From the Laboratory of Immunology, Medical Research
Center, College of Medicine, Yonsei University, CPO Box 8044, Seoul,
South Korea, the § Department of Biochemistry and Molecular
Biology, College of Medicine, Yonsei University, CPO Box 8044, Seoul,
South Korea, and the ¶ Laboratoire de Biochimie et Biologie
Moléculaire des Insectes, Institut Pasteur, 75724, Paris Cedex
15, Paris, France
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ABSTRACT |
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A mitogen-activated protein kinase (MAPK) has
been cloned and sequenced from a Drosophila neoplasmic
l(2)mbn cell line. The cDNA sequence analysis showed
that this Drosophila kinase is a homologue of mammalian p38
MAPK and the yeast HOG1 gene and thus was referred to as
Dp38. A distinguishing feature of all MAPKs is the conserved sequence
TGY in the activation domain. Dp38 was rapidly tyrosine
186-phosphorylated in response to osmotic stress, heat shock, serum
starvation, and H2O2 in Drosophila
l(2)mbn and Schneider cell lines. However, unlike mammalian p38
MAPK, the addition of lipopolysaccharide (LPS) did not significantly
affect the phosphorylation of Dp38 in the LPS-responsive
l(2)mbn cell line. Following osmotic stress, tyrosine
186-phosphorylated forms of Dp38 MAPK were detected exclusively in
nuclear regions of Schneider cells. Yeast complementation studies
demonstrated that the Saccharomyces cerevisiae HOG1 mutant
strain JBY10 (hog1-1) was functionally complemented by
Dp38 cDNA in hyperosmolar medium. These findings demonstrate that
similar osmotic stress-responsive signal transduction pathways are
conserved in yeast, Drosophila, and mammalian cells, whereas LPS signal transduction pathways appear to be different.
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INTRODUCTION |
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Various forms of cellular stress are known to activate a distinct subfamily of mammalian mitogen-activated protein kinases (MAPKs),1 termed p38 MAPK/reactivating kinase (RK) (1, 2). This kinase was found to be most homologous to the Saccharomyces cerevisiae HOG1 kinase that is involved in host protection from hyperosmotic stress (3). In mammals, p38 MAPK is activated by osmotic stress, inflammatory cytokines, LPS, and UV irradiation (4). Once activated, p38 is known to phosphorylate mitogen-activated protein kinase-activated protein kinase-2, which in turn phosphorylates a small heat shock protein (2, 5). Other substrates such as activating transcription factor-2 and Max have also been identified as targets of p38 kinase (4, 6). Furthermore, p38 MAPK was also discovered as an intracellular target for molecules that act as cytokine-suppressive anti-inflammatory drugs (7). The activation of p38 MAPK requires phosphorylation of both threonine and tyrosine residues in subdomain VIII of the activation domain. The p38/HOG1 subfamily enzymes are distinguished by the dual phosphorylation site motif (TGY) in the activation domain (1), which differs from TPY for c-Jun amino-terminal kinase (JNK) (8) and TEY for growth factor-activated MAPK, termed extracellular signal-regulated kinase (ERK) (9). JNK is activated in response to stimuli similar to those for p38 MAPK and phosphorylates a c-Jun NH2-terminal domain. The third subgroup of the MAPKs is ERK, which is activated by epidermal growth factor and nerve growth factor. The ERK subgroup plays a central roles in growth and differentiation (9, 10). Several upstream kinases that are responsible for MAPK activation have been recently characterized and were shown to have specificity for the these three subfamilies of MAPKs. MKK1 and MKK2 activate ERK; MKK3, MKK4, and MKK6 activate p38; and MKK4 activates JNK (11-14).
In Drosophila, ERK and JNK have been cloned, and their functional roles in development have been well studied (15-23). Drosophila ERK-A, encoded by rolled was found to mediate receptor kinase signal transduction pathways including torso, DER, and sevenless pathways (16, 17). Drosophila JNK was shown to participate in dorsal closure and LPS signal transduction (18-23). A partial deduced amino acid sequence of a Drosophila p38 MAPK from a PCR product has been previously reported by Biggs and Zipursky (15). However, no information is available with regard to Drosophila p38 cDNA(s), and its function in Drosophila cell physiology and development remains to be elucidated. In this work, we describe the full cDNA of a Drosophila homologue of p38 MAPK and certain functional roles in Drosophila cells.
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EXPERIMENTAL PROCEDURES |
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Materials-- Reagents were purchased from following sources: Thermus aquaticus polymerase (Takara); Mega prime DNA labeling system (Amersham); Cy5 Autocycle sequencing kit and Cy5 Autoread sequencing kit (Pharmacia Biotech Inc.); Immobilon-P membrane (Millipore Corp.); Nylon filter (Amersham Hybond-N+); PCR machine (Perkin-Elmer); A.L.F. express DNA sequencer (Pharmacia); enhanced chemiluminescence (ECL) reagent (Amersham Corp.); phosphospecific p38 MAPK antibody (New England Biolabs). Water was of Milli Q grade. All other chemicals were of the highest commercial grade.
Insect Cell Cultures-- Gateff's Drosophila l(2)mbn hemocyte cell line (24) and Schneider cell line (25) were grown at 23 °C in Corning flasks in Schneider medium (Life Technologies, Inc.) supplemented with 5 or 10% fetal bovine serum (Biowhittaker) heat-inactivated at 56 °C for 30 min.
PCR Amplification of Dp38 MAPK--
Except when specially
mentioned, all DNA and RNA manipulations were carried out using
standard techniques (26). Two pairs of degenerate oligonucleotide
primers were synthesized, sense primer-1 (SP-1; 5-GTN GCN ATH AAR AAR
YT-3
) and sense primer-2 (SP-2; 5
-GTN GCN ATH AAR AAR AT-3
), which
included all possible codons specifying conserved subdomain II
sequences VAIKKL and VAIKKI, respectively. Antisense primers were
synthesized; antisense-1 (AP-1; 5
-GCN-ACR-TAN-CCN-GTC-AT-3
) and
antisense-2 (AP-2; 5
-GCN ACR-TAN CCN GTN AR-3
) correspond to the
complementary sequences MTGYVA and LTGYVA, respectively, both of which
contain the conserved phosphorylation sites located in subdomain VIII.
An aliquot of the Drosophila l(2)mbn cDNA library (2.5 µl) was amplified with 1 µM of each primer in pairs
(four combinations: SP-1/AP-1, SP-2/AP-1, SP-1/AP-2, and SP-2/AP-2)
with the following thermal cycle profile: 94 °C for 3 min; 35 cycles
of 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 1 min. The amplified mixtures were separated on 8% polyacrylamide gels,
and each band was purified and cloned into pBlueScript SK(
).
Screening of the Drosophila l(2)mbn cDNA Library--
A
Drosophila cDNA library in Zap II (Stratagene) was
made as described (27) and kindly provided by Dr. Dan Hultmark
(University of Stockholm, Sweden). A full-length Dp38 cDNA was
isolated from ~ 2.5 × 105 independent clones
previously transferred onto Hybond N+ membranes (Amersham) using a
specific 420-bp PCR product as a probe. The 420-bp PCR product was
random-primed using fluorescein-11-dUTP. The membranes were
prehybridized at 42 °C in a solution containing 5 × SSC,
5 × Denhardt's solution, 0.1% SDS, 5% dextran sulfate at
60 °C for 2 h. Hybridization was carried out overnight at
60 °C in this solution containing fluorescein-11-dUTP-labeled probe, washed, and detected using anti-fluorescein-horseradish
peroxidase-conjugated antibody according to the manufacturer's
instructions (Amersham). Positive plaques were purified by two rounds
of screening, and the cDNA inserts were subcloned into pBlueScript
SK(
) plasmid by in vivo excision from the recombinant
Zap II as described by Stratagene. The cDNA inserts were sequenced
across both strands using an A.L.F. express automatic sequencer
(Pharmacia).
Western Blot Analysis-- Insect cells (~106), exposed to various stress conditions, were solubilized in SDS-PAGE sample buffer. The total cellular proteins (100 µg/lane) were separated by SDS-PAGE (28) and transferred onto polyvinylidene difluoride membrane (29). The antibody used for this study was rabbit anti-human p38 phosphospecific antibody, purchased from New England Biolabs. This antibody specifically recognizes the phosphorylated active form of Tyr182 of human p38 MAPK and cross-reacts with Drosophila p38 and murine p38 MAPKs. According to the manufacturer's information, it does not cross-react with either inactive p38 or other MAPKs (JNK and ERK).
The anti-p38 phosphospecific antibody was diluted to 1:1000 and incubated with the membrane for 1 h at room temperature (25 °C). After washing, the blot was subsequently incubated for 1 h with goat anti-rabbit IgG conjugated to horseradish peroxidase diluted 1:3000. Detection was carried out using the ECL Western blot detection kit (Amersham).Northern Blot Analysis--
Total RNA from Drosophila
l(2)mbn cells and Schneider cells was extracted (30) and was
separated by using electrophoresis on a denaturating
formaldehyde-agarose gel. The RNA was capillary-transferred to a
Hybond-N membrane, baked at 80 °C for 2 h, and cross-linked by
UV irradiation. The membrane was prehybridized at 42 °C for 1 h
in 5 × SSC, 50% formamide, 0.1% SDS, 100 µg/ml of sonicated salmon sperm DNA. Hybridization was carried out in this solution containing [-32P]dCTP random-primed Dp38 cDNA
insert (106 cpm/ml). The blot was briefly washed at
50 °C in 2 × SSC, 0.1% SDS at room temperature and then
washed twice at 50 °C in 2 × SSC, 0.1% SDS for 20 min and
twice at 55 °C in 2 × SSC, 0.1% SDS for 5 min and
autoradiographed at
70 °C using intensifying screens.
Complementation of the hog1 Mutant by Drosophila p38 MAPK-- The S. cerevisiae hog1 mutant (JBY10) and HOG1 plasmid (pJB30; HOG1 URA3 CEN) as described by Brewter et al. (3) were kindly provided by Dr. C. Gustin (Rice University, Houston, TX). The yeast strain was transformed with Dp38-sense (Dp38-sense URA3 CEN), Dp38-antisense (Dp38-antisense URA3 CEN), or pJB30 (HOG1 URA3 CEN) plasmid as described previously (31). The structures of all plasmids were confirmed by DNA sequencing. The transformants were selected on synthetic complete (SC)-Ura medium (32), and osmotic cell growth was tested on a YPD plate containing 0.6 or 0.9 M NaCl (32).
Cell Growth and Preparation of Yeast Extracts--
Yeast
transformants were cultured 15 h in 5 ml of SC-Ura medium at
30 °C with vigorous shaking. The cultured cells were inoculated into
80 ml of fresh SC-Ura medium, and cells were grown further under the
same condition. When A600 reached 1.0, the cells
were harvested and resuspended into 200 ml of the same medium
containing 0.6 M NaCl. The cells were further incubated for
1.5 h at 30 °C with vigorous shaking. The cells were harvested
by centrifugation at 2,000 rpm for 10 min and were washed one time with
water. The cell pellets were stored at 70 °C for later use.
Transfection Experiment-- Epitope (6 × His) tag was added to the amino-terminal region of Dp38 by subcloning of Dp38 cDNA into His-tagged bacterial expression vector PQE-30 (Qiagen). The His-tagged Dp38 insert was subsequently subcloned into Drosophila transfection vector, pPacPL that contains the actin 5C promotor (34). The transfection vector pPacPL-luciferase was constructed to control transfection efficiency. Cells were transfected by using the calcium phosphate precipitation method (35). The cells were used 24 h post-transfection.
Immunoprecipitation and Kinase Assay--
Schneider cells
expressing His-tagged proteins were activated with 300 mM
NaCl for 0, 5, 15, and 60 min. The cells were washed twice in
Tris-buffered saline and solubilized in lysis buffer (20 mM
HEPES, pH 7.4, 2 mM EDTA, 50 mM
-glycerophosphate, 1 mM dithiothreitol, 1 mM
sodium orthovanadate, 1% Triton X-100, 10% glycerol, 25 µg/ml
aprotinin, and 40 µg/ml phenylmethylsulfonyl fluoride) on ice for 30 min and subsequently centrifuged at 15,000 × g for 15 min at 4 °C. The epitope-tagged protein kinases were immunoprecipitated by incubation for 1 h at 4 °C with the
monoclonal anti-His antibody (Qiagen) prebound to protein G-agarose for
15 min at room temperature. The immunoprecipitates were washed twice successively with decreasing concentrations of NETF buffer,
i.e. 6, 3, and 0% NETF (6, 3, and 0% Nonidet P-40,
respectively, in 100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, 50 mM NaF) and were
finally washed once with kinase assay buffer (25 mM HEPES, pH 7.4, 25 mM
-glycerophosphate, 25 mM
MgCl2, 2 mM dithiothreitol, 0.1 mM
sodium orthovanadate). The immunocomplexes were resuspended in 25 µl
of kinase buffer containing 20 µM ATP and 10 µg of
myelin basic protein, and the kinase assays were initiated by the
addition of 1 µl of [
-32P]ATP (3000 Ci/mmol). The
reactions were terminated after 30 min at 30 °C by the addition of
Laemmli sample buffer. The phosphorylation of the substrate proteins
was examined after SDS-PAGE by autoradiography and phosphor-imaging
(Fuji Bas 2500) analysis.
Immunocytochemistry-- Cells were grown in 24-well culture dishes at a density of 2 × 106 cells/ml and then were fixed for 30 min in 3% formaldehyde, washed, and blocked in a Tris-buffered saline solution containing 0.1% Triton X-100 and 5% normal goat antibody for 1 h at room temperature. Cells were incubated at 4 °C overnight in a Tris-buffered saline solution containing Triton X-100, 5% bovine serum albumin, and a 1:2000 dilution of p38 phosphospecific antibody. The cells were washed with Tris-buffered saline plus 0.1% Triton X-100 and incubated with biotinylated anti-rabbit antibody for 1 h. Detection was carried out with the ABC reagent kit using diaminobenzidine tetrahydrochloride according to the manufacturer's instructions. The cells were mounted in glycerol and examined with a Olympus BX microscope.
Computer Analysis for Sequence Homology-- The Fasta, TFASTA, WORDSEARCH, and BLAST algorithms were used to determine nucleic acid and protein sequence homology between Dp38 and other known proteins (36).
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RESULTS AND DISCUSSION |
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Cloning, Sequence Analysis, and Expression of Dp38 MAPK in
Drosophila Cells--
To isolate a Drosophila homologue of
p38 MAPK, primers were synthesized based on conserved subdomains II and
VIII of known MAPKs. One of the primer pairs (sense primer-1 and
antisense primer-1) gave a specific 420-bp PCR product that was
subsequently cloned, reamplified, and used to screen a Drosophila
l(2)mbn cDNA library. From approximately 2.5 × 105 clones, one positive plaque was obtained. This plaque
was purified, and the insert was excised into pBlueScript SK() and
sequenced. The insert of this clone contained a 5
-noncoding region and
an open reading frame of 1098 bp corresponding to 366 amino acid residues (Fig. 1). An untranslated
flanking sequence of 187 bp was present between the terminal codon and
the poly(A) site. The calculated molecular mass of the deduced protein
was 42,151 Da.
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Induction of Tyr186 Phosphorylation of Dp38-- To address stimulus specificity of Dp38 kinase, Tyr186 phosphorylation of Dp38 was examined using p38 Tyr186-specific antibody following various forms of extracellular stress. This antibody is specific for the Tyr186-phosphorylated "active" form of Dp38 and does not recognize unphosphorylated Dp38. Fig. 3A shows that Tyr186 of Dp38 was intensely phosphorylated in both Drosophila Schneider and l(2)mbn cells in response to osmotic shock, heat shock, and H2O2, whereas unstimulated cells contained only trace levels of Tyr186 phosphorylation. In addition, we also observed that when l(2)mbn cells were cultured at the recommended concentration of 5% fetal bovine serum (24), Dp38 (Tyr186) was constitutively and intensely phosphorylated; however, if the concentration of fetal bovine serum in the culture medium was increased to 10%, as in the present study, the phosphorylated state was not detectable without stimulus (Fig. 3B). We find this result very interesting, since this is the first time a p38 MAPK has been shown to be phosphorylated under conditions of serum starvation. This result suggests the involvement of Dp38 in a wide range of physiological stress conditions. It is important to note that up until now, almost all studies carried out with l(2)mbn cells cultured in Schneider medium were supplemented with 5% fetal bovine serum according to Gateff's instructions (24). The fact that Dp38 is constitutively activated when cells are cultured in 5% fetal bovine serum reflects their state of cellular stress. Hence, one should take into consideration this physiological state when conducting experiments on these cells.
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Tyr186 Phosphorylation Causes Increased Dp38 Enzymatic Activity-- To confirm that the Tyr186 phosphorylation status is directly proportional to the Dp38 MAPK activity, Dp38 kinase activity was assayed using myelin basic protein as a substrate. Schneider cells transfected with epitope (His)-tagged Dp38 were treated with 300 mM NaCl from 0 to 60 min and then immunoprecipitated with monoclonal anti-His antibody bound to protein G agarose. The kinase assay was performed as described under "Experimental Procedures." Fig. 4 shows that there is a good correlation between kinase activity and the Tyr186 phosphorylation status shown in Fig. 3D. While at time 0 cells simply showed background levels of activity, the osmotic stressed Dp38-transfected cells showed highest MAPK activity after 5 min, which then gradually decreased (Fig. 4). However, no kinase activity was detected after osmotic shock in the untransfected control cells (Fig. 4). These results demonstrate that Dp38 Tyr186 phosphorylation is necessary for kinase activity. Furthermore, this result is consistent with the observation that mammalian p38 with mutations in the dual phosphorylation motif (Ala-Gly-Phe instead of Thr-Gly-Tyr) could not be phosphorylated on Tyr and Thr after a stress stimulus and was catalytically inactive (4).
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Dp38 Partially Complemented the Function of HOG1 in HOG1-1
Mutant Cells--
The high sequence homology and similar activation
mechanism induced by hyperosmolarity between Dp38 and HOG1
led us to investigate whether Dp38 is a functional homologue of the
HOG1 gene. HOG1-
1 mutant yeasts were
transformed with HOG1, Dp38-sense, or Dp38-antisense constructs. The viability of transformed yeast cells was then tested on
medium containing 0.6 M NaCl and 0.9 M NaCl.
The results demonstrated that while Dp38-antisense-transformed cells
failed to grow on 0.6 M NaCl medium, whereas
HOG1 and Dp38-sense-transformed cells grew well (Fig.
5). However, Dp38-sense-transformed cells failed to grow on the medium containing 0.9 M NaCl (data
not shown). These results demonstrate that Dp38 partially complemented
the function of HOG1 in a hyperosmotic environment. Han
et al. (1) reported that murine p38 MAPK could complement
the HOG1-
1 mutant strain on 0.9 M medium with
lower efficacy than that of the HOG1 gene. To verify the
Tyr186 phosphorylation status of Dp38 under hyperosmotic
conditions, overnight cultures of Dp38-sense- and
Dp38-antisense-transformed yeast cells were harvested and treated with
medium containing 0.6 M NaCl for 1.5 h at 30 °C. An
aliquot of each lysate (Dp38-sense- and Dp38-antisense-transformed
cells) was subjected to Western blot analysis using Tyr186
phosphospecific antibody (Fig. 6). The
results demonstrate that Dp38-sense-transformed cells contain the
Tyr186-phosphorylated form of Dp38 in the presence of 0.6 M NaCl. However, no phosphorylation band was detected in
Dp38-antisense-transformed cells (Fig. 6). This result leads us to
believe that the Tyr186-phosphorylated active form of Dp38
in complemented cells is an important event for yeast cell survival
under hyperosmotic media.
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Subcellular Distribution of Tyr186-phosphorylated Dp38 MAPK-- The subcellular distribution of Tyr186-phosphorylated Dp38 MAPK was examined by immunocytochemistry. In the control experiment, no reliable staining was detected, which indicates that the level of the Tyr186-phosphorylated form of Dp38 is not significant (data not shown). However, very specific staining was detected in the nuclear region of hyperosmotically stimulated cells (Fig. 7). These data suggest that Dp38 was specifically Tyr186-phosphorylated in response to hyperosmotic stress and exclusively located in the nucleus.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Elisabeth Gateff (Johannes Gutenberg-Universität, Mainz, Germany) for providing the l(2)mbn cell line and to Dr. Dan Hultmark (University of Stockholm, Sweden) for providing the Drosophila l(2)mbn cDNA library. We also thank Dr. J.-K. Seong for assistance in immunocytochemistry, Y.-H. Kim for DNA sequencing analysis and Dr. C. Gustin (Rice University, Houston, TX) for providing the hog1 mutant and hog1 plasmid.
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FOOTNOTES |
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* This work has been supported by Genetic Engineering Research Grants GE 96-128 and GE 97-114 from the Korea Ministry of Education (to W.-J. L.) and partly by the Yonsei University College of Medicine Faculty Grant (1996), the Yonsei University Research Fund (1996), a grant from the Hallym Academy of Science (to K.-Y. C.), and grants from Institut Pasteur and the Pasteur-Weizmann Joint Research Program.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) U86867.
To whom correspondence should be addressed: Laboratory of
Immunology, Medical Research Center, College of Medicine, Yonsei University, CPO Box 8044, Seoul, South Korea. Tel.: 82-23618339; Fax:
82-23628647; E-mail: wjlee1{at}yumc.yonsei.ac.kr.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; HOG1, high osmolarity glycerol protein kinase in S. cerevisiae; LPS, lipopolysaccharide; RK, reactivating kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); SC, synthetic complete.
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REFERENCES |
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