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INTRODUCTION |
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
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.
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EXPERIMENTAL PROCEDURES |
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
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 [
-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
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 [
-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
-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 [
-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-
-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 hog1
Mutant--
S. cerevisiae
hog1
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.
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RESULTS |
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
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
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
[
-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).
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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).
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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.
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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.
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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.
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Failure to Complement Yeast hog1
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 hog1
mutation. When tested
on medium containing 1 M sorbitol, the parental
hog1
strain TM232 (35) failed to grow, while TM232
expressing a plasmid-borne HOG1 cDNA (pJB30) did grow. However,
growth of this hog1
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).
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DISCUSSION |
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
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
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, I
B
, 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 hog1
mutant, presumably because it does
not have the necessary conserved Tyr residue within the 3-residue
phosphorylation site. In contrast, the hog1
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 hog1
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
Tyr mutant,
which creates a p38-type sequence, also failed to rescue the
hog1
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
hog1
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.