Molecular Cloning and Expression of a Stress-responsive Mitogen-activated Protein Kinase-related Kinase from Tetrahymena Cells*

Shigeru NakashimaDagger §, Shulin WangDagger , Naoki Hisamoto, Hideki Sakaiparallel , Masataka AndohDagger , Kunihiro Matsumoto, and Yoshinori NozawaDagger

From the Departments of Dagger  Biochemistry and parallel  Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan and the  Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

    ABSTRACT
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To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids. A homolog of this kinase with 90% amino acid sequence identity was also found in T. pyriformis. Both kinases contain 11 subdomains typical of protein kinases. Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stress-activated protein kinase which are known to be involved in various stress responses. However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr226-Gly-His228) in this MAPK-related kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr226 residue is essential for kinase activity. The MRK mRNA transcript in T. thermophila increased markedly upon temperature downshift from 35 to 15 °C (0.8 °C/min). Interestingly, osmotic shock either by sorbitol (100-200 mM) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T. pyriformis. In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress. This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity. The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Cells must respond appropriately to extracellular stimuli and changes in their environment. The unicellular eukaryotic protozoan Tetrahymena is a potentially useful animal cell model system for studying the molecular mechanism of adaptation to the environment. In the natural environment Tetrahymena is subjected to fluctuations in temperature, pH, amounts of nutrients, and concentration of dissolved gases. Changes in environmental conditions have profound effects on the activities of the cell at the physiological, morphological, and biochemical levels (1, 2). For example, Tetrahymena cells adapt to cold environments by increasing the amounts of unsaturated fatty acids in membrane phospholipids, thus maintaining proper membrane fluidity (3, 4). This is accomplished by an increase in fatty acid desaturase activity in response to a downshift in temperature (5, 6). We have recently cloned the gene encoding Delta 9 fatty acid desaturase that is involved in this process and demonstrated that its mRNA expression is increased in response to cold (7). However, at present the signal transduction mechanism by which this response occurs is not clearly understood.

Differential display (8, 9) and RNA fingerprinting by arbitrarily primed PCR1 (10) are two potentially useful methods to detect altered gene expression in complex RNA populations. In both methods, differential gene expression can be detected relatively easily as differences in the intensities of cDNA fragments following PCR amplification and electrophoretic separation on a gel. To this end, we have used the differential display method to analyze mRNA expression following temperature downshift of Tetrahymena thermophila. In the present study we successfully identify an mRNA which is expressed in response to cold stress. By homology analysis, its predicted amino acid sequence was found to be similar to those of mitogen-activated protein kinases (MAPKs) (11-20), especially to p38 and stress-activated protein kinase (SAPK, also known as c-Jun NH2-terminal kinase JNK) which have been known to be involved in various stress responses. Expression of this MAPK-related kinase (MRK) gene and kinase activity was also enhanced by osmotic stress in Tetrahymena cells. We will discuss its possible roles in response to environmental stress.

    EXPERIMENTAL PROCEDURES
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Materials-- Restriction enzymes and other nucleic acid modifying enzymes were obtained from Boehringer Mannheim, Toyobo, and Nippon gene. Taq DNA polymerase was obtained from Takara. Myelin basic protein (MBP) was purchased from Sigma. Arbitrary primers (AP-1~AP-10) were obtained from GenHunter. Radiolabeled nucleotides, Sequenase version 2 DNA sequencing kit, Multiprime DNA labeling system, ECL Western blot detection system, pGEX-4T-1 prokaryotic gene fusion vector, and glutathione-Sepharose were from Amersham Pharmacia Biotech. Immobilon-P membrane filters were from Millipore. BCA protein assay kit was from Pierce. T-vector was from Novagen. GeneScreen plus hybridization filters were from NEN Life Science Products Inc. T. thermophila and Tetrahymena pyriformis lambda gt10 cDNA libraries (21, 22) were kindly provided by Dr. O. Numata (University of Tsukuba, Japan).

Cell Culture-- T. thermophila was axenically cultivated at 35 °C with constant shaking (90 strokes/min) in a medium containing 2% protease peptone, 0.2% yeast extract, and 0.5% dextrose. Cells were exposed to temperature downshift to 15 °C at a rate of 0.8 °C/min, as described previously (7, 23). T. pyriformis was cultured at 28 °C.

Differential Display-- Total RNA was prepared from T. thermophila by the guanidine thiocyanate method (24). Total RNA (0.2 µg) was reverse transcribed with SuperScript II reverse transcriptase, GT15MN (M = A, C or G; N = A, C, G, or T) oligo(dT) primer (1 µM) and dNTP mixture (20 µM each) at 42 °C for 60 min. The polymerase chain reaction (PCR), recovery, and reamplification of cDNAs obtained were performed as described previously (8, 9) with slight modifications. Reverse transcriptase products from 20 ng of total RNA were amplified by PCR in 20 µl of reaction mixture containing 1 unit of Taq polymerase, 0.5 µM arbitrary primer (AP-1 ~ AP-10), 0.5 µM of the same GT15MN oligo(dT) primer used for reverse transcription, dNTP (2 µM), and [alpha -35S]dCTP (10 µCi). PCR reactions were carried out for one cycle of 3 min at 94 °C, 5 min at 40 °C, and 3 min at 72 °C followed by 40 cycles of 30 s at 94 °C, 2 min at 40 °C, and 1 min at 72 °C (15 min for the last cycle). The amplified cDNAs were separated on 6% denaturing sequence gels followed by autoradiography. cDNA bands specifically amplified in cold-adapted Tetrahymena cells were excised from the gels and eluted by boiling. The eluted DNA was reamplified by PCR using the same set of primers and its nucleotide sequence was determined by the dideoxy nucleotide termination method (25) using Sequenase. Homology search was performed using the BLAST algorithm (26) at the National Center for Biotechnology Information (NCBI), National Library of Medicine (NLM), National Institute of Health (Bethesda, MD), and DNA analysis program DNASIS.

Screening of cDNA Library and DNA Sequencing-- Amplified cDNA fragments were cloned into the pT7Blue T-vector. One of the cDNA fragments tentatively designated number 23 was subjected to further analysis. T. thermophila and T. pyriformis lambda gt10 cDNA libraries (21, 22) were screened by plaque hybridization using a 32P-labeled oligonucleotide probe. The cDNA inserts of positive phage clones were digested with EcoRI and then subcloned into the EcoRI site of pBluscriptII plasmid. Size-fractionated unidirectional deletion of the insert was performed using exonulcease III and mung bean nuclease (7). Amino acid alignment was performed using the computer program ClustalX (27). A phylogenic tree was constructed by the neighbor-joining method (28). Results of the molecular phylogenic analysis were analyzed by the PHYLIP software package (29).

Southern and Northern Blot Analyses-- Genomic DNA was prepared according to the method described by Gall (30). Briefly, DNA was prepared in 10 mM Tris-HCl, pH 9.5, 0.5 M EDTA, 1% SDS at 55 °C for 1 h, and subjected to phenol extraction and ethanol precipitation. About 10 µg of DNA was digested with restriction endonucleases (BamHI, EcoRI, HindIII, KpnI, PstI, SalI, XbaI, and XhoI) and the resultant fragments were separated using a 0.7% agarose gel. DNA fragments were transferred to GeneScreen Plus membrane and fixed by baking at 80 °C for 2 h. The membrane was then probed with [alpha -32P]dCTP-labeled probe generated from the cloned DNA. The blots were then visualized by exposure of Kodak X-Omat film. For Northern blotting, 20 µg of total RNA was subjected to electrophoresis on a 1.2% formaldehyde-agarose gel and transferred to GeneScreen Plus filters. Northern hybridization was performed as follows. After prehybridization for 4 h at 42 °C in hybridization buffer containing 50% formamide, 1% SDS, 1 M NaCl, 50 mM Tris-HCl, pH 7.0, 0.1 mg/ml denatured salmon sperm DNA, Denhardt's solution (0.1% bovine serum albumin, 0.1% Ficoll 400, and 0.1% polyvinylpyrrolidone), filters were incubated with 32P-labeled probe for 16 h at 42 °C. Probe was made by random primer labeling using the Multiprime DNA labeling system according to the manufacture's protocol. Filters were washed twice in 2 × SSC (0.3 M NaCl, 0.03 M trisodium citrate, pH 7.0) containing 0.1% SDS at 45 °C for 30 min and in 0.2 × SSC containing 0.1% SDS at 50 °C for 20 min, and exposed to Kodak O-Xmat AR film. The density of each band was measured by a densitometer (Atto, Densitograph, Tokyo, Japan).

Plasmids-- In order to express T. pyriformis MRK in Escherichia coli or Saccharomyces cerevisiae, the codons TAA and TAG, which are recognized as glutamine in Tetrahymena (21, 31, 32), were first mutated to the universal glutamine codons CAA and CAG, respectively, by in vitro mutagenesis as reported (33). Substitution of Thr226 by Ala was also performed by in vitro mutagenesis. The nucleotide sequence was confirmed by sequencing with the dideoxy nucleotide termination method. The T. pyriformis MRK cDNAs were subcloned into the pGEX-4T-1 vector to generate glutathione S-transferase (GST)-conjugated proteins in E. coli. For experiments using S. cerevisiae, cDNAs were subcloned into the yeast expression vector pNV7s (34).

Kinase Assay-- The peptide RLRELFIEEIKRYH, corresponding to the COOH-terminal peptide of MRK was synthesized and conjugated with keyhole limpet hemocyanin. Polyclonal antiserum was raised in rabbits and affinity purified against the peptide conjugated to Sepharose 6B. Tetrahymena proteins were extracted in buffer containing 25 mM Hepes, pH 7.4, 1% Triton X-100, 50 mM NaCl, 5 mM EDTA, 10 mM sodium fluoride, 1 mM Na3VO4, 0.2 mM Na2MoO4, 10 mM beta -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and centrifuged at 12,000 × g for 15 min at 4 °C. The resulting supernatant (100 µg/400 µl) was mixed with 5 µg of the purified antibody and incubated for 4 h at 4 °C. 25 µl of protein A-Sepharose was added to the tubes and incubation was continued for another 1 h. The precipitate was washed three times in 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5 M NaCl, 0.1% Triton X-100, and twice in 20 mM Hepes, pH 7.4, 10 mM MgCl2, 5 mM EGTA. Kinase reactions were performed in 20 µl of a buffer consisting of 20 mM Hepes, pH 7.4, 10 mM MgCl2, 2 mM EGTA, 1 mM dithiothreitol, 100 µM ATP, 0.5 mg/ml MBP, and 2 µCi of [gamma -32P]ATP. After incubation for 5 min at 30 °C, the reaction was stopped by the addition of Laemmli's sample buffer. Phosphorylated MBP was resolved by SDS-PAGE and detected by autoradiography. The bands on the autoradiogram were quantified by an image analyzer (Atto Densitograph, Atto, Tokyo). The wild type (Thr226) and mutant (Ala226) recombinant MRKs were generated as GST fusions in E. coli NM522 by incubating with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h. The expressed proteins were purified as described above or with glutathione-Sepharose, and essentially the same results were obtained with both purification methods. The activities of GST-conjugated MRKs were measured with MBP as a substrate, as described above.

Western Blot Analysis-- Tetrahymena proteins were extracted as described above. Extracted proteins were separated by SDS-PAGE on 9% polyacrylamide gels and electrophoretically transferred onto Immobilon-P membranes. Blocking was performed in Tris-buffered saline containing 5% skimmed milk powder and 0.05% Tween 20. The membranes were probed with antibodies against the COOH-terminal peptide of MRK. Detection was performed with the ECL system. Protein content was determined by the BCA protein assay using BSA as a standard.

Complementation of Yeast hog1Delta Mutant-- S. cerevisiae hog1Delta mutant strain TM232 and a plasmid pJB30 containing the HOG1 gene were described elsewhere (12, 35). Yeasts were transformed with plasmids and transformants were selected as described previously (36). The transformants were tested for growth in the presence of 1 M sorbitol at 30 °C for 2 days.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Isolation of MRK cDNA by Differential Display-- RNA was extracted from T. thermophila cells grown at 35 °C and cells harvested at 1 h after shifting to 15 °C. These RNA samples were subjected to differential display analysis using 10 arbitrary primers (AP-1 ~ AP-10) and two anchored primers (GT15MG and GT15MA). Approximately 1,500 bands ranging from 100 to 400 base pairs in size were successfully amplified. Most of the bands did not display significant changes in intensity between the cells cultured at 35 °C and 15 °C, but several bands did show differences (data not shown). Differential expression of these latter mRNAs was confirmed by Northern blot analysis using the cDNA fragments as probes. A cDNA fragment (number 23, 348 base pairs) amplified with GT15MA and AP-7 recognized about 1.7 kilobase pairs mRNA which was markedly expressed in cells shifted to 15 °C. Using this cDNA fragment as a probe, a T. thermophila lambda gt10 cDNA library (21, 22) was screened and the full-length cDNA was isolated. Its counterpart in T. pyriformis was also obtained by screening a T. pyriformis lambda gt10 cDNA library (21, 22) with the same probe. The entire nucleotide and deduced amino acid sequences are shown in Fig. 1. An open reading frame encodes a protein of 430 amino acids, with a predicted molecular mass of 49 kDa in both T. thermophila and T. pyriformis. Their deduced amino acid sequences showed 90% identity and contained 11 conserved amino acid domains (I-XI) characteristic of protein kinases (37). Comparison of the amino acid sequences with the NIH Protein Bank data base using blastx search (26) revealed that they were similar to MAPKs (Fig. 2). The deduced amino acid sequence of T. thermophila gene showed 40% sequence identity with human ERK1 (11), 39% with Arabidopsis thaliana ATMPK3 (17), 39% with Nicotinia tabacum NTF3 (18), 38% with yeast HOG1 (12), and 38% with human p38 (13). It has been reported that the amino acid sequences of MAPKs are divergent between subdomains VII and VIII. Subdomains VII and VIII are separated by a gap of 6 amino acids in p38 and HOG1. However, this gap is 8 amino acids in JNK and 12 amino acids in ERK. In Tetrahymena, this gap covers 57 amino acids. In addition, the Tetrahymena kinase has another gap between subdomains IX and X. Whereas most of the MAPKs consist of a total of 360 ~ 380 amino acids (11-20), these additional amino acids between VII and VIII, and IX and X make the Tetrahymena proteins larger than most of the MAPKs. A phylogenic tree, constructed by the neighbor-joining method, showed that Tetrahymena kinase is more closely related to the stress-activated MAPKs such as p38 and JNK/SAPK than to the ERKs (Fig. 3). Members of the MAPK family possess three conserved residues Thr-X-Tyr in the subdomain VIII and are enzymatically activated by phosphorylation of the Thr and Tyr residues therein (38, 39). However, in Tetrahymena Thr is conserved but Tyr is replaced by His. The partial sequence of a gene described as a "Plasmodium falciparum MAPK-like protein" found in the NIH Protein Bank data base (GenBank accession number X98689) also has a His at the corresponding position (Thr-Ser-His). Based on these sequence comparisons we designate this Tetrahymena kinase as a MRK. Southern blot hybridization was performed in order to ascertain whether only a single homolog of this MRK is present in Tetrahymena. Genomic DNA (10 µg) from T. pyriformis was digested with restriction endonucleases and separated by agarose gel electrophoresis. After transfer to nylon membrane, the membrane was probed with the [alpha -32P]dCTP-labeled starting DNA clone (number 23, as above). A single band was recognized in each lane under high-stringency conditions (data not shown).


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Fig. 1.   Restriction maps and nucleotide sequences and predicted amino acid sequences of Tetrahymena MRKs. A and B, T. thermophila. C and D, T. pyriformis. A and C, the coding region is indicated by an open box. B and D, nucleotides are numbered in the 5' to 3' direction and amino acids are shown in single-letter code below the nucleotide sequence. The in-frame termination codon is marked by an asterisk.


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Fig. 2.   Sequence comparison of 11 conserved kinase domains of the two Tetrahymena MRK and other MAPK family kinases. Amino acids were aligned and gaps were introduced to maximize homology using the computer program ClustalX (27). Roman numerals indicate the 11 conserved subdomains of protein kinases according to Hanks and Quinn (37). The single fully conserved and highly conserved amino acid residues are indicated by asterisks and double dots, respectively. S. cerevisiae HOG1 (GenBank accession number, L06279), A. thaliana ATMPK3 (D21839), N. tabacum NFT3 (X69971), human ERK1 (Z11694), human p38 (AF015256), human JNK1 (A53063), and human SAPK4 (Y10488).


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Fig. 3.   Phylogenic tree analysis of MAPK-related kinases. A phylogenic tree was constructed by the neighbor-joining method (28). The results were analyzed by the PHYLIP package (29). The following kinases were included in the analysis: S. cerevisiae HOG1 (GenBank accession number, L06279), FUS3 (M31132), A. thaliana ATMPK3 (D21839), N. tabacum NFT3 (X69971), Dictyostelium discoideum (U11077), T. brucei (Z54341), Xenopus laevis (X59813), human ERK1 (Z11694), human ERK2 (Z11695), human JNK1 (A53063), human p38 (AF015256), rat SAPK 3 (S68680), and human SAPK4 (Y10488).

Kinase Activity of the T. pyriformis MRK-- In order to assess kinase activity, T. pyriformis MRK cDNA was subcloned into the prokaryotic expression vector pGEX-4T-1 and GST-conjugated protein was expressed in E. coli NM522. In unicellular ciliates including Tetrahymena, TAA and TAG codons are translated as glutamine (21, 31, 32). However, these codons are universal stop codons in E. coli and most other organisms, and thus they were converted to CAA and CAG, respectively, for expression in NM522. In vitro kinase assay revealed that the protein phosphorylated MBP and became autophosphorylated (Fig. 4). Activation of MAPK requires phosphorylation of a tyrosine and a threonine residue (38, 39). These sites lie in the phosphorylation lip between subdomains VII and VIII of the protein kinase. In Tetrahymena MRK the residue corresponding to Thr is Thr226, while that corresponding to Tyr is His228. To test whether Thr226 is important for kinase activity, we generated a mutation leading to an Ala substitution for Thr226 (AGH). Western blot analysis revealed that wild-type (TGH) and mutated (AGH) proteins conjugated with GST were almost equally expressed in E. coli NM522 (Fig. 4). The wild-type protein showed slower mobility on SDS-PAGE compared with the mutant protein. The mutant protein was no longer able to phosphorylate MBP and lost its ability to autophosphorylate (Fig. 4). These results suggest that this Thr residue may be a key factor in the activity of Tetrahymena MRK.


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Fig. 4.   Kinase activity of the GST/MRK fusion expressed in E. coli. GST-conjugated proteins were expressed in the E. coli strain NM 522. A, autoradiogram of in vitro kinase assays. Expressed proteins were purified by immunoprecipitation (from starting E. coli extracts containing 100 µg of proteins). Kinase activity was measured as described under "Experimental Procedures." Typical results from three separate experiments are shown. The positions of molecular mass markers in kDa are shown on the left. B, the protein levels of the recombinant kinases were analyzed by Western blotting with anti-MRK antibody. Essentially the same results were obtained with anti-GST antibody. 25 µg of E. coli protein extracts were separated by SDS-PAGE on 9% polyacrylamide gels and electrophoretically transferred onto Immobilon-P membrane. Right, TGH, wild-type (Thr226 and His228); left, AGH, mutant (Ala226).

Expression of MRK mRNA upon Temperature Downshift-- Expression of MRK upon temperature downshift was examined by Northern blot analysis. A single band of 1.7 kilobase pairs was recognized by hybridization with the 32P-labeled MRK cDNA BglII fragment (544-871) as a probe. Although the amount of mRNA detected in cells grown at 35 °C was low, cold stress increased mRNA expression of the kinase and this increased expression was detectable immediately after downshift from 35 to 15 °C (time 0 in Fig. 5). The maximal level was obtained 1 h after the temperature shift and was maintained for at least 2 h.


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Fig. 5.   Northern blotting analysis of MRK mRNA following temperature downshift. T. thermophila cells were grown at 35 °C to the logarithmic phase and cooled from 35 to 15 °C over 25 min (0.8 °C/min). After downshift to 15 °C, total RNA was extracted at the indicated time points by the guanidine thiocyanate method as described under "Experimental Procedures." 20 µg of total RNA was separated by electrophoresis on 1.2% formaldehyde-agarose gel and transferred to GeneScreen Plus membranes. The membranes were hybridized with the 32P-labeled MRK cDNA BglII fragment (544-871) as a probe. Bands were detected by autoradiography. A, typical autoradiogram from three experiments. B, the densities of the bands were monitored with a densitometer (Atto Densitograph, Tokyo, Japan). The results shown (-fold increases from the control value) are mean ± S.D. from three separate experiments.

Expression of MRK mRNA upon Osmotic Stress-- JNK/SAPK-type MAPKs are known to be activated in response to environmental stresses, such as temperature change and osmotic stress. In the plant A. thaliana, expression of ATMPK3 mRNA (A. thaliana MAPK) increases in response to cold stress as well as to stress caused by high salinity (40). Therefore, expression of T. pyriformis MRK by osmotic stress was examined. As expected, its mRNA increased when cells were exposed either to NaCl (25-100 mM) or sorbitol (100-200 mM) (Fig. 6). Increases in mRNA expression were detectable at 1 h when cells were incubated with 25 mM NaCl or 100 mM sorbitol. At 100 mM NaCl the increase in mRNA was seen at 30 min (data not shown).


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Fig. 6.   Expression of MRK gene in response to osmotic stress. T. pyriformis cells were incubated in control medium (a) or medium containing 100 mM sorbitol (b), 200 mM sorbitol (c), 25 mM NaCl (d), 50 mM NaCl (e), or 100 mM NaCl (f) for 1 h. The expression level of MRK mRNA was examined by Northern blotting as described in the legend to Fig. 5. The membranes were hybridized with the 32P-labeled MRK cDNA BglII fragment (569-895) as a probe. A, typical autoradiogram from three experiments. B, the densities of the bands were monitored with a densitometer (Atto Densitograph, Tokyo, Japan). The results shown (-fold increases from the control value) are mean ± S.D. from three separate experiments.

Activation of MRK upon Osmotic Stress-- Changes in MRK activity were measured by immune complex kinase assay. A polyclonal rabbit antibody against the COOH-terminal 14 amino acids (RLRELFIEEIKRYH) of MRK was obtained. Western blot analysis showed that the antibody, but not preimmune serum, recognized a protein of about 50 kDa in T. pyriformis (data not shown). The antibody was also found to immunoprecipitate kinase activity directed against MBP as a substrate. Activation of MRK by osmotic stress was detectable within 15 min and lasted for at least 60 min. After 30 min exposure to 50 mM NaCl or 100 mM sorbitol, MBP phosphorylation increased 2.5- or 1.8-fold, respectively, over baseline (Fig. 7). Western blotting after 60 min detected no increase in the amount of MRK (Fig. 7), indicating post-translational activation of the kinase.


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Fig. 7.   Activation of MRK in response to osmotic stress. T. pyriformis cells were incubated in control medium or medium containing 100 mM sorbitol or 50 mM NaCl for the indicated periods of time. A, the kinase activity in immunoprecipitate with antibody against MRK was determined with MBP as a substrate. Phosphorylated MBP was resolved by SDS-PAGE and detected by autoradiography. B, the protein levels were analyzed by Western blotting. The results shown are the typical data from three separate experiments.

Failure to Complement Yeast hog1Delta Mutant by MRK-- The amino acid sequence similarity to stress-activated MAP kinase families and its activation by osmotic stress strongly suggest that MRK functions as a stress-activated kinase in Tetrahymena. Therefore, we introduced a cDNA encoding T. pyriformis MRK into the yeast expression vector pNV7s to examine whether it was able to complement the S. cerevisiae hog1Delta mutation. When tested on medium containing 1 M sorbitol, the parental hog1Delta strain TM232 (35) failed to grow, while TM232 expressing a plasmid-borne HOG1 cDNA (pJB30) did grow. However, growth of this hog1Delta strain was not rescued by expression of T. pyriformis MRK (data not shown). Moreover, the p38-type mutant kinase (TGY) was unable to complement TM232 in this assay (data not shown).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

MAPKs are serine/threonine kinases that are activated by extracellular stimuli such as growth factors and differentiation factors (for reviews, see Refs. 38, 39, 41-43). They are thought to play key roles in integrating multiple intracellular signals transmitted by various second messengers. MAP kinases are unique in that they become activated when both Thr and Tyr residues are phosphorylated. Members of the MAPK superfamily are thought to be important signal transducing molecules that transmit signals from the cell surface to the nucleus. In addition to classical MAPKs (also called to as ERKs), recent studies have identified two other members of the MAPK superfamily: p38 (also known as CSBP/RK/MPK2) (13, 44) and SAPK (also referred to as c-Jun NH2-terminal kinase JNK) (14, 45). p38 and SAPK are activated by inflammatory cytokines and cellular stresses such as ultraviolet light and high osmolarity (14, 45-47). The phosphorylation cascades made up of MAPKK kinase, MAPK kinase, and MAP kinase are conserved among various signal transduction pathways from yeast to vertebrates (38, 39, 41-43). However, very little information has been obtained regarding these kinases in protozoa. Tetrahymena is a useful animal cell model system for studying the molecular mechanism by which organisms adapt to changes in their environments. Tetrahymena possess the ability to adapt to fluctuations in temperature, pH, amounts of nutrients, and concentration of dissolved gases by inducing physiological, morphological and/or biochemical changes (1, 2). For example, Tetrahymena cells exhibit marked changes in membrane lipid composition when exposed to altered growth temperatures. Thus, this cell has proved to be a potentially suitable model system for studying the molecular mechanisms by which the thermo-adaptive control of membrane lipids is achieved (3, 4). We have previously demonstrated that the gene encoding Delta 9 fatty acid desaturase, which is essential for membrane homeoviscous adaptation, is induced by temperature downshift in Tetrahymena (7).

In the present study we have successfully cloned a putative protein kinase similar to MAPKs from Tetrahymena. This kinase shows nearly 40% amino acid identity to other members of the MAPK family. As discussed above, in yeast and mammalian cells, three types of MAPKs have been identified and they are differentially activated by different kinds of signals (40-45). In the higher plant species A. thaliana, nine MAPK genes have been identified and their functions extensively characterized (48). However, there has been very limited information about MAPKs in protozoa. Molecular cloning of MAPK-like proteins from Trypanosoma brucei (GenBank accession number Z54341) and P. falciparum (X82646 and X98689) have been reported. The P. falciparum MAPK-like kinase (X82646, 826 amino acids) has a long carboxyl-terminal tail consisting of nearly 460 amino acids (49). Yeast HOG1 (435 amino acids) also has an additional carboxyl terminus consisting of about 70 amino acids (12). Due to these long COOH-terminal tails, these two MAPKs are larger than most of the MAPKs, which are typically made up of 360-380 amino acids. The deduced amino acid sequences of the cloned Tetrahymena genes contain additional amino acids inserted between the kinase subdomains VII and VIII, and between IX and X. These inserts make the Tetrahymena kinase larger than most of MAPKs, although the sizes of their COOH-terminal tails are almost the same as those of other members of the MAPK family except HOG1 and P. falciparum MAPK-like kinase.

This MRK gene was initially isolated using the differential display technique to compare mRNA expression from control and cold-stressed Tetrahymena cells. The kinase mRNA increases in response to low-temperature shift as well as osmotic stress. It has also been observed in the plant A. thaliana that the ATMPK3 mRNA is transcriptionally up-regulated by cold and osmotic stresses (40). Thus, it was conceivable that this MRK in Tetrahymena cells would also be involved in the stress-signaling pathway. As shown previously by us (7), the Delta 9 desaturase mRNA is induced in response to a temperature down-shift from 35 to 15 °C and peaks immediately thereafter. By contrast, peak induction of the MRK mRNA was observed 1 h after the temperature shift. This finding led us to speculate that the initial response to physical stress is associated with a phosphorylation cascade including MRK in Tetrahymena cells. Indeed, the activity of this kinase increased in response to osmotic stress.

In addition to the long insertions between the kinase subdomains, the Tetrahymena MRK is distinct in that whereas most MAPKs have a Tyr residue at Thr-X-Tyr, phosphorylation of which is prerequisite for activation, this is replaced by His (Thr-Gly-His) in the Tetrahymena kinase. A recombinant GST fusion to the Tetrahymena MRK expressed in E. coli was shown to phosphorylate MBP and itself. This is not surprising given that recombinant p38 expressed in E. coli was reported to phosphorylate MBP, Ikappa Balpha , and itself (13). However, Tetrahymena MRK activity was lost when Thr226 was replaced by Ala, suggesting that the Thr226 residue is essential for kinase activity. Alternatively, Thr226 may be an autophosphorylation site and its phosphorylation may result in activation of MRK. A similar finding was recently reported for Nlk (50), a murine homologue of Drosophila Nemo MAPK-related protein (51). The amino acid sequence of this kinase is homologous to the MAPKs but Nlk contains the sequence Thr-Gln-Glu in place of Thr-X-Tyr. Mutation of the Thr residue to Val, Asp, or Glu abolishes its kinase activity. The partial sequence of a P. falciparum MAPK-like protein found in the Genbank data base (accession number X98689) also has a His at the corresponding position (Thr-Ser-His), suggesting that this protozoan has a kinase(s) of this type. In prokaryotes there is a histidine kinase autophosphorylation system, known as the two-component system (His-Asp phospho-transfer mechanism), which is widely distributed (52). Recently, the HOG1 pathway, one of the MAPK cascades in yeast that mediates the response to osmotic stress, appears to be initiated by a similar two-component osmosensing system, namely the SLN1-YPD1-SSK1 pathway (53). A similar two-component signal transduction pathway that mediates the ethylene response is also found in higher plants (18). Therefore, it is tempting to speculate that His228 may be involved in the regulation of Tetrahymena MRK, although further work is necessary to confirm this. It would also be of great interest to elucidate the mechanism by which Tetrahymena MRK is activated by identifying upstream regulator(s).

Tetrahymena MRK failed to complement the S. cerevisiae hog1Delta mutant, presumably because it does not have the necessary conserved Tyr residue within the 3-residue phosphorylation site. In contrast, the hog1Delta mutant is complemented by mammalian p38 (13) or JNK1 (46, 54). When the conserved Thr183 and Tyr185 phosphorylation sites of JNK1 were replaced with Ala and Phe, respectively, it no longer complemented the hog1Delta mutant for growth under high osmolarity conditions (46), consistent with the idea that these conserved amino acids are required for activation by the upstream activator MAPK kinase PBS2. The Tetrahymena MRK His228 right-arrow Tyr mutant, which creates a p38-type sequence, also failed to rescue the hog1Delta mutant. Recent studies indicate that scaffold proteins that create multienzyme complexes determine the specificity of activation and function of MAP kinases (reviewed in Ref. 55). In the HOG1 pathway, PBS2 functions as a scaffold protein and forms a multiprotein complex that includes HOG1 (56). This suggests that the Tetrahymena MRK may not bind to PBS2. Another possibility is that His228 is essential for Tetrahymena MRK activity, as discussed above.

In summary, we have cloned a MRK gene from the unicellular eukaryotic protozoan Tetrahymena. Sequence analysis shows it is closely related to stress-responsive MAPKs found in other cell types. In fact, its mRNA level was up-regulated in response to cold and osmotic stresses. Moreover, the activity of the kinase was increased in response to osmotic stresses. Therefore, it is conceivable that this gene is involved in stress signal transduction and that there exists a stress-responsive protein phosphorylation cascade in protozoa. However, Tetrahymena MRK failed to complement S. cerevisiae hog1Delta mutant. This is probably due to the fact that the consensus Thr-X-Tyr phosphorylation site found in most MAPKs is replaced by Thr-Gly-His in Tetrahymena MRK. In order to elucidate the stress-signaling pathway in Tetrahymena, the mechanism by which this MRK is activated is currently under study.

    ACKNOWLEDGEMENTS

We are grateful to Dr. E. Nishida (Kyoto University) and Dr. H. Fukushi (Gifu University) for helpful discussions, and E. Matsukuma, I. Katayama, and S. Ito for assistance. We also thank Dr. O. Numata (Tsukuba University) for the gift of the Tetrahymena lambda gt10 libraries.

    FOOTNOTES

* This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture of Japan and the Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB008977 and AB008979.

§ To whom all correspondence should be addressed: Dept. of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan. Tel.: 81-58-267-2230; Fax: 81-58-265-9002; E-mail: bio{at}cc.gifu-u.ac.jp.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MRK, MAPK-related kinase; SAPK, stress-activated protein kinase; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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