Cold induces stress-activated protein kinase-mediated response in the fission yeast Schizosaccharomyces pombe Teresa Soto 1 , Francisco F. Beltra ´ n 1 , Vanessa Paredes 1 , Marisa Madrid 1 , Jonathan B. A. Millar 2 , Jero Vicente-Soler 1 , Jose ´ Cansado 1 and Mariano Gacto 1 1 Departamento de Gene ´ tica y Microbiologı ´ a, Facultad de Biologı ´ a, Universidad de Murcia, Spain; 2 Division 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 28 °Cto15°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 during the 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 inducing the 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 living organisms. 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 favouring efficient 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 sensing and 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-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) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03214.x [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 following stress [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 lacking Pcr1p, 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-binding properties [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 resulting in 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. MATERIALS AND METHODS Strains and culture media The S. pombe strains employed in this study are listed in Table 1. They were routinely grown with shaking at 28 °C 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.) 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. 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 37 °C in Luria–Bertani medium plus 50 lgÆmL )1 ampi- cillin. Strains TS-1, TS-2, TS-3 and TS-4 were constructed by mating the appropriate parental strains (see Table 1), and selecting diploids 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 (D 600 ¼ 0.7–1) at 28 °C were subjected to heat (48 °C), osmotic (0.75 M NaCl), oxidative (1 m M H 2 O 2 ), or cold (15 °C) stresses. At different times, the cells from 30 mL of culture were collected in Falcon tubes containing ice (equivalent to 10 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 Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5057 4 °C. Under these conditions, the previously described Sty1p phosphorylation due to centrifugation [28] was not observed in unstressed cells. After washing with NaCl/P i buffer, 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 employing chilled acid-washed glass beads and lysis buffer (10% glycerol, 50 m M Tris/HCl pH 7.5, 150 m M NaCl, 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 using Ni 2 -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 denaturing lysis buffer (6 M Guanidine HCl, 0.1 M sodium phosphate, 50 m M Tris HCl, pH 8.0) and the Atf1p protein isolated by affinity precipitation on Ni 2 -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. pombe mutants and wild-type strains were grown in YES liquid medium to mid-log phase and, after appropriate dilutions, different number of cells were spotted per duplicate on YES agar plates and incubated either at 28 °C 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], andusedtoprobeforleu1 + 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). RESULTS Sty1p activation following a cold stress The effects of low temperature in the fission yeast have been scarcely investigated [34]. Because in S. pombe arangeof environmental stresses activates Sty1 MAP kinase through phosphorylation [28] we examined if Sty1p was also activated following a 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 employing Ni 2 -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-tag in order to normalize the protein level during the course of the experiment. As shown in Fig. 1, a thermal downshift from 28 to 15 °C 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 during cold shock (Fig. 1). These results show that phos- phorylation of Sty1 kinase of S. pombe can be fully induced by low temperature at 15 °C, although with a delayed kinetics as compared to the effect of other previously characterized stimuli. Cold shocks performed in a range from 10 to 25 °C 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 25 °C, which is relatively close to optimal temperature for growth (28–30 °C), 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 15 °C(Fig.2A). These analyses revealed that Wis1 MAPK kinase is essential for Sty1p activation during a cold stress in S. pombe. 5058 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Wis1p-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 during an 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 15 °Cin 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. Sensing of 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 during oxidative stress Mcs4p acts in a conserved phospho-relay system initiated by two PAS/ PAC domain-containing histidine 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 during cold stress in strain VB1700, where mcs4 + is replaced by a mutant allele bearing a 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 Among the 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 during a 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 15 °C 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 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 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- noblotting with anti-Ha antibody as loading control. Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5059 always with the same apparent size, corresponding to the unphosphorylated form (Fig. 3). These results clearly indi- cate that Sty1p is the only MAP kinase that phosphorylates Atf1p in vivo following a 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 + , encoding glycerol-3 phosphate dehydroge- 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 – ,andatf1 – strains of S. pombe during a 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 cellssubjectedtoacold 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 and Mcs4p 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 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 followingacoldstress.Wild-type (JM1821) and Dsty1 (TS-4) strains carrying a chro- mosomal copy of Ha-tagged atf1 + gene were grown at 28 °C(0time)andshiftedto15°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 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) Ó FEBS 2002 regulated by Atf1p [24,26], was induced in a Sty1p-Atf1p- dependent manner during exposure 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. pombe is 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 studying the 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 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 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 28 °Cresultedingrowthresump- 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 during cold stress in the absence of the Sty1 kinase that channels the stress signal (Fig. 4), the role of Atf1p/Pcr1pingrowthatlowtemperatureislikely 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)weregrowninYESmediumtomid-logphaseandshiftedto15°Cforthetimes 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 with 32 P-labelled probes of gpd + ,ctt1 + , tps1 + , ntp1 + and leu1 + (loading control). Numbers below each frame indicate normalized relative values of expression. Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5061 DISCUSSION The main aim of this study was to analyse the cold shock response in S. pombe,ayeastthatdisplaysacascadeof 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 37 °C, although the optimum temperature forgrowthis28–30°C [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 during a thermal downshift to 15 °C, with maximal level within 2–3 h of exposure. This cold-induced activation is unnoticed unless the span of sampling to 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 interesting observation concerns the delay in the kinetics of phosphorylation and dephosphorylation of Sty1p 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 to the freezing of 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 during sensing and transduction of the cold shock signal could be that membrane fluidity decreases greatly upon temperature downshifts, thereby slowing down 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 during a 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 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 °Cor15 °C for 3 and 10 days, respectively, prior to being photographed. 5062 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (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 MAPKKKWin1pasanalternativeviatoactivatetosome 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 expressing a D412N version of Mcs4p indicated that the Mak2p and Mak3p phosphorelay system is not involved in Sty1p phosphorylation following a cold stress, which is similar to what happens during other environmental stresses except during oxidative 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 during cold 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. Following a 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 underlying crossed 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 15 °C (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 28 °C, 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 acting as 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 15 °C. Altogether, these data are consistent with a new role for these factors in transcriptional events sustaining specific development programs in the cold. 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