 h. Interferon-gamma (200 units/ml) was then added to the cultures. KFR1 protein was immunoprecipitated from cells before serum starvation (lane +), after starvation but before interferon-gamma addition (lane 0), and 10, 30, and 60 min after interferon-gamma addition. The immunoprecipitated KFR1 samples were then assayed for kinase activity with myelin basic protein (A) or histone H-1 (B) as substrates in the presence of [gamma -32P]ATP. The 32P-labeled substrates were separated by SDS-PAGE, blotted onto membrane, and detected by autoradiography. The radioactive bands were also excised, and their radioactivities were counted (C). Panel D is the KFR1 kinase activity assay using histone H-1 substrate after treatment with bovine serum albumin (BSA) as control. Identical blots were also immunostained with antibody against KFR1 (E), indicating that approximately an equal amount of KFR1 protein was present in these samples.
[View Larger Version of this Image (49K GIF file)]


It has been reported that T. brucei invasion stimulates interferon-gamma 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-gamma (500 units/ml) in HMI-10 medium for 24 h (approximately 15-20% increase in cell numbers over the control without interferon-gamma ) (data not shown). In contrast, growth of procyclic cells was not at all promoted by the interferon-gamma treatment.

KFR1 Is a Serine Protein Kinase

Phosphoamino acid analysis of the KFR1-phosphorylated substrates, histone H-1, myelin basic protein, and beta -casein, was performed. The substrates were labeled with [gamma -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.


Fig. 6. Phosphoamino acid analysis. A, three substrates, histone H-1 (H-1), myelin basic protein (MBP), and beta -casein (beta -C), were labeled by immunoprecipitated KFR1 from bloodstream-form cells and [gamma -32P]ATP, separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and then hydrolyzed by HCl. The hydrolyzed products were separated by electrophoresis on a TLC plate and visualized by autoradiography. B, phosphoamino acid analysis of 32P-labeled myelin basic protein by KFR1 upon interferon-gamma treatment as illustrated in Fig. 5A is shown. C, phosphoamino acid analysis of phosphorylated myelin basic protein by KFR1 before (lane 1) and after treatment of GST (lane 2) or GST-HVH2 (lane 3) (also see Fig. 3B). Positions of phosphoamino acids detected by ninhydrin are indicated by arrows. Ori, origin.
[View Larger Version of this Image (30K GIF file)]


We also followed the time course of changes in KFR1 activity upon interferon-gamma 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-gamma 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-gamma -stimulated KFR1 must be in the phosphorylated form in the bloodstream form of T. brucei.

KFR1 Substrates in T. brucei Cell Lysate

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.


Fig. 7. KFR1 substrates in T. brucei lysate. T. brucei bloodstream-form cell lysates were first treated by boiling and sonication. The lysates were then used as substrates in the kinase assay (see "Experimental Procedures"). Lane 1, immunoprecipitants by regular rabbit IgG as control; lane 2, immunoprecipitated KFR1. The reaction mixtures were fractionated by SDS-PAGE, and the radioactive bands were visualized by autoradiography. The KFR1 substrates in T. brucei lysate are indicated by arrowheads. F, the dye front of SDS-PAGE.
[View Larger Version of this Image (50K GIF file)]



DISCUSSION

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-gamma . It remains to be examined, however, what the link is between interferon-gamma , 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-beta and interferon-gamma 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-beta and interferon-gamma in two different ways. First, it has been reported that production of TGF-beta 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-gamma 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-beta and interferon-gamma may allow the parasite to evade host immune responses. Second, as we found in the current studies, interferon-gamma 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-gamma for the bloodstream form of T. brucei contrasts with that for another trypanosome parasite, T. cruzi, in which interferon-gamma -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-gamma are different. It is also intriguing to notice that interferon-gamma exerts opposite effects on the bloodstream forms of T. brucei and certain mammalian cells. In fibroblasts and macrophages, interferon-gamma 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-gamma would thus be an interesting endeavor.


FOOTNOTES

*   This investigation was supported by National Institutes of Health Grant AI-21786.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303-4230. Tel.: 415-424-8222; Fax: 415-354-0776.
1   The abbreviations used are: TGF-beta , transforming growth factor beta ; PDGF, platelet-derived growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; PCR, polymerase chain reaction; PARP, procyclic acidic repetitive protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
2   S.-B. Hua and C. C. Wang, unpublished data.

ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Ming, M., Ewen, M. E., and Pereira, M. E. (1995) Cell 82, 287-296 [Medline] [Order article via Infotrieve]
  2. Cross, G. A. (1990) Annu. Rev. Immunol. 8, 83-110 [CrossRef][Medline] [Order article via Infotrieve]
  3. Mansfield, J. M. (1994) Parasitol. Today 10, 267-270 [Medline] [Order article via Infotrieve]
  4. Bakhiet, M., Olsson, T., van der Meide, P., and Kristensson, K. (1990) Clin. Exp. Immunol. 81, 195-199 [Medline] [Order article via Infotrieve]
  5. Bakhiet, M., Mix, E., Kristensson, K., Wigzell, H., and Olsson, T. (1993) Eur. J. Immunol. 23, 1535-1539 [Medline] [Order article via Infotrieve]
  6. Olsson, T., Bakhiet, M., Edlund, C., Hojeberg, B., Van der Meide, P., and Kristensson, K. (1991) Eur. J. Immunol. 21, 2447-2454 [Medline] [Order article via Infotrieve]
  7. Olsson, T., Bakhiet, M., Hojeberg, B., Ljungdahl, A., Edlund, C., Andersson, G., Ekre, H. P., Fung, L. W., Mak, T., and Wigzell, H. (1993) Cell 72, 715-727 [Medline] [Order article via Infotrieve]
  8. Bakhiet, M., Olsson, T., Edlund, C., Hojeberg, B., Holmberg, K., Lorentzen, J., and Kristensson, K. (1993) Scand. J. Immunol. 37, 165-178 [Medline] [Order article via Infotrieve]
  9. Pestka, S., Langer, J. A., Zoon, K. C., and Samuel, C. E. (1987) Annu. Rev. Biochem. 56, 727-777 [CrossRef][Medline] [Order article via Infotrieve]
  10. De Maeyer, E., and De Maeyer-Guignard, J. (1988) Interferons and Other Regulatory Cytokines, John Wiley & Sons, Inc., New York
  11. Sen, G. C., and Lengyel, P. (1992) J. Biol. Chem. 267, 5017-5020 [Free Full Text]
  12. Pearse, R. N., Feinman, R., Shuai, K., Darnell, J. E., Jr., and Ravetch, J. V. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4314-4318 [Abstract]
  13. Farrar, M. A., and Schreiber, R. D. (1993) Annu. Rev. Immunol. 11, 571-611 [CrossRef][Medline] [Order article via Infotrieve]
  14. Xu, J., Rockow, S., Kim, S., Xiong, W., and Li, W. (1994) Mol. Cell. Biol. 14, 8018-8027 [Abstract]
  15. Xu, J., Kim, S., Chen, M., Rockow, S., Yi, S. E., Wagner, A. J., Hay, N., Weichselbaum, R. R., and Li, W. (1995) Blood 86, 2774-2788 [Abstract/Free Full Text]
  16. Hua, S. B., and Wang, C. C. (1994) J. Cell. Biochem. 54, 20-31 [Medline] [Order article via Infotrieve]
  17. Hirumi, H., and Hirumi, K. (1989) J. Parasitol. 75, 985-989 [Medline] [Order article via Infotrieve]
  18. Cunningham, I. (1977) J. Protozool. 24, 325-329 [Medline] [Order article via Infotrieve]
  19. Trueheart, J., Boeke, J. D., and Fink, G. R. (1987) Mol. Cell. Biol. 7, 2316-2328 [Medline] [Order article via Infotrieve]
  20. Elion, E. A., Brill, J. A., and Fink, G. R. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 41-49 [Medline] [Order article via Infotrieve]
  21. Sommer, J. M., Peterson, G., Keller, G. A., Parsons, M., and Wang, C. C. (1993) FEBS Lett. 316, 53-58 [CrossRef][Medline] [Order article via Infotrieve]
  22. Sommer, J. M., Cheng, Q. L., Keller, G. A., and Wang, C. C. (1992) Mol. Biol. Cell. 3, 749-759 [Abstract]
  23. Hua, S. B., Li, X., Coffino, P., and Wang, C. C. (1995) J. Biol. Chem. 270, 10264-10271 [Abstract/Free Full Text]
  24. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Cooper, J. A., Sefton, B. M., and Hunter, T. (1983) Methods Enzymol. 99, 387-402 [Medline] [Order article via Infotrieve]
  26. Guan, K. L., and Butch, E. (1995) J. Biol. Chem. 270, 7197-7203 [Abstract/Free Full Text]
  27. Agabian, N. (1990) Cell 61, 1157-1160 [Medline] [Order article via Infotrieve]
  28. Li, F., Hua, S.-B., Wang, C. C., and Gottesdiener, K. (1996) Mol. Biochem. Parasitol. 78, 227-236 [CrossRef][Medline] [Order article via Infotrieve]
  29. Hoshi, M., Nishida, E., and Sakai, H. (1989) Eur. J. Biochem. 184, 477-486 [Abstract]
  30. Sanghera, J. S., Paddon, H. B., Bader, S. A., and Pelech, S. L. (1990) J. Biol. Chem. 265, 52-57 [Abstract/Free Full Text]
  31. Ray, L. B., and Sturgill, T. W. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1502-1506 [Abstract]
  32. Nishida, E., and Gotoh, Y. (1993) Trends Biochem. Sci. 18, 128-131 [CrossRef][Medline] [Order article via Infotrieve]
  33. Blumer, K. J., and Johnson, G. L. (1994) Trends Biochem. Sci. 19, 236-240 [CrossRef][Medline] [Order article via Infotrieve]
  34. Cano, E., and Mahadevan, L. C. (1995) Trends Biochem. Sci. 20, 117-122 [CrossRef][Medline] [Order article via Infotrieve]
  35. Boulikas, T. (1995) Crit. Rev. Eukaryotic Gene Expression 5, 1-77 [Medline] [Order article via Infotrieve]
  36. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846 [Free Full Text]
  37. Huang, J., Mohammadi, M., Rodrigues, G. A., and Schlessinger, J. (1995) J. Biol. Chem. 270, 5065-5072 [Abstract/Free Full Text]
  38. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249-252 [CrossRef][Medline] [Order article via Infotrieve]
  39. Nishizuka, Y. (1984) Nature 308, 693-698 [Medline] [Order article via Infotrieve]
  40. Nishizuka, Y. (1984) Science 225, 1365-1370 [Medline] [Order article via Infotrieve]
  41. Noh, D. Y., Shin, S. H., and Rhee, S. G. (1995) Biochim. Biophys. Acta 1242, 99-113 [CrossRef][Medline] [Order article via Infotrieve]
  42. Cardoso de Almeida, M. L., and Turner, M. J. (1983) Nature 302, 349-352 [Medline] [Order article via Infotrieve]
  43. Ferguson, M. A., Homans, S. W., Dwek, R. A., and Rademacher, T. W. (1988) Science 239, 753-759 [Medline] [Order article via Infotrieve]
  44. Bülow, R., and Overath, P. (1986) J. Biol. Chem. 261, 11918-11923 [Abstract/Free Full Text]
  45. Bülow, R., and Overath, P. (1985) FEBS Lett. 187, 105-110 [CrossRef][Medline] [Order article via Infotrieve]
  46. Keith, K., Hide, G., and Tait, A. (1990) Mol. Biochem. Parasitol. 43, 107-116 [Medline] [Order article via Infotrieve]
  47. Maudlin, I., and Welburn, S. C. (1988) Trop. Med. Parasitol. 39, 56-58 [Medline] [Order article via Infotrieve]
  48. Welburn, S. C., Maudlin, I., and Molyneux, D. H. (1994) Med. Vet. Entomol. 8, 81-87 [Medline] [Order article via Infotrieve]
  49. Welburn, S. C., and Maudlin, I. (1990) Med. Vet. Entomol. 4, 43-48 [Medline] [Order article via Infotrieve]
  50. Kaibuchi, K., Takai, Y., and Nishizuka, Y. (1985) J. Biol. Chem. 260, 1366-1369 [Abstract]
  51. Matsuo, T., Hazeki, K., Hazeki, O., Katada, T., and Ui, M. (1996) Biochem. J. 315, 505-512 [Medline] [Order article via Infotrieve]
  52. Zeng, F. Y., Benguria, A., Kafert, S., Andre, S., Gabius, H. J., and Villalobo, A. (1995) Mol. Cell. Biochem. 142, 117-124 [Medline] [Order article via Infotrieve]
  53. Karpus, W. J., and Swanborg, R. H. (1991) J. Immunol. 146, 1163-1168 [Abstract/Free Full Text]
  54. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 421-425 [Abstract]
  55. Mabbott, N. A., Sutherland, I. A., and Sternberg, J. M. (1995) Parasite Immunol. (Oxf.) 17, 143-150
  56. Sternberg, J. M., and Mabbott, N. A. (1996) Eur. J. Immunol. 26, 539-543 [Medline] [Order article via Infotrieve]
  57. Munoz-Fernandez, M., Fernandez, M. A., and Fresno, M. (1992) Eur. J. Immunol. 22, 301-307 [Medline] [Order article via Infotrieve]
  58. Silva, J. S., Morrissey, P. J., Grabstein, K. H., Mohler, K. M., Anderson, D., and Reed, S. G. (1992) J. Exp. Med. 175, 169-174 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.