Cold induces stress-activated protein kinase-mediated responsein the fission yeast Schizosaccharomyces pombe

Teresa Soto1, Francisco F. Beltran1, Vanessa Paredes1, Marisa Madrid1, Jonathan B. A. Millar2,Jero Vicente-Soler1, Jose Cansado1 and Mariano Gacto1

1Departamento de Genetica y Microbiologıa, Facultad de Biologıa, 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 transcriptionalmachinery and modulates gene expression via a cascade ofprotein phosphorylation. Cells ofS. pombe subjected to coldshock (transfer from 28 �C to 15 �C) transiently activatedthe Sty1p mitogen-activated protein kinase (MAPK) byphosphorylation. Induction of this response was completelyabolished in cells disrupted in the upstream response regu-lator Mcs4p. The cold-triggered Sty1p activation was par-tially dependent onWak1pMAPKKK and fully dependenton Wis1p MAPKK suggesting that the signal transmissionfollows abranchedpathway,with the redundantMAPKKKWin1p as alternative transducer to Wis1p, which subse-quently activates the effector Sty1p MAPK. Also, the bZIP

transcription factor Atf1p became phosphorylated in aSty1p-dependent way during the cold shock and this phos-phorylation was found responsible for the increasedexpression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strainsdeleted in transcription factors Atf1p or Pcr1p were unableto grow upon incubation at low temperature whereas thosedisrupted in anymember of the SAPKpathwaywere able todo so. These data reveal that S. pombe responds to cold byinducing the SAPK pathway. However, such activation isdispensable for yeast growth in cold conditions, supportingthat the presence of Atf1/Pcr1 heterodimers, rather than anoperative SAPK pathway, is critical to ensure yeast growthat low temperature by an as yet undefined mechanism.

Keywords: cold; SAPK pathway; fission yeast.

Low temperature is an important environmental signal forall living organisms. Adaptive response to cold stressinvolves synthesis of several types of proteins. In bacteria,thermal downshifts induce cold-shock proteins (Csp) thatfunction as RNA chaperones favouring efficient translationof mRNAs at low temperature [1]. However, in eukaryotesno proteins homologous to bacterial Csp’s have beenisolated and cold shock-inducible proteins range fromstructural components involved in ribosomal biogenesis totranscriptional regulation factors that activate gene expres-sion in response to a drop in temperature [2,3].

The mitogen-activated protein kinase (MAPK) signallingpathways are critical for the sensing and response ofeukaryotic cells to changes in the external environment [4].These MAPK cascades are highly conserved throughevolution and serve to transduce signals to the nucleus,which result in new patterns of gene expression [5,6]. EachMAPK module comprises at least three protein kinases: aMAP kinase is activated through phosphorylation on

specific threonine and tyrosine residues by a MAPK kinase(MAPKK or MEK) which is in turn activated byphosphorylation in one or several serine and threonineresidues by a MAPKK kinase (MAPKKK or MEKK).Recently, different studies have revealed a key role forMAPK cascades in the response of metazoan cells toosmotic changes, heat shock, oxidative stress and UVradiation, as well as to treatment with inflammatorycytokines, DNA damaging agents and vasoactive neuro-peptides [7–13]. In mammalian cells the c-Jun N-terminalkinase (JNK) and p38/RK/CSBP kinases have beencharacterized 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] andElk-1 [17,18], which regulate gene expression in response tovarious conditions.

The identification of a highly conserved SAPK pathwayin the fission yeast Schizosaccharomyces pombe allows toanalyse the precise mechanisms by which SAPKs areactivated in a systemmore amenable than higher eukaryoticcells [19–22]. In this yeast, the central element of SAPKcascade is the MAP kinase Sty1p (also known as Spc1p orPhh1p), which is highly homologous to mammalian p38kinase and becomes activated by a similar series of stresses[20,21,23–25]. Deletion of sty1+ brings about partiallysterile elongated cells that are sensitive to osmostress, heatshock, oxidative treatment, and UV injury. Sty1pMAPK isdirectly phosphorylated by Wis1p MAPKK in S. pombecells subjected to such stresses, and no Sty1p phosphory-lation is detected in the absence of Wis1p under any stresscondition [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 Genetica y

Microbiologıa, Facultad de Biologıa, 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-activated protein kinase;

Csp, cold-shock proteins.

(Received 27 May 2002, revised 22 August 2002,

accepted 29 August 2002)

Eur. J. Biochem. 269, 5056–5065 (2002) � FEBS 2002 doi:10.1046/j.1432-1033.2002.03214.x

[20]. The transmission pathway of the stress signal toWis1pis dual, and either the MAPKKKs Wak1p (also known asWis4p or Wik1p) or Win1p are responsible for Wis1pphosphorylation [26–28]. A response regulator protein,Mcs4p, associates with Wak1p, and probably with Win1p,to regulateMAPKKKactivity in response to several stimuli[25,29]. In S. pombe different transcription factors functiondownstream of the Sty1p MAP kinase cascade, amongwhich Atf1p, Pcr1p and Pap1p have been extensivelystudied. Interestingly, Atf1p and Pcr1p were originallyreported as Mts1 and Mts2, respectively, and shown to beinvolved in meiotic homologous recombination [30]. Atf1p(also known as Gad7p) is a mammalian ATF-2 homologueb-ZIP protein that associates to and is phosphorylated bySty1p following stress [31–33]. In fact, Sty1p is the onlyknown kinase involved in Atf1p phosphorylation so thatS. pombe mutants lacking atf1+ gene show many of thephenotypes previously described for sty1– cells [24]. Tran-scription of several stress-response genes is controlled byAtf1p [32,33]. On the other hand, strains lacking Pcr1p,which forms heterodimers with Atf1p [30], display abehaviour similar to atf1– cells [34]. Finally, Pap1ptranscription factor, with high homology to mammalianc-Jun and similar DNA-binding properties [35], is a targetfor Sty1p MAPK under oxidative stress [36]. However, incontrast to Atf1p, Pap1p is neither phosphorylated nor asubstrate for Sty1p upon stress conditions. S. pombe cellsdeleted in pap1+ gene show high sensitivity to oxidativestress but not to osmotic stress or nutrient deprivation [36].

Although different stimuli have been used in S. pombe toreveal signalling routes that control cell adaptation, theeffect of low temperature has received no attention. In thiswork we have dissected the SAPK cascade in cells of thefission yeast subjected to a thermal downshift. We reportthat cold activates the Wak1p/Win1p-Wis1p-Sty1p path-way resulting in Atf1p phosphorylation and increasedexpression of selected genes. Activation of the SAPKcascade, however, is not essential for yeast growth in thecold. Our data provide evidence for the existence of a novel

Atf1p/Pcr1p -mediated, SAPK-independent pathway that isinvolved 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 inTable 1. They were routinely grown with shaking at 28 �Cin yeast extract plus supplements (YES) [37] or Edinburghminimal medium (EMM2). Culture media were supple-mented with adenine, leucine, histidine or uracil(100 mgÆL)1, all obtained from Sigma Chemical Co.)depending on the requirements for each particular strain.Solid media were made by the addition of 2% (w/v) bacto-agar (Difco Laboratories). Transformation of S. pombestrains was performed by the lithium acetate method asdescribed elsewhere [37]. Escherichia coli DH5a wasemployed as a host to propagate plasmids. It was grownat 37 �C in Luria–Bertani medium plus 50 lgÆmL)1 ampi-cillin. Strains TS-1, TS-2, TS-3 and TS-4 were constructedby mating the appropriate parental strains (see Table 1),and selecting diploids in EMM2medium with histidine plusleucine (strains TS-1 and TS-2), or leucine (strains TS-3 andTS-4). Sporulation was performed in malt extract liquid(MEL) medium [37] and the spores purified by glusulasetreatment [38] were allowed to germinate in YES medium.Strains with the desired genotype were identified bySouthern and immunoblot analysis with anti-Ha antibodies(see below).

Stress treatments

Yeast cultures grown to mid-log phase (D600 ¼ 0.7–1) at28 �C were subjected to heat (48 �C), osmotic (0.75 M

NaCl), oxidative (1 mM H2O2), or cold (15 �C) stresses.At different times, the cells from 30 mL of culture werecollected in Falcon tubes containing ice (equivalent to10 mL of distilled water) and harvested by centrifugation at

Table 1. S. pombe strains used in this study.

Strain Genotype Source/Reference

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

TK003 h– leu 1–32 T. Kato

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

TS-4 h– 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

� FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5057

4 �C.Under these conditions, the previously describedSty1pphosphorylation due to centrifugation [28] was not observedin unstressed cells. After washing with NaCl/Pi buffer, yeastpellets 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 preparedunder native conditions employing chilled acid-washed glassbeads and lysis buffer (10% glycerol, 50 mM Tris/HClpH 7.5, 150 mM NaCl, 0.1% Nonidet NP-40, plus specificprotease and phosphatase inhibitor cocktails for fungal andyeast extracts obtained from Sigma Chemical Co.). Thelysates were removed and cleared by centrifugation at10 000 g for 15 min. Ha6H-tagged Sty1p was purified byusing Ni2-nitrilotriacetic acid agarose beads (Qiagen Inc.),as reported previously [39]. The purified proteins wereresolved in 10% SDS/PAGE gels, transferred to nitrocel-lulose filters (Amersham Pharmacia), and incubated witheither a mouse anti-Ha (Roche Molecular Biochemicals,clone 12CA5) or mouse anti-(phospho-p38) (New EnglandBiolabs) antibodies. The immunoreactive bands wererevealed with an HRP-conjugated anti-(mouse Ig) Igsecondary antibody (Sigma Chemical Co.) and the ECLsystem (Amersham-Pharmacia). For Atf1p-Ha6H purifica-tion, the pelleted cells were lysed into denaturing lysis buffer(6 M Guanidine HCl, 0.1 M sodium phosphate, 50 mM TrisHCl, pH 8.0) and the Atf1p protein isolated by affinityprecipitation on Ni2-nitrilotriacetic acid agarose beads aspreviously described [40]. The purified proteins wereresolved in 6% SDS/PAGE gels, transferred to nitrocellu-lose filters (Amersham Pharmacia), and incubated with amouse anti-Ha antibody (12CA5). The immunoreactivebands were detected as described above.

Plate assay of cold sensitivity for growth

S. pombemutants and wild-type strains were grown in YESliquid medium to mid-log phase and, after appropriatedilutions, different number of cells were spotted perduplicate on YES agar plates and incubated either at28 �C for 3 days, or 15 �C for 10 days.

RNA isolation and hybridization

Total RNA preparations from cold-shocked strains wereobtained essentially as described in [39] and resolvedthrough 1.5% agarose-formaldehyde gels. Northern(RNA)-hybridization analyses were performed as describedby Sambrook et al. [41]. A 1.2Kb fragment of the gpd1+

gene [42] was amplified by PCR with the 5¢ oligonucleotideTGGATATGGTCAACAAGG and the 3¢ oligonucleotideGTTTCAGTACCGCCCTCG, and used to probe forgpd1+ mRNA, while a 1 Kb fragment of the ctt1+ gene[43] was amplified with the 5¢ oligonucleotide CGTCCCTGTTTACAC and the 3¢ oligonucleotide GCTTCCTTGGAACAT. Probes for tps1+ and ntp1+ were prepared aspreviously reported [44,45]. An approximately 900 bpfragment of the leu1+ gene was amplified by PCR [46],and used to probe for leu1+mRNA as an internal standardfor the RNA amount loaded in each lane. To establish

quantitative conclusions, the level of mRNAs was quanti-fied in a Phosphorimager (Molecular Dynamics) andcompared 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 beenscarcely investigated [34]. Because in S. pombe a range ofenvironmental stresses activates Sty1 MAP kinase throughphosphorylation [28] we examined if Sty1p was alsoactivated following a cold stress. To this end, we usedstrain JM1521 which harbors a genomic copy of sty1+

tagged with two copies of the Ha epitope and six histidineresidues. Exponentially growing cultures of this strain weresubjected to either cold, heat shock, osmotic or oxidativestresses. Samples were collected from 0 to 360 min, andSty1p-Ha6H protein was purified by affinity chromatogra-phy employing Ni2-nitrilotriacetic acid agarose beads. Theactivation status of Sty1p was analysed by Westernimmunoblotting, using antiphospho-p38 antibodies,whereas duplicated samples were probed with amonoclonalantibody to the Ha-tag in order to normalize the proteinlevel during the course of the experiment. As shown inFig. 1, a thermal downshift from 28 to 15 �C provoked aclear and largely maintained activation of Sty1p, withsignificant phosphorylation level within 1 h of treatment, amaximum at 2–3 h, and a rather slight decrease afterwards.The kinetics of cold-induced Sty1p activation was markedlydifferent from the quick and transient activation achievedunder heat shock [28] (Fig. 1). Osmotic stress also produceda relatively quick activation of Sty1 kinase followed by aslow decrease in Sty1p phosphorylation (Fig. 1). Oxidativestress promoted a similar Sty1p rapid activation patternwhich, however, was maintained for even longer time thanduring cold shock (Fig. 1). These results show that phos-phorylation of Sty1 kinase of S. pombe can be fully inducedby low temperature at 15 �C, although with a delayedkinetics as compared to the effect of other previouslycharacterized stimuli. Cold shocks performed in a rangefrom 10 to 25 �C indicated that the kinetics of cold-inducedphosphorylation of Sty1p is rather sensitive and dependenton the particular temperature chosen for stress. Weobserved a rather rapid response at 25 �C,which is relativelyclose to optimal temperature for growth (28–30 �C), andsignificant longer lags as the temperature of exposuredropped to lower values (results not shown).

Wis1p is the MAPK kinase that activates Sty1p duringcold stress

Earlier studies have demonstrated that the presence ofWis1p is critical to ensure Sty1p phosphorylation duringheat shock and osmotic or oxidative stresses [19–22]. Toasses whether cold-induced activation of Sty1p takes placein a Wis1p-dependent manner we constructed strain TS-3,which expresses Ha6H-tagged Sty1 protein in a wis1–

background. In contrast towis1+ control strain JM1521, nosignal of phosphorylated Sty1p was detected when strainTS-3 was subjected to a cold shock at 15 �C (Fig. 2A).These analyses revealed thatWis1MAPKkinase is essentialfor Sty1p activation during a cold stress in S. pombe.

5058 T. Soto et al. (Eur. J. Biochem. 269) � FEBS 2002

Wis1p-Sty1p activation during cold stress is largelydependent on Wak1 MAPKKK and Mcs4p

The MAPKKK homologue Wak1p/Wis4p/Wik1p is anessential regulator of the Wis1p-Sty1p cascade in S. pombe.Shiozaki et al. [28] demonstrated that Sty1p activation isgreatly reduced in wak1– cells during an osmotic stress,whereas oxidative stress or heat shock still induced a strongactivation of Sty1p kinase. In this context, we tried toestimate the levels of Sty1p phosphorylation in strain TS-2(wak1–, Sty1p-Ha6H) to determine the role ofWak1p in thecold-induced activation process. As shown in Fig. 2A, onlya slight increase in Sty1p activationwas observed at 15 �C instrain TS-2 as compared to control strain JM1521. In fact,we detected Sty1p phosphorylation in strain TS-2 only at180 min, which corresponds to the maximum of Sty1pactivation in wild-type strains (see Fig. 1). Also, we studiedthe level of Sty1p activation in strain TS-2 subjected to anosmotic shock to confirm that, as previously described [28],only a weak increase in Sty1p phosphorylation was likewiseoccurring (compare Fig. 2B with Fig. 1). Taken together,

these results indicate that, similar to osmostress, Wak1p is akey element in the regulation of Sty1p activation by cold inS. pombe. However, the existence of some Sty1p activationin the absence of Wak1p reveals that to some extent otherMEKK, likely Win1p, can transmit the cold stress signal tothe Wis1p-Sty1p cascade as an alternative branch of thepathway.

Sensing of multiple stresses through the SAPK pathwayleads to Mcs4p phosphorylation, that alters the activity ofWak1p, and probably also the Win1p MAPKKK, topromote sequential phosphorylation of Wis1p MAPKKand Sty1p MAPK [29]. We examined Sty1p phosphory-lation in strain TS-1 which shows mcs4+ disrupted. Upona cold shock Sty1p was not phosphorylated in strain TS-1(Fig. 2A). Thus, the lack of interaction between Mcs4pand either Wak1p or Win1p totally blocks the transmis-sion of the signal induced by low temperature that resultsin phosphorylation of MAPK Sty1p. Recently, it has beenreported that during oxidative stress Mcs4p acts in aconserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p adMak3p [29]. Mcs4p phosphorylation at aspartate 412appears critical for Sty1p activation in response tohydrogen peroxide, but not to other environmentalstresses [29]. Hence, we performed a time-course studyof Sty1p phosphorylation during cold stress in strainVB1700, where mcs4+ is replaced by a mutant allelebearing a nonphosphorylable asparagine at residue 412.As shown in Fig. 2C, mcs4(D412N) cells displayed apattern of Sty1p phosphorylation similar to wild-type cells(Fig. 2A), suggesting that phosphorylation of Mcs4pD412 is not required for activation of Sty1p by coldstress.

Atf1p is phosphorylated in a Sty1p-dependent wayand regulates gene expression during cold stress

Among the several bZIP transcription factors that appear tofunction downstream of the Sty1 MAP kinase cascade,Atf1p has been investigated in some detail during the lastyears. Atf1p is phosphorylated by Sty1p both in vivo andin vitro under different stress conditions and induces theexpression of different stress-response genes [33]. Theexistence of aWis1p-Sty1p-mediated response to cold stressin S. pombe, led us to explore the phosphorylation status ofAtf1p during a thermal downshift. To this purpose we usedstrain JM1821, which carries a genomic copy of the atf1+

gene tagged with two copies of the Ha epitope and sixhistidine residues, and took advantage of previous findingsdemonstrating that Atf1p of unstressed cells migrates in gelas a single protein band of approximately 85 kDa thatundergoes a Sty1p-dependent band shift due to phosphory-lation under different stresses [29,32]. Cold treatment ofwild-type strain JM1821 of S. pombe induced Atf1p phos-phorylation in vivo (Fig. 3). This response was evident upon90 min of treatment at 15 �C and was maintained for atleast 3 h. Besides, the kinetics of Atf1p phosphorylationmatched closely with Sty1p activation (see Fig. 1). Also, thelevel of Atf1p increased at longer treatment times, an effectthat has been interpreted by others as due to Atf1pstimulation of its own expression under stress [24,47].Contrary to these results, Atf1p purified from sty1– strainTS-4 subjected to the low temperature treatment migrated

Fig. 1. Kinetics of cold-induced activation of Sty1p in S. pombe.Wild-

type strain JM1521 carrying a 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 mM H2O2) 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-

noblotting with anti-Ha antibody as loading control.

� FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5059

always with the same apparent size, corresponding to theunphosphorylated form (Fig. 3). These results clearly indi-cate that Sty1p is the only MAP kinase that phosphorylatesAtf1p in vivo following a cold stress in S. pombe.

A number of stress-responsive genes have been shownto be targets of the Atf1p transcription factor. One ofthese, gpd1+, encoding glycerol-3 phosphate dehydroge-nase, is involved in the synthesis of glycerol, whoseintracellular accumulation is important in response to highosmolarity 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 levelof expression of gpd1+ gene in wild-type, sty1–, and atf1–

strains of S. pombe during a cold stress. As shown inFig. 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 increasewas absent in sty1– and atf1– strains under the sameconditions (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 carrying a 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 inmunoblotting was used as a control to determine loaded Sty1p.Wis1p andMcs4p function is critical in the cold-

induced activation of Sty1p while Wak1p plays an important role. (B) Osmostress-induced Sty1p activation is largely dependent on Wak1p. The

Dwak1mutant strain TS-2 was subjected to an osmotic stress with 0.5 MNaCl in YESmedium. Aliquots were processed and analyzed as described

in (A). (C) D412 of Mcs4p is not required for the activation of Sty1pMAP kinase in response to cold stress. Themcs4 (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 following a cold stress. Wild-type (JM1821) and Dsty1 (TS-4) strains carrying a 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 withNi2-nitrilotriacetic acid beads and analyzed by SDS/PAGE followed by inmunoblotting with anti-Ha

antibodies. Samples marked with asterisk (*) show a typical Atf1p shift due to phosphorylation.

5060 T. Soto et al. (Eur. J. Biochem. 269) � FEBS 2002

regulated by Atf1p [24,26], was induced in a Sty1p-Atf1p-dependent manner during exposure of S. pombe to a coldshock (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 thatS. pombe is able to transduce a cold-induced stress signalthrough the Wis1-Sty1 MAP kinase pathway, to promoteAtf1p phosphorylation and to trigger the subsequentexpression of stress-responsive genes.

Sensitivity of mutants in SAPK pathway to cold stress

The experiments described above demonstrate that theSAPK pathway of S. pombe can be fully activated inresponse to low temperatures. We next addressed thequestion of the biological significance of such a response bystudying the ability of different mutants affected in thispathway to grow at low temperatures in YES solid medium.As shown in Fig. 5, all the strains assayed displayed normalgrowth when incubated at 28 �C for 3 days. However,significant differences were observed in cell viability among

different mutants after incubation at 15 �C for 10 days.S. pombe strains lacking Mcs4p, Wak1p, Wis1p or Sty1pkinases were slightly more sensitive to cold stress than theirwild-type counterparts (Fig. 5), whereas atf1–, pcr1–, andatf1– pcr1– strains were unable to grow at these conditions.On the contrary, the strain TP108–3C, which lackstranscription factor pap1–, did not exhibit the cold tem-perature defective phenotype and exhibited a growth similarto wild-type cells. The failure of atf1– and pcr1– mutants togrow at low temperature is not related to a marked decreasein the viability of these strains because a shift of cold-stressed cultures back to 28 �C resulted in growth resump-tion (not shown). Taken together, these results indicate thatthe SAPK pathway plays a discrete role in the survival ofS. pombe to low temperatures and that cell viability(measured as growth) appears to be fully dependent onthe existence of Atf1p-Pcr1p heterodimers. As Atf1p is notphosphorylated during cold stress in the absence of the Sty1kinase that channels the stress signal (Fig. 4), the role ofAtf1p/Pcr1p in growth at low temperature is likelyindependent on the existence of an operative SAPKpathway.

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 grown 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 nylonmembranes and hybridized with 32P-labelled probes of gpd+, ctt1+, tps1+, ntp1+ and leu1+ (loading control). Numbers below

each frame indicate normalized relative values of expression.

� FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5061

D I S C U S S I O N

The main aim of this study was to analyse the cold shockresponse in S. pombe, a yeast that displays a cascade ofstress activated protein kinases homologous to the SAPKpathway present in higher eukaryotes [20–25]. In richmedium, wild-type S. pombe strains are able to growth in arange from 15 to 37 �C, although the optimum temperaturefor growth is 28–30 �C [48]. The key element of the SAPKpathway in this yeast is the Sty1p MAP kinase, thatbecomes phosphorylated by different stressful conditions.We have demonstrated that Sty1p from S. pombe isspecifically phosphorylated at threonine and tyrosine resi-dues during a thermal downshift to 15 �C, with maximallevel within 2–3 h of exposure. This cold-induced activationis unnoticed unless the span of sampling to detect signal ofSty1p phosphorylation extends for longer times than thoseusually considered for other stress stimuli. Our data providethe first evidence that low temperature is an activator of theSAPK pathway and that this signal transduction systemcontrols gene transcription in response to cold environ-ments.

An interesting observation concerns the delay in thekinetics of phosphorylation and dephosphorylation ofSty1p during cold stress as compared to other stimuli.Unexpectedly, we detected Sty1 activation even at temper-atures well below 15 �C, for example, immediately prior tothe freezing of cell samples at )20 �C (data not shown).Indeed, this pattern of activation is slower than thedescribed for heat shock, osmostress, treatment withreactive oxygen species or UV radiation [23,24], but similarto the kinetics observed in response to nitrogen starvation[32]. It thus appears that the cold stress signal is transducedthrough the SAPK cascade in S. pombe with a rate specificof this type of stress and relatively dependent of the severityof 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 cellularreceptors that trigger activation of Sty1p but the nature ofthe upstream components involved in this signalling remainfor most part unknown [25]. A possible reason for the slowresponse in Sty1p phosphorylation during sensing andtransduction of the cold shock signal could be thatmembrane fluidity decreases greatly upon temperaturedownshifts, thereby slowing down membrane associatedcellular functions. Also, a decreased rate in general meta-bolism would limit the generation of second messengers.

The level of Sty1p phosphorylation increasedmarkedly inresponse to cold shock and was maintained for at least 6 h(Fig. 1). Contrariwise, a severe heat shock induced a quickand transient phosphorylation of Sty1p that becamedephosphorylated within 60–90 min. This is likely due tothe activity of threonine-phosphatases Ptc1p and Ptc3p andtyrosine-phosphatase Pyp1 [49]. Other treatments, such asosmotic 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 inthe dephosphorylation of the osmotic stress-activated Sty1p[20]. Interestingly, however, the Sty1p activation induced byoxidative stress, although rather rapid, was followed by avery slow decrease in Sty1p phosphorylation, as during acold shock (see Fig. 1). This differential behaviour mayreflect that one or several of the protein phosphatases aremaintained partially inactive when S. pombe is subjected toany of these two stresses.

The fact that Wis1p is apparently the only MAPKK thatactivates Sty1p during cold stress (Fig. 2) is not surprising,as this is the case for other stresses [20,21,23,24]. Wis1p isthus able to integrate the transmission of different stresssignals to Sty1pMAP kinase, including thermal downshifts.From the two MAPKKKs that are known to activateWis1p (i.e.Win1p or Wak1p), Wak1p appears to be themain responsible for Sty1p activation during cold 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 being photographed.

5062 T. Soto et al. (Eur. J. Biochem. 269) � FEBS 2002

(Fig. 2A), similar to what happens during osmostress [28].The Win1p mitotic regulator, which controls the activationof Sty1p kinase under multiple stressful conditions [27], islikely responsible for the slight cold-induced activation ofSty1p observed in the absence of Wak1p (Fig. 2B). Thus,the signal transmission for cold appears to follow abranched pathway, with either Wak1p or the redundantMAPKKKWin1p as an alternative via to activate to someextent the Sty1p kinase through Wis1p. On the other hand,our results with cells disrupted in the upstream responseregulator Mcs4p indicate that the signal for cold does notreach in such cells the level of MAPKKs, which stronglysupports the suggestion that Mcs4p likely interacts withboth Wak1p and Win1p [27,29]. Also, the occurrence ofSty1p phosphorylation in S. pombe cells expressing aD412N version of Mcs4p indicated that the Mak2p andMak3p phosphorelay system is not involved in Sty1pphosphorylation following a cold stress, which is similar towhat happens during other environmental stresses exceptduring oxidative stress. In any case, the control of SAPKpathways by two-component systems appears exclusive oflower eukaryotes under specific stresses, as no structuralhomologues of phospho-relay proteins have been identifiedin mammals [29]. Thus, because such a system is notoperative in S. pombe during cold induced activation of theSAPK pathway, this model may be relevant to study andcharacterize the transduction of temperature downshiftssignals in cells from higher eukayotes, including mammals.

Following a shift to low temperature, the bZIP trancrip-tion factor Atf1p becomes phosphorylated in vivo in aSty1p-dependent manner (Fig. 3). In coincidence withprevious studies, our data confirm that Sty1p is the onlykinase able to phosphorylate Atf1p at any stress condition[32,33]. As Atf1p becomes phosphorylated under coldtemperature, one might anticipate changes in the expressionof Atf1p-regulated genes upon incubation of S. pombe atlow 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-triggeredexpression of gpd1+ might be interpreted in terms ofsynthesis of a cryoprotectant metabolite [50]. Catalase mayalso act as a protectant as it has been shown in plants andyeast cells that cold involves oxidative stress [51,52].Additionally, we have observed a retarded cold-inducedincrease in the expression of tps1+ and ntp1+, which codefor enzymes involved in trehalose metabolism. This iscongruent with accumulation and turnover of the lowmolecular mass carbohydrate trehalose, a well knownstabilizer of macromolecular components [53]. As a whole,it appears that the induction of compatible solutes anddefences against oxidant species forms part of the responseto low temperature and that the expression of a conservedset of stress-responsive genes is the basis of the general stressresponse underlying crossed stress tolerance. In this respect,pretreatment with low temperature induces a significantadaptive response to osmostress in S. pombe cells (F.F.Beltran and J. Cansado, unpublished results). It is worth tomention that transcription factor Pap1p is neither activatednor translocated from the cytoplasm to the cell nucleus atlow temperatures and that ctt1+ expression was induced atlow temperatures in a pap1-deficient strain (data not

shown). Thus, in analogy to what happens during osmoticstress [33], the expression of ctt1+ at cold temperatureappears to be mostly dependent on Atf1p/Pcr1p.

Although the MAP kinase pathway confers a slight, butconsistent, growth advantage at 15 �C (Fig. 5), this path-way is not essential for growth at such temperature.Instead, the transcription factors Atf1p and Pcr1p, whichare key effectors of the SAPK phosphorylation cascaderequired for a variety of developmental decisions [31–34]and dispensable for growth at 28 �C, were needed forgrowth in the cold. Surprisingly, however, the cold sensitivephenotype in atf1– or pcr1– strains is not shared by mutantsdisrupted in either sty1+ or wis1+ (Fig. 5), indicating thatthe role of these factors at low temperature is independentof their function as SAPK-driven multifunctional switchesthat activate specific responses against extracellular condi-tions. Hence, although cold stress in S. pombe induces theSAPK pathway, the function of this cascade does notguarantee far more than a slight better adjustment of cellgrowth to cold conditions. On the contrary, the presence ofAtf1p and Pcr1p (presumably acting as a heterodimer) isvital for growth al low temperature by a mechanismunrelated to the SAPK pathway. Preliminary observationsindicate that there is not specific cell cycle block in Datf1cells arrested at 15 �C. Altogether, these data are consistentwith a new role for these factors in transcriptional eventssustaining specific 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 Fundacion

Seneca (Region de Murcia) and the Fundacion Ramon Areces,

respectively. This work was supported in part by grant BMC

2001–0135 from MCYT, Spain.

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