Millar2, Jero Vicente-Soler1, Jose´ Cansado1and Mariano Gacto1 1 Departamento de Gene´tica y Microbiologı´a, Facultad de Biologı´a, Universidad de Murcia, Spain;2Division of Yeast Geneti
Trang 1Cold induces stress-activated protein kinase-mediated response
Teresa Soto1, Francisco F Beltra´n1, Vanessa Paredes1, Marisa Madrid1, Jonathan B A Millar2,
Jero Vicente-Soler1, Jose´ Cansado1and Mariano Gacto1
1
Departamento de Gene´tica y Microbiologı´a, Facultad de Biologı´a, Universidad de Murcia, Spain;2Division of Yeast Genetics, National Institute for Medical Research, London, UK
In the fission yeast Schizosaccharomyces pombe the Wak1p/
Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK)
pathway relays environmental signals to the transcriptional
machinery and modulates gene expression via a cascade of
protein phosphorylation Cells of S pombe subjected to cold
shock (transfer from 28C to 15 C) transiently activated
the Sty1p mitogen-activated protein kinase (MAPK) by
phosphorylation Induction of this response was completely
abolished in cells disrupted in the upstream response
regu-lator Mcs4p The cold-triggered Sty1p activation was
par-tially dependent on Wak1p MAPKKK and fully dependent
on Wis1p MAPKK suggesting that the signal transmission
follows a branched pathway, with the redundant MAPKKK
Win1p as alternative transducer to Wis1p, which
subse-quently activates the effector Sty1p MAPK Also, the bZIP
transcription factor Atf1p became phosphorylated in a Sty1p-dependent way duringthe cold shock and this phos-phorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+and ntp1+genes Strains deleted in transcription factors Atf1p or Pcr1p were unable
to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to
do so These data reveal that S pombe responds to cold by inducingthe SAPK pathway However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth
at low temperature by an as yet undefined mechanism Keywords: cold; SAPK pathway; fission yeast
Low temperature is an important environmental signal for
all livingorganisms Adaptive response to cold stress
involves synthesis of several types of proteins In bacteria,
thermal downshifts induce cold-shock proteins (Csp) that
function as RNA chaperones favouringefficient translation
of mRNAs at low temperature [1] However, in eukaryotes
no proteins homologous to bacterial Csp’s have been
isolated and cold shock-inducible proteins range from
structural components involved in ribosomal biogenesis to
transcriptional regulation factors that activate gene
expres-sion in response to a drop in temperature [2,3]
The mitogen-activated protein kinase (MAPK) signalling
pathways are critical for the sensingand response of
eukaryotic cells to changes in the external environment [4]
These MAPK cascades are highly conserved through
evolution and serve to transduce signals to the nucleus,
which result in new patterns of gene expression [5,6] Each
MAPK module comprises at least three protein kinases: a
MAP kinase is activated through phosphorylation on
specific threonine and tyrosine residues by a MAPK kinase (MAPKK or MEK) which is in turn activated by phosphorylation in one or several serine and threonine residues by a MAPKK kinase (MAPKKK or MEKK) Recently, different studies have revealed a key role for MAPK cascades in the response of metazoan cells to osmotic changes, heat shock, oxidative stress and UV radiation, as well as to treatment with inflammatory cytokines, DNA damaging agents and vasoactive neuro-peptides [7–13] In mammalian cells the c-Jun N-terminal kinase (JNK) and p38/RK/CSBP kinases have been characterized as stress-activated protein kinases (SAPKs) [7,10–13] able to phosphorylate (and therefore activate) transcription factors such as c-Jun [7,11], ATF2 [14–16] and Elk-1 [17,18], which regulate gene expression in response to various conditions
The identification of a highly conserved SAPK pathway
in the fission yeast Schizosaccharomyces pombe allows to analyse the precise mechanisms by which SAPKs are activated in a system more amenable than higher eukaryotic cells [19–22] In this yeast, the central element of SAPK cascade is the MAP kinase Sty1p (also known as Spc1p or Phh1p), which is highly homologous to mammalian p38 kinase and becomes activated by a similar series of stresses [20,21,23–25] Deletion of sty1+ brings about partially sterile elongated cells that are sensitive to osmostress, heat shock, oxidative treatment, and UV injury Sty1p MAPK is directly phosphorylated by Wis1p MAPKK in S pombe cells subjected to such stresses, and no Sty1p phosphory-lation is detected in the absence of Wis1p under any stress condition [20,21,23,24] Activation of Sty1p is also con-trolled by the action of two phosphatases, Pyp1p and Pyp2p
Correspondence to J Cansado, Departamento de Gene´tica y
Microbiologı´a, Facultad de Biologı´a, University of Murcia,
30071 Murcia, Spain Fax: + 34 68 363963, Tel.: + 34 68 364953,
E-mail: jcansado@um.es
Abbreviations: YES, yeast extract plus supplements; MEL, malt
extract liquid; EMM2, Edinburgh minimal medium; Ha6H,
hemagglutinin antigen epitope and six histidines; MAPK,
mitogen-activated protein kinase; SAPK, stress-mitogen-activated protein kinase;
Csp, cold-shock proteins.
(Received 27 May 2002, revised 22 August 2002,
accepted 29 August 2002)
Trang 2[20] The transmission pathway of the stress signal to Wis1p
is dual, and either the MAPKKKs Wak1p (also known as
Wis4p or Wik1p) or Win1p are responsible for Wis1p
phosphorylation [26–28] A response regulator protein,
Mcs4p, associates with Wak1p, and probably with Win1p,
to regulate MAPKKK activity in response to several stimuli
[25,29] In S pombe different transcription factors function
downstream of the Sty1p MAP kinase cascade, among
which Atf1p, Pcr1p and Pap1p have been extensively
studied Interestingly, Atf1p and Pcr1p were originally
reported as Mts1 and Mts2, respectively, and shown to be
involved in meiotic homologous recombination [30] Atf1p
(also known as Gad7p) is a mammalian ATF-2 homologue
b-ZIP protein that associates to and is phosphorylated by
Sty1p followingstress [31–33] In fact, Sty1p is the only
known kinase involved in Atf1p phosphorylation so that
S pombe mutants lacking atf1+ gene show many of the
phenotypes previously described for sty1–cells [24]
Tran-scription of several stress-response genes is controlled by
Atf1p [32,33] On the other hand, strains lackingPcr1p,
which forms heterodimers with Atf1p [30], display a
behaviour similar to atf1– cells [34] Finally, Pap1p
transcription factor, with high homology to mammalian
c-Jun and similar DNA-bindingproperties [35], is a target
for Sty1p MAPK under oxidative stress [36] However, in
contrast to Atf1p, Pap1p is neither phosphorylated nor a
substrate for Sty1p upon stress conditions S pombe cells
deleted in pap1+ gene show high sensitivity to oxidative
stress but not to osmotic stress or nutrient deprivation [36]
Although different stimuli have been used in S pombe to
reveal signalling routes that control cell adaptation, the
effect of low temperature has received no attention In this
work we have dissected the SAPK cascade in cells of the
fission yeast subjected to a thermal downshift We report
that cold activates the Wak1p/Win1p-Wis1p-Sty1p
path-way resultingin Atf1p phosphorylation and increased
expression of selected genes Activation of the SAPK
cascade, however, is not essential for yeast growth in the
cold Our data provide evidence for the existence of a novel
Atf1p/Pcr1p -mediated, SAPK-independent pathway that is involved in growth determination at low temperature
M A T E R I A L S A N D M E T H O D S
Strains and culture media The S pombe strains employed in this study are listed in Table 1 They were routinely grown with shaking at 28C
in yeast extract plus supplements (YES) [37] or Edinburgh minimal medium (EMM2) Culture media were supple-mented with adenine, leucine, histidine or uracil (100 mgÆL)1, all obtained from Sigma Chemical Co.) dependingon the requirements for each particular strain Solid media were made by the addition of 2% (w/v) bacto-agar (Difco Laboratories) Transformation of S pombe strains was performed by the lithium acetate method as described elsewhere [37] Escherichia coli DH5a was employed as a host to propagate plasmids It was grown
at 37C in Luria–Bertani medium plus 50 lgÆmL)1 ampi-cillin Strains TS-1, TS-2, TS-3 and TS-4 were constructed
by matingthe appropriate parental strains (see Table 1), and selectingdiploids in EMM2 medium with histidine plus leucine (strains TS-1 and TS-2), or leucine (strains TS-3 and TS-4) Sporulation was performed in malt extract liquid (MEL) medium [37] and the spores purified by glusulase treatment [38] were allowed to germinate in YES medium Strains with the desired genotype were identified by Southern and immunoblot analysis with anti-Ha antibodies (see below)
Stress treatments Yeast cultures grown to mid-log phase (D600¼ 0.7–1) at
28C were subjected to heat (48 C), osmotic (0.75M NaCl), oxidative (1 mM H2O2), or cold (15C) stresses
At different times, the cells from 30 mL of culture were collected in Falcon tubes containingice (equivalent to
10 mL of distilled water) and harvested by centrifugation at
Table 1 S pombe strains used in this study.
JM1059 h –
ade6-M216 his7–366 leu 1–32 ura4-D18 J.B.A Millar JM1368 h – ade6-M216 his7–336 leu 1–32 ura4-D18 mcs4 + ::ura4 + J.B.A Millar VB1700 h– ade6-M210 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4+)mcs4+(D412N) [29]
JM1478 h– ade6-M216 his7–366 leu 1–32 ura4-D18 wak1::ura4+ J.B.A Millar JM1521 h + ade6-M210 his7–366 leu 1–32 ura4-D18 sty1:Ha6H (ura4 + ) J.B.A Millar JM1821 h– ade6-M216 leu 1–32 ura4-D18 atf1+:Ha6H (ura4+) J.B.A Millar
TK102 h – his1–102 leu 1–32 ura4-D18 wis1 + :: his1 + T Kato TK107 h – leu 1–32 ura4-D18 sty1 + :: ura4 + T Kato TK108 h+leu 1–32 ura4-D18 sty1+:: ura4+ T Kato TS-1 h – ade6-M216 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4 + ) mcs4 + ::ura4 + This work TS-2 h – ade6-M216 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4 + ) wak1 + ::ura4 + This work TS-3 h+ade6-M210 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4+) wis1+::his1+ This work
ade6-M216 leu 1–32 ura4-D18 atf1:Ha6H (ura4 + ) sty1 +
::ura4+ This work WSP547 h – ade6-M210 his3-D1 leu 1–32 ura4-D18 W.P Wahls WSP643 h– ade6-M210 his3-D1 leu 1–32 ura4-D18 atf1+:: ura4+ W.P Wahls WSP643 h –
ade6-M210 his3-D1 leu 1–32 ura4-D18 pcr1+:: his3+ W.P Wahls WSP672 h – ade6-M210 his3-D1 leu 1–32 ura4-D18 atf1 + :: ura4 + pcr1 + ::his3 + W.P Wahls TP108–3c h– leu 1–32 ura4-D18 pap1+:: ura4+ T Toda
Trang 34C Under these conditions, the previously described Sty1p
phosphorylation due to centrifugation [28] was not observed
in unstressed cells After washingwith NaCl/Pibuffer, yeast
pellets were immediately frozen in liquid nitrogen
Purification and detection of activated
Sty1-hemagglu-tinin antigen epitope and six histidines (Ha6H)
and Atf1-Ha6H proteins
To analyse Sty1p, total cell homogenates were prepared
under native conditions employingchilled acid-washed glass
beads and lysis buffer (10% glycerol, 50 mM Tris/HCl
pH 7.5, 150 mMNaCl, 0.1% Nonidet NP-40, plus specific
protease and phosphatase inhibitor cocktails for fungal and
yeast extracts obtained from Sigma Chemical Co.) The
lysates were removed and cleared by centrifugation at
10 000 g for 15 min Ha6H-tagged Sty1p was purified by
usingNi2-nitrilotriacetic acid agarose beads (Qiagen Inc.),
as reported previously [39] The purified proteins were
resolved in 10% SDS/PAGE gels, transferred to
nitrocel-lulose filters (Amersham Pharmacia), and incubated with
either a mouse anti-Ha (Roche Molecular Biochemicals,
clone 12CA5) or mouse anti-(phospho-p38) (New England
Biolabs) antibodies The immunoreactive bands were
revealed with an HRP-conjugated anti-(mouse Ig) Ig
secondary antibody (Sigma Chemical Co.) and the ECL
system (Amersham-Pharmacia) For Atf1p-Ha6H
purifica-tion, the pelleted cells were lysed into denaturinglysis buffer
(6MGuanidine HCl, 0.1Msodium phosphate, 50 mMTris
HCl, pH 8.0) and the Atf1p protein isolated by affinity
precipitation on Ni2-nitrilotriacetic acid agarose beads as
previously described [40] The purified proteins were
resolved in 6% SDS/PAGE gels, transferred to
nitrocellu-lose filters (Amersham Pharmacia), and incubated with a
mouse anti-Ha antibody (12CA5) The immunoreactive
bands were detected as described above
Plate assay of cold sensitivity for growth
S pombemutants and wild-type strains were grown in YES
liquid medium to mid-logphase and, after appropriate
dilutions, different number of cells were spotted per
duplicate on YES agar plates and incubated either at
28C for 3 days, or 15 C for 10 days
RNA isolation and hybridization
Total RNA preparations from cold-shocked strains were
obtained essentially as described in [39] and resolved
through 1.5% agarose-formaldehyde gels Northern
(RNA)-hybridization analyses were performed as described
by Sambrook et al [41] A 1.2Kb fragment of the gpd1+
gene [42] was amplified by PCR with the 5¢ oligonucleotide
TGGATATGGTCAACAAGG and the 3¢ oligonucleotide
GTTTCAGTACCGCCCTCG, and used to probe for
gpd1+mRNA, while a 1 Kb fragment of the ctt1+gene
[43] was amplified with the 5¢ oligonucleotide CGTCCCTG
TTTACAC and the 3¢ oligonucleotide GCTTCCTTGGA
ACAT Probes for tps1+ and ntp1+ were prepared as
previously reported [44,45] An approximately 900 bp
fragment of the leu1+gene was amplified by PCR [46],
and used to probe for leu1+mRNA as an internal standard
for the RNA amount loaded in each lane To establish
quantitative conclusions, the level of mRNAs was quanti-fied in a Phosphorimager (Molecular Dynamics) and compared with the internal control (leu1+mRNA)
R E S U L T S
Sty1p activation following a cold stress The effects of low temperature in the fission yeast have been scarcely investigated [34] Because in S pombe a rang e of environmental stresses activates Sty1 MAP kinase through phosphorylation [28] we examined if Sty1p was also activated followinga cold stress To this end, we used strain JM1521 which harbors a genomic copy of sty1+ tagged with two copies of the Ha epitope and six histidine residues Exponentially growing cultures of this strain were subjected to either cold, heat shock, osmotic or oxidative stresses Samples were collected from 0 to 360 min, and Sty1p-Ha6H protein was purified by affinity chromatogra-phy employingNi2-nitrilotriacetic acid agarose beads The activation status of Sty1p was analysed by Western immunoblotting, using antiphospho-p38 antibodies, whereas duplicated samples were probed with a monoclonal antibody to the Ha-tagin order to normalize the protein level duringthe course of the experiment As shown in Fig 1, a thermal downshift from 28 to 15C provoked a clear and largely maintained activation of Sty1p, with significant phosphorylation level within 1 h of treatment, a maximum at 2–3 h, and a rather slight decrease afterwards The kinetics of cold-induced Sty1p activation was markedly different from the quick and transient activation achieved under heat shock [28] (Fig 1) Osmotic stress also produced
a relatively quick activation of Sty1 kinase followed by a slow decrease in Sty1p phosphorylation (Fig 1) Oxidative stress promoted a similar Sty1p rapid activation pattern which, however, was maintained for even longer time than duringcold shock (Fig 1) These results show that phos-phorylation of Sty1 kinase of S pombe can be fully induced
by low temperature at 15C, although with a delayed kinetics as compared to the effect of other previously characterized stimuli Cold shocks performed in a range from 10 to 25C indicated that the kinetics of cold-induced phosphorylation of Sty1p is rather sensitive and dependent
on the particular temperature chosen for stress We observed a rather rapid response at 25C, which is relatively close to optimal temperature for growth (28–30C), and significant longer lags as the temperature of exposure dropped to lower values (results not shown)
Wis1p is the MAPK kinase that activates Sty1p during cold stress
Earlier studies have demonstrated that the presence of Wis1p is critical to ensure Sty1p phosphorylation during heat shock and osmotic or oxidative stresses [19–22] To asses whether cold-induced activation of Sty1p takes place
in a Wis1p-dependent manner we constructed strain TS-3, which expresses Ha6H-tagged Sty1 protein in a wis1– background In contrast to wis1+control strain JM1521, no signal of phosphorylated Sty1p was detected when strain TS-3 was subjected to a cold shock at 15C (Fig 2A) These analyses revealed that Wis1 MAPK kinase is essential for Sty1p activation duringa cold stress in S pombe
Trang 4Wis1p-Sty1p activation during cold stress is largely
dependent on Wak1 MAPKKK and Mcs4p
The MAPKKK homologue Wak1p/Wis4p/Wik1p is an
essential regulator of the Wis1p-Sty1p cascade in S pombe
Shiozaki et al [28] demonstrated that Sty1p activation is
greatly reduced in wak1– cells duringan osmotic stress,
whereas oxidative stress or heat shock still induced a strong
activation of Sty1p kinase In this context, we tried to
estimate the levels of Sty1p phosphorylation in strain TS-2
(wak1–,Sty1p-Ha6H) to determine the role of Wak1p in the
cold-induced activation process As shown in Fig 2A, only
a slight increase in Sty1p activation was observed at 15C in
strain TS-2 as compared to control strain JM1521 In fact,
we detected Sty1p phosphorylation in strain TS-2 only at
180 min, which corresponds to the maximum of Sty1p
activation in wild-type strains (see Fig 1) Also, we studied
the level of Sty1p activation in strain TS-2 subjected to an
osmotic shock to confirm that, as previously described [28],
only a weak increase in Sty1p phosphorylation was likewise
occurring (compare Fig 2B with Fig 1) Taken together,
these results indicate that, similar to osmostress, Wak1p is a key element in the regulation of Sty1p activation by cold in
S pombe However, the existence of some Sty1p activation
in the absence of Wak1p reveals that to some extent other MEKK, likely Win1p, can transmit the cold stress signal to the Wis1p-Sty1p cascade as an alternative branch of the pathway
Sensingof multiple stresses through the SAPK pathway leads to Mcs4p phosphorylation, that alters the activity of Wak1p, and probably also the Win1p MAPKKK, to promote sequential phosphorylation of Wis1p MAPKK and Sty1p MAPK [29] We examined Sty1p phosphory-lation in strain TS-1 which shows mcs4+disrupted Upon
a cold shock Sty1p was not phosphorylated in strain TS-1 (Fig 2A) Thus, the lack of interaction between Mcs4p and either Wak1p or Win1p totally blocks the transmis-sion of the signal induced by low temperature that results
in phosphorylation of MAPK Sty1p Recently, it has been reported that duringoxidative stress Mcs4p acts in a conserved phospho-relay system initiated by two PAS/ PAC domain-containinghistidine kinases, Mak2p ad Mak3p [29] Mcs4p phosphorylation at aspartate 412 appears critical for Sty1p activation in response to hydrogen peroxide, but not to other environmental stresses [29] Hence, we performed a time-course study
of Sty1p phosphorylation duringcold stress in strain VB1700, where mcs4+ is replaced by a mutant allele bearinga nonphosphorylable asparagine at residue 412
As shown in Fig 2C, mcs4(D412N) cells displayed a pattern of Sty1p phosphorylation similar to wild-type cells (Fig 2A), suggesting that phosphorylation of Mcs4p D412 is not required for activation of Sty1p by cold stress
Atf1p is phosphorylated in a Sty1p-dependent way and regulates gene expression during cold stress Amongthe several bZIP transcription factors that appear to function downstream of the Sty1 MAP kinase cascade, Atf1p has been investigated in some detail during the last years Atf1p is phosphorylated by Sty1p both in vivo and
in vitro under different stress conditions and induces the expression of different stress-response genes [33] The existence of a Wis1p-Sty1p-mediated response to cold stress
in S pombe, led us to explore the phosphorylation status of Atf1p duringa thermal downshift To this purpose we used strain JM1821, which carries a genomic copy of the atf1+ gene tagged with two copies of the Ha epitope and six histidine residues, and took advantage of previous findings demonstrating that Atf1p of unstressed cells migrates in gel
as a single protein band of approximately 85 kDa that undergoes a Sty1p-dependent band shift due to phosphory-lation under different stresses [29,32] Cold treatment of wild-type strain JM1821 of S pombe induced Atf1p phos-phorylation in vivo (Fig 3) This response was evident upon
90 min of treatment at 15C and was maintained for at least 3 h Besides, the kinetics of Atf1p phosphorylation matched closely with Sty1p activation (see Fig 1) Also, the level of Atf1p increased at longer treatment times, an effect that has been interpreted by others as due to Atf1p stimulation of its own expression under stress [24,47] Contrary to these results, Atf1p purified from sty1–strain TS-4 subjected to the low temperature treatment migrated
Fig 1 Kinetics of cold-induced activation of Sty1p in S pombe
Wild-type strain JM1521 carryinga Ha6H-tagged chromosomal version of
the sty1 + gene was grown in YES medium to mid-log phase and
subjected to a cold stress (15 C), heat shock (40 C), osmotic shock
(0.5 M Na Cl) or oxidative stress (1 m M H 2 O 2 ) for the times indicated.
Aliquots were harvested and Sty1p was purified by affinity
chroma-tography Activated Sty1p was detected by inmunoblotting with
anti(phospho p38) antibodies Total Sty1p was determined by
inmu-noblottingwith anti-Ha antibody as loadingcontrol.
Trang 5always with the same apparent size, correspondingto the
unphosphorylated form (Fig 3) These results clearly
indi-cate that Sty1p is the only MAP kinase that phosphorylates
Atf1p in vivo followinga cold stress in S pombe
A number of stress-responsive genes have been shown
to be targets of the Atf1p transcription factor One of
these, gpd1+, encodingglycerol-3 phosphate dehydrog
e-nase, is involved in the synthesis of glycerol, whose
intracellular accumulation is important in response to high
osmolarity conditions The expression of gpd1+is induced
in S pombe via the Wis1p-Sty1p-Atf1p pathway [23,25,32,33] With these precedents, we studied the level
of expression of gpd1+gene in wild-type, sty1–, and atf1– strains of S pombe duringa cold stress As shown in Fig 4, a modest but reproducible increase of gpd1+ expression was evident after 4–5 h of treatment in wild-type strain induced by cold stress However, this increase was absent in sty1– and atf1– strains under the same conditions (Fig 4) Also, the expression of the cytoplas-mic catalase gene ctt1+, which is characteristically
Fig 2 Functional SAPK pathway is required for cold stress activation of Sty1p (A) Sty1p phosphorylation in S pombe cells subjected to a cold stress is dependent on Wis1p, Wak1p and Mcs4p Wild-type (JM1521), Dwis1 mutant (TS-3), Dwak1 mutant (TS-2) and Dmcs4 mutant (TS-1) strains carryinga Ha6H-tagged chromosomal version of the sty1 + gene were subjected to a cold stress at 15 C for the times indicated Aliquots were harvested and Sty1p was purified by affinity chromatography Activated Sty1p was detected by inmunoblotting with anti-(phospho p38) antibodies and anti-Ha antibody inmunoblottingwas used as a control to determine loaded Sty1p Wis1p and Mcs4p function is critical in the cold-induced activation of Sty1p while Wak1p plays an important role (B) Osmostress-cold-induced Sty1p activation is largely dependent on Wak1p The Dwak1 mutant strain TS-2 was subjected to an osmotic stress with 0.5 M NaCl in YES medium Aliquots were processed and analyzed as described
in (A) (C) D412 of Mcs4p is not required for the activation of Sty1p MAP kinase in response to cold stress The mcs4 (D412N) strain VB1700 was subjected to a cold stress at 15 C in YES medium, and aliquots were processed as described in (A).
Fig 3 Sty1p-dependent phosphorylation of Atf1p in vivo follow ing a cold stress Wild-type (JM1821) and Dsty1 (TS-4) strains carryinga chro-mosomal copy of Ha-tagged atf1+gene were grown at 28 C (0 time) and shifted to 15 C for the times indicated Aliquots were harvested and the Atf1p-Ha6H tagged protein was purified with Ni 2 -nitrilotriacetic acid beads and analyzed by SDS/PAGE followed by inmunoblottingwith anti-Ha antibodies Samples marked with asterisk (*) show a typical Atf1p shift due to phosphorylation.
Trang 6regulated by Atf1p [24,26], was induced in a
Sty1p-Atf1p-dependent manner duringexposure of S pombe to a cold
shock (Fig 4) Similarly, we observed a retarded
cold-induced increase in the expression of tps1+ and ntp1+,
which code for trehalose-6-phosphate synthase and
neut-ral trehalase, respectively (Fig 4) These results show that
S pombeis able to transduce a cold-induced stress signal
through the Wis1-Sty1 MAP kinase pathway, to promote
Atf1p phosphorylation and to trigger the subsequent
expression of stress-responsive genes
Sensitivity of mutants in SAPK pathway to cold stress
The experiments described above demonstrate that the
SAPK pathway of S pombe can be fully activated in
response to low temperatures We next addressed the
question of the biological significance of such a response by
studyingthe ability of different mutants affected in this
pathway to grow at low temperatures in YES solid medium
As shown in Fig 5, all the strains assayed displayed normal
growth when incubated at 28C for 3 days However,
significant differences were observed in cell viability among
different mutants after incubation at 15C for 10 days
S pombe strains lackingMcs4p, Wak1p, Wis1p or Sty1p kinases were slightly more sensitive to cold stress than their wild-type counterparts (Fig 5), whereas atf1–, pcr1–, and atf1–pcr1–strains were unable to grow at these conditions
On the contrary, the strain TP108–3C, which lacks transcription factor pap1–, did not exhibit the cold tem-perature defective phenotype and exhibited a growth similar
to wild-type cells The failure of atf1–and pcr1–mutants to grow at low temperature is not related to a marked decrease
in the viability of these strains because a shift of cold-stressed cultures back to 28C resulted in g rowth resump-tion (not shown) Taken together, these results indicate that the SAPK pathway plays a discrete role in the survival of
S pombe to low temperatures and that cell viability (measured as growth) appears to be fully dependent on the existence of Atf1p-Pcr1p heterodimers As Atf1p is not phosphorylated duringcold stress in the absence of the Sty1 kinase that channels the stress signal (Fig 4), the role of Atf1p/Pcr1p in g rowth at low temperature is likely independent on the existence of an operative SAPK pathway
Fig 4 Sty1p MAP kinase regulates the induction of gpd1+, ctt1+, tps1+and ntp1+genes during cold stress through the Atf1p transcription factor Strains TK003 (WT), TK107 (Dsty1) and WSP 643 (Datf1) were g rown in YES medium to mid-log phase and shifted to 15 C for the times indicated Total RNA was extracted from each sample and 20 lg were resolved in 1.5% agarose-formaldehyde gels The denatured RNAs were transferred to nylon membranes and hybridized with32P-labelled probes of gpd+, ctt1+, tps1+, ntp1+and leu1+(loadingcontrol) Numbers below each frame indicate normalized relative values of expression.
Trang 7D I S C U S S I O N
The main aim of this study was to analyse the cold shock
response in S pombe, a yeast that displays a cascade of
stress activated protein kinases homologous to the SAPK
pathway present in higher eukaryotes [20–25] In rich
medium, wild-type S pombe strains are able to growth in a
range from 15 to 37C, although the optimum temperature
for g rowth is 28–30C [48] The key element of the SAPK
pathway in this yeast is the Sty1p MAP kinase, that
becomes phosphorylated by different stressful conditions
We have demonstrated that Sty1p from S pombe is
specifically phosphorylated at threonine and tyrosine
resi-dues duringa thermal downshift to 15C, with maximal
level within 2–3 h of exposure This cold-induced activation
is unnoticed unless the span of samplingto detect signal of
Sty1p phosphorylation extends for longer times than those
usually considered for other stress stimuli Our data provide
the first evidence that low temperature is an activator of the
SAPK pathway and that this signal transduction system
controls gene transcription in response to cold
environ-ments
An interestingobservation concerns the delay in the
kinetics of phosphorylation and dephosphorylation of
Sty1p duringcold stress as compared to other stimuli
Unexpectedly, we detected Sty1 activation even at
temper-atures well below 15C, for example, immediately prior to
the freezingof cell samples at )20 C (data not shown)
Indeed, this pattern of activation is slower than the
described for heat shock, osmostress, treatment with
reactive oxygen species or UV radiation [23,24], but similar
to the kinetics observed in response to nitrogen starvation
[32] It thus appears that the cold stress signal is transduced
through the SAPK cascade in S pombe with a rate specific
of this type of stress and relatively dependent of the severity
of the thermal downshift, with longer delays at lower
tem-peratures It has been proposed that different environmental
stresses may be sensed by specific membrane-bound cellular receptors that trigger activation of Sty1p but the nature of the upstream components involved in this signalling remain for most part unknown [25] A possible reason for the slow response in Sty1p phosphorylation duringsensingand transduction of the cold shock signal could be that membrane fluidity decreases greatly upon temperature downshifts, thereby slowingdown membrane associated cellular functions Also, a decreased rate in general meta-bolism would limit the generation of second messengers The level of Sty1p phosphorylation increased markedly in response to cold shock and was maintained for at least 6 h (Fig 1) Contrariwise, a severe heat shock induced a quick and transient phosphorylation of Sty1p that became dephosphorylated within 60–90 min This is likely due to the activity of threonine-phosphatases Ptc1p and Ptc3p and tyrosine-phosphatase Pyp1 [49] Other treatments, such as osmotic stress, induced an early burst of Sty1p phosphory-lation which, even under continuous stress, returned sub-sequently close to the basal level only after 60 min (Fig 1) Pyp1p and Pyp2p phosphatases appear to play a key role in the dephosphorylation of the osmotic stress-activated Sty1p [20] Interestingly, however, the Sty1p activation induced by oxidative stress, although rather rapid, was followed by a very slow decrease in Sty1p phosphorylation, as duringa cold shock (see Fig 1) This differential behaviour may reflect that one or several of the protein phosphatases are maintained partially inactive when S pombe is subjected to any of these two stresses
The fact that Wis1p is apparently the only MAPKK that activates Sty1p during cold stress (Fig 2) is not surprising,
as this is the case for other stresses [20,21,23,24] Wis1p is thus able to integrate the transmission of different stress signals to Sty1p MAP kinase, including thermal downshifts From the two MAPKKKs that are known to activate Wis1p (i.e.Win1p or Wak1p), Wak1p appears to be the main responsible for Sty1p activation duringcold shock
Fig 5 The requirement of Atf1p and Pcr1p for S pombe growth at low temperature is not dependent on the existence of an operative SAPK pathway The indicated number of cells from wild-type and mutants in the SAPK pathway were spotted onto YES plates and incubated at 28 C or 15 C for 3 and 10 days, respectively, prior to beingphotographed.
Trang 8(Fig 2A), similar to what happens during osmostress [28].
The Win1p mitotic regulator, which controls the activation
of Sty1p kinase under multiple stressful conditions [27], is
likely responsible for the slight cold-induced activation of
Sty1p observed in the absence of Wak1p (Fig 2B) Thus,
the signal transmission for cold appears to follow a
branched pathway, with either Wak1p or the redundant
MAPKKK Win1p as an alternative via to activate to some
extent the Sty1p kinase through Wis1p On the other hand,
our results with cells disrupted in the upstream response
regulator Mcs4p indicate that the signal for cold does not
reach in such cells the level of MAPKKs, which strongly
supports the suggestion that Mcs4p likely interacts with
both Wak1p and Win1p [27,29] Also, the occurrence of
Sty1p phosphorylation in S pombe cells expressinga
D412N version of Mcs4p indicated that the Mak2p and
Mak3p phosphorelay system is not involved in Sty1p
phosphorylation followinga cold stress, which is similar to
what happens duringother environmental stresses except
duringoxidative stress In any case, the control of SAPK
pathways by two-component systems appears exclusive of
lower eukaryotes under specific stresses, as no structural
homologues of phospho-relay proteins have been identified
in mammals [29] Thus, because such a system is not
operative in S pombe duringcold induced activation of the
SAPK pathway, this model may be relevant to study and
characterize the transduction of temperature downshifts
signals in cells from higher eukayotes, including mammals
Followinga shift to low temperature, the bZIP
trancrip-tion factor Atf1p becomes phosphorylated in vivo in a
Sty1p-dependent manner (Fig 3) In coincidence with
previous studies, our data confirm that Sty1p is the only
kinase able to phosphorylate Atf1p at any stress condition
[32,33] As Atf1p becomes phosphorylated under cold
temperature, one might anticipate changes in the expression
of Atf1p-regulated genes upon incubation of S pombe at
low temperatures Indeed, this happens for several
Atf1-regulated genes studied in this work, gpd1+, ctt1+, tps1+
and ntp1+, whose expression rises significantly by a
Sty1p-Atf1p-dependent mechanism after a thermal downshift
(Fig 4) The physiological significance of the cold-triggered
expression of gpd1+ might be interpreted in terms of
synthesis of a cryoprotectant metabolite [50] Catalase may
also act as a protectant as it has been shown in plants and
yeast cells that cold involves oxidative stress [51,52]
Additionally, we have observed a retarded cold-induced
increase in the expression of tps1+and ntp1+, which code
for enzymes involved in trehalose metabolism This is
congruent with accumulation and turnover of the low
molecular mass carbohydrate trehalose, a well known
stabilizer of macromolecular components [53] As a whole,
it appears that the induction of compatible solutes and
defences against oxidant species forms part of the response
to low temperature and that the expression of a conserved
set of stress-responsive genes is the basis of the general stress
response underlyingcrossed stress tolerance In this respect,
pretreatment with low temperature induces a significant
adaptive response to osmostress in S pombe cells (F.F
Beltran and J Cansado, unpublished results) It is worth to
mention that transcription factor Pap1p is neither activated
nor translocated from the cytoplasm to the cell nucleus at
low temperatures and that ctt1+expression was induced at
low temperatures in a pap1-deficient strain (data not
shown) Thus, in analogy to what happens during osmotic stress [33], the expression of ctt1+ at cold temperature appears to be mostly dependent on Atf1p/Pcr1p
Although the MAP kinase pathway confers a slight, but consistent, growth advantage at 15C (Fig 5), this path-way is not essential for growth at such temperature Instead, the transcription factors Atf1p and Pcr1p, which are key effectors of the SAPK phosphorylation cascade required for a variety of developmental decisions [31–34] and dispensable for growth at 28C, were needed for growth in the cold Surprisingly, however, the cold sensitive phenotype in atf1–or pcr1–strains is not shared by mutants disrupted in either sty1+or wis1+(Fig 5), indicating that the role of these factors at low temperature is independent
of their function as SAPK-driven multifunctional switches that activate specific responses against extracellular condi-tions Hence, although cold stress in S pombe induces the SAPK pathway, the function of this cascade does not guarantee far more than a slight better adjustment of cell growth to cold conditions On the contrary, the presence of Atf1p and Pcr1p (presumably actingas a heterodimer) is vital for growth al low temperature by a mechanism unrelated to the SAPK pathway Preliminary observations indicate that there is not specific cell cycle block in Datf1 cells arrested at 15C Altogether, these data are consistent with a new role for these factors in transcriptional events sustainingspecific development programs in the cold
A C K N O W L E D G E M E N T S
We are indebted to W.P Wahls, T Kato and T Toda for kind supply of yeast strains, and to F Garro for technical assistance V Paredes and M Madrid are predoctoral fellows from the Fundacio´n Se´neca (Regio´n de Murcia) and the Fundacio´n Ramo´n Areces, respectively This work was supported in part by grant BMC 2001–0135 from MCYT, Spain.
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