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Molecular and Cellular Biology, May 2005, p. 3945-3955, Vol. 25, No. 10
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.10.3945-3955.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Response of Fission Yeast to Toxic Cations Involves Cooperative Action of the Stress-Activated Protein Kinase Spc1/Sty1 and the Hal4 Protein Kinase

Ling-yu Wang, Koichi Shimada,{dagger} Masayo Morishita, and Kazuhiro Shiozaki*

Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616

Received 13 December 2004/ Returned for modification 19 January 2005/ Accepted 22 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stress-activated protein kinases (SAPKs), members of a mitogen-activated protein kinase (MAPK) subfamily, are highly conserved among eukaryotes. Studies of yeasts demonstrated that SAPKs play pivotal roles in survival responses to high osmolarity, oxidative stress, and heat shock. Here we report a novel physiological role of the fission yeast Spc1 SAPK in cellular resistance to certain cations, such as Na+, Li+, and Ca2+. Strains lacking Spc1 or its activator, Wis1 MAPK kinase, are hypersensitive to these cations. Spc1 positively regulates expression of sod2+ encoding a Na+/H+ antiporter through Atf1 and other transcription factors. In addition, we have identified a novel Spc1-interacting protein, Hal4, which is highly homologous to the budding yeast Sat4/Hal4 protein kinase. Like its budding yeast counterpart, the fission yeast Hal4 kinase is essential for cellular resistance to Na+, Li+, and Ca2+. The hal4-null phenotype is complemented by overexpression of the Trk1 potassium transporter or increased K+ in the growth medium, suggesting that Hal4 promotes K+ uptake, which consequently increases cellular resistance to other cations. Interestingly, the Spc1-Hal4 interaction appears to be required for cellular resistance to Ca2+ but not Na+ and Li+. We propose that Spc1 SAPK and Hal4 kinase cooperatively function to protect cells from the toxic cations.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In response to environmental fluctuations, cells modulate diverse aspects of their physiology for maximal adaptation. In eukaryotes from yeast to humans, stress-activated protein kinases (SAPKs) play key roles in cellular responses to the environment. SAPKs belong to the mitogen-activated protein kinase (MAPK) family, and their prototype, Hog1, was first identified as a component of the signaling pathway that induces cellular response to high-osmolarity stress in Saccharomyces cerevisiae (6). In the fission yeast Schizosaccharomyces pombe, the Hog1 ortholog, Spc1 (also known as Sty1/Phh1), is responsive to multiple forms of environmental stress (23, 30, 50). Osmostress, oxidative stress, UV, and heat shock as well as nutritional limitation induce Spc1 activation, and the spc1-null ({Delta}spc1) mutant is hypersensitive to these stress conditions, indicating the essential roles of Spc1 SAPK in cellular resistance to the stresses (10, 11, 51, 56). The same set of stresses also activate the mammalian SAPKs p38 and JNK, which have been implicated in a variety of biological processes, including inflammation, cellular differentiation, apoptosis, cancer, and immune responses (25). Because of the evolutionary conservation of SAPKs, genetic studies of yeasts have been instrumental for discovery and characterization of SAPK regulation and function (12, 37).

Like other MAPKs, SAPKs are activated through simultaneous phosphorylation of the conserved threonine and tyrosine residues in the activation loop (61). In S. pombe, Spc1 is phosphorylated and activated by Wis1 MAPKK (30, 50). Wis1 is also activated through phosphorylation carried out by MAPKKKs, Wis4 and Win1 (42, 43, 49, 56), and aspartate substitutions for the MAPKKK phosphorylation sites in Wis1 (wis1DD) mimic phosphorylation and constitutively activate Wis1 (55). On the other hand, Spc1 is negatively regulated by dephosphorylation; tyrosine phosphatases Pyp1 and Pyp2 (11, 30, 50) and a serine/threonine phosphatase, PP2C (36, 52), dephosphorylate and inactivate the SAPK. A recent study indicates that Spc1 is also regulated by a molecular chaperone, Cdc37 (59).

One of the known effects of Spc1 activation is to induce expression of genes required for cellular survival of stress conditions. Activated Spc1 translocates from the cytoplasm to the nucleus (15) and regulates gene expression through an ATF/CREB-family transcription factor, Atf1 (51, 62). Recent DNA microarray experiments demonstrated that the Spc1-Atf1 pathway is required for the majority of the core environmental stress response (CESR) genes, ~140 genes that are induced under multiple stress conditions (9). The CESR genes comprise those involved in a variety of cellular processes, including carbohydrate and lipid metabolism, DNA repair, protein folding/degradation, antioxidants, and transcription, illustrating that the cellular stress response encompasses modulation of diverse cellular processes. In addition to Atf1, Spc1 SAPK also interacts and phosphorylates two protein kinases, Cmk2 and Srk1 (4, 44, 57), although the contribution of these kinases to cellular stress resistance is less obvious.

Here we report a novel physiological role of Spc1 SAPK: Spc1 activity is essential for cellular resistance to Na+, Li+, and Ca2+, cations the concentrations of which are normally kept relatively low in the cytoplasm. Spc1 positively regulates expression of the sod2+ gene encoding a plasma membrane Na+/H+ antiporter, which exports Na+ and Li+ (22). In addition, through a yeast two-hybrid screen, we have isolated a novel protein kinase that physically interacts with Spc1. This kinase has been named Hal4, because of its high similarity to the budding yeast Sat4/Hal4 kinase, which is known to play a role in cellular salt tolerance (33). Like {Delta}spc1, the S. pombe {Delta}hal4 mutant shows hypersensitivity to Na+, Li+, and Ca2+. The multicopy suppressor screen of the {Delta}hal4 cation sensitivity has isolated the trk1+ potassium transporter gene, and potassium added to the growth medium complements the {Delta}hal4 phenotypes. These results suggest that Hal4 kinase promotes K+ uptake, which increases the cellular resistance to other toxic cations. We propose that Spc1 SAPK and Hal4 kinase act cooperatively for cellular cation homeostasis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Yeast strains and cultures. S. pombe strains used in this study are listed in Table 1. Growth media and basic techniques for S. pombe have been described previously (2, 32). For construction of the spc1KM strain, Lys-49 of the ATP-binding site in Spc1 was mutated to Met by the QuikChange XL site-directed mutagenesis kit (Stratagene) in the spc1+ DNA fragment, which was used to transform the spc1::ura4+ strain (CA1366). Ura– transformants were selected by 5-fluorootic acid-containing medium, and replacement of the spc1::ura4 locus with spc1KM was confirmed by Southern hybridization. Expression of the Spc1KM protein was also confirmed by anti-Spc1 immunoblotting.


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  		TABLE 1. S. pombe strains used in this study

 
S. pombe cells were grown in YES yeast extract medium and EMM2 synthetic minimal medium. NaCl, KCl, LiCl, and CaCl2 were added in YES medium as indicated. Salt tolerance was determined by the drop test using saturated culture diluted to an optical density at 600 nm of 1.0 and subsequent 10-fold serial dilutions. SD minimal medium used for the growth test was previously described (48).

Purification and detection of the Spc1HA6H protein. KS1376 (spc1:HA6H) cells growing at 30°C in YES medium were stressed with 0.6 M or 0.1 M KCl and 0.1 M NaCl and harvested along the time course by rapid filtration (54). The Spc1HA6H protein was purified under denaturing conditions and analyzed by immunoblotting with anti-phospho-p38 antibodies (54).

Multicopy suppressor screens. To isolate multicopy suppressors that complement the Li sensitivity of the {Delta}spc1 mutant, strain KS1366 ({Delta}spc1) was transformed with an S. pombe genomic library in the pAL-KS vector (58) and plated onto EMM2 agar plates supplemented with uracil, adenine, and histidine. After incubation at 30°C for 4 days, transformants were replica plated onto YES agar plates containing 4 mM LiCl. Colonies that showed plasmid-dependent resistance to LiCl were isolated, and plasmids recovered from these colonies were analyzed by DNA sequencing. The multicopy suppressor screen with the {Delta}hal4 strain was performed similarly, except that a lower concentration of LiCl (2 mM) was used for selection.

Isolation of the hal4+ gene by yeast two-hybrid screens. The spc1TA mutant gene, in which Thr-171 is replaced with Ala, was constructed by the QuikChange XL site-directed mutagenesis kit and used as bait in a yeast two-hybrid screen to isolate Spc1-interacting proteins from the S. pombe two-hybrid cDNA library in the pGADGH vector (18). The yeast two-hybrid assay was carried out in S. cerevisiae HF7c (14) by His+ selection and the ß-galactosidase assay. From ~107 transformants screened, three different plasmids containing the hal4+ cDNA were isolated.

Plasmid construction. Sequences of the PCR primers used in this study are available upon request. All PCR-amplified fragments were confirmed by DNA sequencing.

The hal4+ open reading frame (ORF) was amplified by PCR using S. pombe genomic DNA as template and cloned into the pREP1 (29) and pREP1-KZ (53) vectors. The QuikChange XL site-directed mutagenesis kit was used to introduce a point mutation into the hal4+ gene to construct the hal4KM allele. An internal deletion mutant of hal4, {Delta}(181-230), was constructed by a PCR-based mutagenesis method previously described (24). For the yeast two-hybrid assays to characterize interaction between Hal4 and Spc1, hal4 coding sequences for the full length (1 to 636) or residues 181 to 350, 231 to 636, 181 to 300, 231 to 350, and 181 to 250 were amplified by PCR with S. pombe genomic DNA as template and cloned into the pGADGH vector. The wild-type spc1+ gene was used as bait. To express the full-length and the C-terminally truncated Trk1, the entire ORF (residues 1 to 841) as well as the sequence encoding residues 1 to 803 was amplified by PCR and cloned into the S. pombe expression vector pNMT1 (Invitrogen).

Construction of hal4:myc strain. For constructing strains in which the chromosomal hal4+ gene is tagged with the sequences encoding 12 copies of the Myc epitope, a NotI site was introduced by PCR at the 3' end of the hal4+ ORF. The resultant PCR fragment of the 3'-terminal, 648-bp hal4+ ORF was used to construct the pBluescript-{Delta}NHal4-Myc plasmid with the ura4+ marker gene. The plasmid was linearized at a XbaI site within the hal4 ORF and used to transform a wild-type (PR109) strain, and the integration of the plasmid at the hal4+ locus was confirmed by Southern blotting analysis. The cation resistance of the resultant hal4:myc strain was comparable to that of wild-type cells, indicating that the hal4:myc fusion gene is functional.

Immunoprecipitation. hal4:myc (CA1998), hal4:myc {Delta}spc1 (CA2021), and cmk2:myc (KS2012) cells, grown to mid-log phase at 30°C in YES medium, were harvested before and after stress treatments by rapid filtration (54). Cells were lysed in lysis buffer (50 mM HEPES, pH 7.4, 2 mM EDTA, 2 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 0.1 mM Na3VO4). Anti-Spc1 rabbit polyclonal antibodies conjugated to protein A-Sepharose (Pharmacia Biotech) were used to precipitate Spc1 protein from the lysate, which was followed by immunoblotting with anti-Spc1 and mouse monoclonal anti-Myc antibodies (9E10; Boehringer Mannheim).

Gene disruption. For hal4 gene disruption, the 0.5-kb genomic sequences immediately upstream and downstream of the hal4+ ORF were amplified by PCR with pairs of primers, sp27-f and sp27-fcT7 for the upstream sequence and T3sp27-b and sp27-bc for the downstream sequence. Subsequently, these fragments were ligated to the 5' and 3' ends of the 1.8-kb ura4+ marker gene fragment through another PCR. The resultant PCR product, a ura4+ fragment flanked by the genomic sequences adjacent to hal4+, was used for transformation. Stable Ura+ transformants were isolated, and the disruption of the hal4 locus was confirmed by Southern hybridization experiments. Similar procedures were used in the trk1 and trk2 gene disruptions. The PCR primers used were U5trk1 and U3trk1 to amplify the sequence upstream of trk1+ and D5trk1 and D3trk1 to amplify the sequence downstream of trk1+. The trk2+ upstream sequence was amplified with primers U5trk2 and U3trk2, and the trk2+ downstream sequence was amplified with D5trk2 and D3trk2 primers. Instead of ura4+, kanMX6 (5) was used as the marker gene, and transformants were selected on YES agar plates containing 0.1 mg/ml G418.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inactivation of the Spc1 MAPK cascade brings about hypersensitivity to toxic cations. Mutants lacking functional Spc1 SAPK or Wis1 MAPKK are hypersensitive to high-osmolarity stress, indicating that the Spc1 cascade is essential for cellular responses to high-osmolarity stress (30, 50, 52). During further characterization of the osmostress response of S. pombe cells using various salts as osmolyte, we noticed that the spc1-null ({Delta}spc1) mutant was hypersensitive to even low concentrations of NaCl. While wild-type cells grew in the presence of 0.5 M NaCl (data not shown; 22), even 0.1 M NaCl in the growth medium caused an apparent growth defect in the {Delta}spc1 mutant (Fig. 1A). The same concentration of KCl did not inhibit the growth of {Delta}spc1 cells, suggesting that the observed growth inhibition is due to Na+, rather than Cl– or osmolarity. Because the {Delta}wis1 MAPKK mutant also exhibited very similar Na+ sensitivity (Fig. 1A), the activity of Spc1 MAPK appears to be required for cellular resistance to Na+. The requirement of the Spc1 kinase activity for the cation resistance was further confirmed by the spc1KM strain, which expresses a catalytically inactive Spc1 SAPK with a point mutation in its ATP-binding site; spc1KM mutant cells are sensitive to high osmolarity of 1 M KCl as well as to low concentrations of Na+ (Fig. 1B). We therefore examined whether cells induce activation of Spc1 SAPK in response to Na+ stress. In contrast to high-osmolarity stress by 0.6 M KCl, addition of 0.1 M NaCl to the growth medium brought about only weak, transient Spc1 activation comparable to that by 0.1 M KCl (Fig. 1C), probably due to mild osmostress. Thus, although Spc1 activity is essential for cellular survival of Na+ stress, Na+ is not an activating stimulus for the SAPK.



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  		FIG. 1. The Spc1 MAPK cascade is required for cellular resistance to the toxic cations. (A) Wild-type (KS1598), {Delta}spc1 (KS1366), {Delta}wis1 (JM544), {Delta}atf1 (KS1497), and {Delta}cmk2 {Delta}srk1 (CA2685) strains were grown in liquid YES medium, and serial dilutions were spotted on YES agar plates containing 0.1 M KCl, 0.1 M NaCl, 4 mM LiCl, and 80 mM CaCl2. (B) The spc1KM strain (CA3081), which expresses a catalytically inactive Spc1 mutant kinase, was grown in YES medium, and serial dilutions were spotted on YES plates with 1 M KCl for osmostress, 0.1 M KCl, and 0.1 M NaCl. (C) The spc1:HA6H strain (KS1376) grown to the mid-log phase at 30°C in YES medium was treated with 0.6 M KCl, 0.1 M KCl, and 0.1 M NaCl, and aliquots were harvested at the indicated times. The Spc1HA6H protein was purified under denaturing conditions and analyzed by immunoblotting with anti-phospho-p38 antibodies, which specifically recognize the phosphorylated, active form of Spc1 MAPK. (D) The cation sensitivity of the wis1DD (KS2081) and wis1DD {Delta}atf1 (CA363) strains was tested by the spot test on YES plates with 0.1 M KCl, 0.1 M NaCl, and 4 mM LiCl. These strains express the Wis1DD mutant MAPKK, in which the MAPKKK phosphorylation sites, Ser-469 and Thr-473, were replaced with aspartic acid for constitutive activation. WT, wild type.

 
Survey of other metal ions demonstrated that the {Delta}spc1 mutant is also hypersensitive to Li+ and Ca2+ (Fig. 1A). To further confirm that the Spc1 cascade has an active role in protecting cells from these cations, we examined the cation sensitivity of a strain expressing the mutant Wis1DD MAPKK. Wis1DD has aspartate substitutions at the MAPKKK phosphorylation sites and constitutively activates Spc1 SAPK (55). As shown in Fig. 1D, the wis1DD allele conferred higher resistance to the toxic cations. We also observed elevated cation resistance with a strain lacking the Pyp1 tyrosine phosphatase (data not shown), a negative regulator of Spc1 SAPK (30, 50). These results strongly suggest that the Spc1 cascade positively regulates cellular resistance to the cations.

The Atf1 transcription factor contributes to cellular cation resistance. To understand how Spc1 SAPK regulates cellular responses to the toxic cations, we next examined whether the known Spc1 substrates are required for the cation resistance. The Atf1 transcription factor is phosphorylated by Spc1 upon stress and regulates a number of stress resistance genes (51, 62). The {Delta}atf1 strain was not significantly sensitive to 0.1 M NaCl, although it showed moderate Li+ sensitivity (Fig. 1A). On the other hand, Atf1 is clearly important for the increased Na+ and Li+ resistance of wis1DD cells, because the wis1DD {Delta}atf1 double mutant is no more resistant to these cations than wild-type cells are (Fig. 1D). As previously reported (39), {Delta}atf1 cells are very sensitive to Ca2+, a phenotype severer than that of the {Delta}spc1 strain (Fig. 1A). Thus, the cation-sensitive phenotypes of {Delta}spc1 and {Delta}atf1 are not identical, implying that, in addition to Atf1, Spc1 may function also independently of Atf1 in cellular responses to the toxic cations.

Two protein kinases, Cmk2 and Srk1, have recently been reported as downstream targets of Spc1 SAPK (4, 44, 57). The {Delta}cmk2 {Delta}srk1 double mutant (Fig. 1A) as well as the individual single mutants (data not shown) did not show altered sensitivity to Na+, Li+, and Ca2+, indicating that these Spc1 substrates are not involved in the cellular cation resistance.

Spc1 SAPK regulates expression of sod2+, a Na+/H+ antiporter gene. Aiming to discover how Spc1 affects the cellular resistance to the toxic cations, we performed two screens, a multicopy suppressor screen of the {Delta}spc1 mutant and a yeast two-hybrid screen with Spc1 as bait. In the former screen, we screened an S. pombe genomic library for plasmid clones that rescue the Li+-sensitive phenotype of the {Delta}spc1 mutant (Materials and Methods). Other than the plasmids carrying spc1+ itself, this screen repeatedly isolated plasmids that contain the sod2+ gene encoding a Na+/H+ antiporter in the plasma membrane (22) (Fig. 2A). Sod2 exports Na+ and Li+, and increased expression of Sod2 confers cellular resistance to these cations (22). Indeed, the multicopy sod2+ plasmid complemented both Li+ and Na+ sensitivities of {Delta}spc1 cells (Fig. 2A) but not their Ca2+-sensitive phenotype (data not shown). In order to test the possibility that sod2+ expression is regulated by Spc1 SAPK, the sod2+ mRNA level was quantified by Northern blotting in wild-type and {Delta}spc1 strains. The sod2+ mRNA level in the {Delta}spc1 mutant was reduced to ~50% of that in wild-type cells (Fig. 2B), indicating that Spc1 SAPK positively regulates sod2+ expression. Consistent with this idea, sod2+ expression was increased by ~30% in the wis1DD mutant, which expresses activated Wis1 MAPKK. The sod2+ mRNA level in the {Delta}atf1 strain was ~75% of that in wild-type cells, implying that Atf1 partly contributes to sod2+ transcription. These results suggest that Spc1 SAPK regulates sod2+ expression through Atf1 and other transcription factors and that the reduced sod2+ level in the {Delta}spc1 mutant contributes to its Na+- and Li+-sensitive phenotypes.



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  		FIG. 2. Spc1 positively regulates expression of sod2+ encoding a Na+/H+ antiporter. (A) sod2+ was isolated as a multicopy suppressor of the {Delta}spc1 Li+ sensitivity. The {Delta}spc1 strain (CA1366) carrying an empty vector and a sod2+ plasmid isolated from the multicopy suppressor screen was grown in selective EMM medium, and serial dilutions were spotted on YES plates with 4 mM LiCl and 0.1 M NaCl. (B) sod2+ Northern blotting of wild-type (CA1598), {Delta}spc1 (CA1366), {Delta}atf1 (KS1497), and wis1DD (KS2081) strains. Poly(A)+ RNA was hybridized with 32P-labeled sod2+ probe. Signal levels quantified and normalized by control hybridization with the leu1+ probe are indicated below each lane. WT, wild type.

 
Isolation of Hal4 protein kinase as a novel Spc1-interacting protein. In the yeast two-hybrid screen for proteins interacting with Spc1, the cDNA of Cmk2, a known substrate of Spc1 (4, 44), as well as plasmids carrying the ORF SPAC29A4.16, was isolated. SPAC29A4.16 encodes a putative serine-threonine protein kinase most homologous to the S. cerevisiae Sat4/Hal4 kinase (33); therefore, we termed this S. pombe gene hal4+. The hal4+ gene encodes a 636-amino-acid, 69-kDa protein with a C-terminal kinase catalytic domain. The amino acid sequence similarity between the S. pombe Hal4 and S. cerevisiae Sat4 proteins is mostly limited to their catalytic domains, with 67% identity (Fig. 3A, shaded). Published genome data for other fungi, including Candida, Aspergillus, and Neurospora spp., shows that they also contain protein kinases with high similarities in their catalytic domains (data not shown). On the other hand, little similarity was detected in the N-terminal, noncatalytic domains. In addition to Sat4, S. cerevisiae has another homologous protein kinase, Hal5 (33), but no apparent paralog was found in the S. pombe genome.



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  		FIG. 3. S. pombe Hal4 is highly homologous to the S. cerevisiae Sat4/Hal4 protein kinase. (A) Amino acid sequence alignment between the kinase domains of S. pombe Hal4 and S. cerevisiae Sat4/Hal4. Identical residues are shaded. (B) The Spc1-binding domain within S. pombe Hal4 was determined by testing the interaction of truncated Hal4 constructs with Spc1 in the yeast two-hybrid assay. Residues from 181 to 230 (represented by a hatched box) are required for binding to Spc1.

 
The shortest hal4 cDNA clone isolated in the two-hybrid screen lacked the sequence for the N-terminal 180 residues (181 to 636 in Fig. 3B). We confirmed that the full-length (1 to 636) Hal4 also interacts with Spc1 in the two-hybrid assay. Further truncation analyses of the hal4 sequence in the same assay demonstrated that residues 180 to 230 in the noncatalytic domain are critical for interaction with Spc1 (Fig. 3B). This region does not share any apparent similarity with the previously reported MAPK-docking sequences (21, 47).

Interaction between Spc1 SAPK and Hal4 kinase was also confirmed biochemically. We inserted the Myc epitope sequence at the 3' end of the chromosomal hal4+ ORF, so that the Hal4-Myc fusion protein is expressed from the hal4 locus. Unlike the {Delta}hal4 strain (see below), the resultant hal4:myc strain shows cation resistance comparable to that of hal4+ cells (data not shown), indicating that the Hal4-Myc fusion protein is functional. Immunoprecipitation of Spc1 from the hal4:myc cell lysate copurified Hal4-Myc, while a similar procedure using {Delta}spc1 hal4:myc cells resulted in little precipitation of Hal4-Myc (Fig. 4). Stress conditions that activate Spc1 SAPK, such as osmostress, did not affect the Hal4-Spc1 interaction. Although coprecipitation of Hal4 with Spc1 was not as efficient as that for another Spc1-interacting kinase, Cmk2, under the experimental conditions tested (Fig. 4), these biochemical data corroborated the in vivo interaction between Hal4 and Spc1 SAPK.



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  		FIG. 4. Hal4 kinase physically associates with Spc1 SAPK. The hal4:myc (CA1998) and hal4:myc {Delta}spc1 (CA2021) strains, which express Hal4 fused to the Myc epitope, were grown to the mid-log phase at 30°C in YES medium. Aliquots of cells were harvested before (–) or after 10-min high-osmolarity stress from 0.6 M KCl (Os), and immunoprecipitates by anti-Spc1 antibodies (IP) were analyzed by Western blotting with anti-Myc and anti-Spc1 antibodies. The Hal4 protein levels in the crude lysate used in this experiment were also examined by anti-Myc immunoblotting (bottom panel). The cmk2:myc strain (KS2012) expressing the Cmk2-Myc fusion protein was used as a positive control.

 
Hal4 kinase does not appear to be a regulator of Spc1 activation. Quantification of Spc1 activation in hal4+ and {Delta}hal4 strains showed comparable activation of Spc1 in response to the known Spc1-activating stresses (data not shown).

Hal4 protein kinase is required for cellular resistance to the toxic cations. S. cerevisiae SAT4/HAL4 and its paralog, HAL5, were isolated for their ability to confer halotolerance on wild-type cells upon overexpression, and the null mutants of these genes exhibit hypersensitivity to Na+, Li+, and Ca2+ (33). To test whether S. pombe Hal4 is also required for cellular resistance to these cations, we constructed the hal4-null ({Delta}hal4) mutant by gene disruption. Haploid {Delta}hal4 cells were viable and showed no apparent growth defect in both rich and minimal growth media (data not shown). However, their growth was impaired in the presence of Na+, Li+, and Ca2+, and these {Delta}hal4 phenotypes were even severer than those of the {Delta}spc1 mutant (Fig. 5A). On the other hand, in contrast to {Delta}spc1, the {Delta}hal4 mutant is not sensitive to high-osmolarity stress of 1 M KCl. These results indicate that Hal4 protein kinase plays an important role in cellular resistance to Na+, Li+, and Ca2+. We noticed that the {Delta}spc1 {Delta}hal4 double mutant is more sensitive to those cations than the individual single mutants are (Fig. 5A). This observation suggests that the functional relationship between Spc1 and Hal4 in the cellular cation response is not simply through a linear pathway; both Spc1 activation and Hal4 function are required for proper cellular responses to Na+, Li+, and Ca2+. Consistently, overexpression of Hal4 did not complement the {Delta}spc1 cation sensitivity, and overexpression of Spc1 did not rescue the {Delta}hal4 phenotype (data not shown).



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  		FIG. 5. Hal4 kinase is required for cellular resistance to Na+, Li+, and Ca2+. (A) The growth phenotypes of wild-type (KS1598), {Delta}spc1 (KS1366), {Delta}hal4 (CA1787), and {Delta}spc1 {Delta}hal4 (CA1992) strains were examined as described in Fig. 1, except that lower concentrations of LiCl (2 mM) and CaCl2 (20 mM) were added to YES agar plates. Unlike {Delta}spc1, the {Delta}hal4 mutant is not sensitive to high osmolarity from 1 M KCl. (B) The protein kinase activity of Hal4 is required for the toxic cation resistance. The {Delta}hal4 (CA1787) strain expressing GST (pGST) alone or wild-type (pGST-hal4) or kinase-inactive (pGST-hal4KM) Hal4 proteins fused to GST was grown on YES plates containing 0.1 M NaCl and 2 mM LiCl. Expression of the GST-Hal4 and GST-Hal4KM proteins was confirmed by glutathione bead precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie blue staining (right panel). (C) Wild-type and mutant Hal4 proteins lacking the Spc1-binding domain (residues 181 to 230) were expressed in the {Delta}hal4 (CA1787) strain using the pREP1 expression vector (29) with the C-terminal hemagglutinin (HA) epitope, and cell growth was tested on YES plates containing 0.1 M NaCl, 2 mM LiCl, and 80 mM CaCl2. Hal4{Delta}(180-230) complemented the Na+- and Li+-, but not Ca2+-, sensitive phenotypes of the {Delta}hal4 mutant. The expression of the full-length and {Delta}(181-230) Hal4 proteins was confirmed by anti-HA immunoblotting (right panels); antitubulin staining of the same membrane served as a loading control. WT, wild type.

 
The importance of the protein kinase activity of Hal4 for its function was confirmed by constructing a catalytically inactive Hal4 mutant. Lys-385 within the ATP-binding domain of Hal4 was replaced with methionine by site-directed mutagenesis. Expression of this Hal4KM protein as a fusion with a glutathione S-transferase (GST) tag did not rescue the cation sensitivity of the {Delta}hal4 strain, while wild-type GST-Hal4 complemented the {Delta}hal4 defect (Fig. 5B). Thus, Hal4 functions as a protein kinase to protect cells from the cation stress.

To test whether the interaction with Spc1 is important for the Hal4 function, the mutant Hal4 lacking the Spc1-binding domain ({Delta}181-230, Fig. 3B) was expressed in the {Delta}hal4 mutant. Like the wild-type hal4+ gene, the mutant hal4{Delta}(181-230) gene complemented the Na+ and Li+ sensitivity of {Delta}hal4 cells, indicating that the mutant Hal4 kinase is active even without the Spc1-binding domain (Fig. 5C). Strikingly, however, the hal4{Delta}(181-230)-expressing cells were found to be sensitive to Ca2+, indicating that this mutant kinase cannot replace the Hal4 function in cellular resistance to Ca2+. These results imply that the Spc1-Hal4 interaction is required for cell viability in the presence of Ca2+ but dispensable for resistance to Na+ and Li+.

Hal4 protein kinase is involved in cellular potassium homeostasis. Aiming to obtain a clue for the physiological function of Hal4 kinase, we next performed a multicopy suppressor screen to isolate genes whose overexpression rescues the cation sensitivity of {Delta}hal4 cells (Materials and Methods). Screening of 6 x 104 genomic library transformants identified 76 plasmids containing hal4+ itself and one plasmid carrying the trk1 gene encoding a major potassium transporter (7, 27). The isolated trk1 clone lacked the 3' end of the ORF for the C-terminal 38 amino acid residues but complemented the cation-sensitive phenotypes of the {Delta}hal4 strain to the levels comparable to the complementation by the hal4+ plasmid (Fig. 6A). In order to test the effect of the C-terminal 38-residue truncation, the full-length and the C-terminally truncated trk1 ORFs were amplified by PCR and cloned into an expression vector. As shown in Fig. 6B, expression of the full-length Trk1 only weakly complemented the hal4 phenotypes, compared to the strong suppression by the Trk1 lacking its C-terminal 38 residues. Trk1 has 12 membrane-spanning sequences, with its N and C termini predicted to be in the cytoplasm (7, 27), and these results may suggest a role of the C-terminal cytoplasmic domain in the regulation of Trk1. On the other hand, the cation sensitivity of {Delta}spc1 cells was not significantly rescued by plasmids expressing the full-length (data not shown) or C-terminally truncated Trk1 (Fig. 6C), implying that Hal4 and Spc1 contribute to the toxic cation resistance through a distinct mechanism.



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  		FIG. 6. The cation sensitivity phenotype of the {Delta}hal4 mutant is rescued by overexpression of the Trk1 potassium transporter. (A) trk1+ was isolated as a multicopy suppressor of the {Delta}hal4 Li+ sensitivity. Wild-type (KS1598) and {Delta}hal4 (CA1787) strains transformed with the empty vector plasmid and the same multicopy vector carrying hal4+ (pHAL4) or trk1 (pTRK1) were grown in EMM minimal medium and subjected to the spot test on YES plates with 0.1 M NaCl, 2 mM LiCl, or 30 mM CaCl2. The trk1 genomic fragment in pTRK1 isolated in the multicopy screen was found to be truncated, expressing Trk1 lacking the C-terminal 38 residues. (B) Wild-type (KS1598) and {Delta}hal4 (CA1787) strains transformed with the empty pNMT1 expression vector or the same vector to express the full-length Trk1 (pNMT1-TRK1) and a truncated Trk1 lacking the C-terminal 38 residues (pNMT1-TRK1{Delta}C) were subjected to the spot test as in panel A. Expression of the truncated Trk1 efficiently complemented the {Delta}hal4 phenotype. (C) Trk1 overexpression does not complement the {Delta}spc1 phenotypes. Wild-type (KS1598) and {Delta}spc1 (KS1366) strains carrying the empty vector or the spc1+ (pSPC1) or trk1 (pTRK1) plasmid were subjected to the spot test as in panel A, using YES plates containing 0.1 M NaCl, 4 mM LiCl, and 80 mM CaCl2. Overexpression of trk1 in {Delta}spc1 cells from the pTRK1 plasmid was confirmed by Northern blotting (right panel). Twenty-five micrograms of total RNA from the {Delta}trk1 mutant (lane {Delta}; CA2227) and {Delta}spc1 strains carrying the vector or pTRK1 was hybridized with 32P-labeled trk1+ probe. WT, wild type.

 
The observation above suggests that the Hal4 function may be related to K+ transport. Interestingly, studies of budding yeast demonstrated that cellular sensitivity to the toxic cations is significantly affected by cellular potassium homeostasis. The plasma membrane H+-ATPase generates electrical membrane potential, negative inside, which is counteracted by K+ uptake (46). The plasma membrane is hyperpolarized in mutants defective in K+ transport, leading to higher rates of other cation uptake across the electrical gradient (28); therefore, those mutants are sensitive to even low concentrations of Na+, Li+, and Ca2+. Based on genetic analyses, it was proposed that S. cerevisiae SAT4 positively regulates K+ uptake through two major potassium transporters, Trk1 and Trk2 (33). Therefore, we further examined whether S. pombe Hal4 kinase is involved in cellular potassium uptake.

We found that the {Delta}hal4 mutant grows poorly in synthetic SD medium, which contains only 7 mM of K+ (Fig. 7A). This growth defect was significantly rescued by supplementing the growth medium with 0.2 M KCl, an observation consistent with the idea that Hal4 plays a role in K+ uptake. In contrast, {Delta}spc1 cells exhibited no apparent growth defect in SD medium (data not shown), suggesting that Spc1 SAPK is not required for K+ uptake. Furthermore, addition of K+ to the growth medium was found to rescue the cation-sensitive phenotypes of {Delta}hal4 cells. In the presence of 50 mM KCl, the resistance of the {Delta}hal4 mutant to the toxic cations was comparable to that of wild-type cells (Fig. 7B). The {Delta}trk1 mutant is also hypersensitive to Na+ (7) and Li+ (Fig. 7B), but this phenotype was rescued by additional K+ in the growth medium. Together, these results strongly suggest that, also in S. pombe, Hal4 kinase positively regulates K+ uptake.



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  		FIG. 7. (A) The growth defect of the {Delta}hal4 mutant under K+-limited conditions. Wild-type (KS1598) and {Delta}hal4 (CA1787) strains were grown at 30°C in SD medium supplemented with 0.2 M KCl to the mid-log phase. Cells were washed with SD medium and then diluted at time zero to an optical density at 600 nm (OD600) of 0.2 in SD medium with (+ KCl) or without 0.2 M KCl, and their growth at 30°C was monitored. (B) Growth of wild-type (KS1598), {Delta}hal4 (CA1787), {Delta}trk1 (CA2227), and {Delta}spc1 (CA1366) cells was examined by the spot test on YES agar plates containing 0.1 M NaCl and 2 mM LiCl, with (+ KCl) or without supplementation with 50 mM KCl. The cation sensitivity phenotypes of the {Delta}hal4 and {Delta}trk1 mutants were completely rescued by increased K+ in the growth medium. The {Delta}spc1 cation sensitivities were also partially complemented by addition of KCl. WT, wild type.

 
As in S. cerevisiae, the S. pombe genome contains two genes, trk1+ and trk2+, encoding evolutionarily conserved TRK-family K+ transporters (7, 27). To test whether the S. pombe Hal4 kinase regulates K+ uptake through the TRK transporters as proposed for S. cerevisiae Sat4, we examined genetic interactions between the hal4+ and trk+ genes. Genetic crosses between hal4::ura4+ ({Delta}hal4) and trk1::kanR ({Delta}trk1) produced no viable progeny of {Delta}hal4 {Delta}trk1; spores expected to carry both mutations often failed to germinate or ceased to grow immediately after germination (data not shown). Therefore, by crossing the {Delta}trk1 mutant with a {Delta}hal4 strain transformed by a hal4+ plasmid, viable {Delta}hal4 {Delta}trk1 double mutant cells carrying the hal4+ plasmid were isolated. Loss of the hal4+ plasmid was observed only when these cells were grown in rich medium supplemented with high concentrations of K+, such as 1 M KCl (data not shown), indicating the suppression of the {Delta}hal4 {Delta}trk1 lethality by high levels of external K+. In contrast, the {Delta}trk1 {Delta}trk2 double mutant was found to be viable even in regular growth media, as previously reported (8), although its growth was significantly slower than that of wild-type cells unless the growth medium was supplemented with 0.2 M KCl (data not shown). These observations do not negate Trk regulation by Hal4, but the severer growth defect of the {Delta}hal4 {Delta}trk1 double mutant than the {Delta}trk1 {Delta}trk2 mutant indicates that S. pombe Hal4 may also have a function independent of Trk1 and Trk2.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Alkali cations, such as K+, Na+, and Ca2+, are vital elements that play multiple roles in all living cells, whereas these cations must be maintained within a limited concentration range to avoid toxicity. For example, the concentration of Na+ is kept relatively low in many cell types, while K+ is found at much higher concentrations, since some metabolic enzymes are known to be sensitive to Na+ as well as Li+ ions (1, 13, 34, 35). Although NaCl was used as an osmolyte to isolate and characterize mutants of the osmosensing Hog1 SAPK in S. cerevisiae, we noticed that the homologous SAPK in S. pombe, Spc1, is required for cellular resistance to Na+, independently of its role in the osmostress response. Cells lacking functional Spc1 are hypersensitive to Na+, Li+, and Ca2+, while strains with increased Spc1 activity exhibit enhanced resistance to these cations. The crucial roles of SAPK in cellular responses to osmostress, oxidative stress, and heat shock have been well studied; however, as far as we are aware, this is the first to clearly demonstrate the requirement of SAPK for cellular salinity resistance.

One of the major functions of Spc1 SAPK is to induce expression of stress resistance genes through the Atf1 transcription factor (51, 62). Indeed, the {Delta}atf1 mutant is hypersensitive to Ca2+ and also shows mild sensitivity to Na+ and Li+. In addition, the hyperresistance to Na+ and Li+ in the wis1DD strain was compromised in the {Delta}atf1 background. These data suggest that transcriptional regulation by Atf1 partly contributes to cellular resistance to the cations. Previous DNA microarray studies demonstrated that the Spc1-Atf1 pathway regulates the majority of the CESR genes inducible in response to multiple forms of stress (9). However, from their sequence information, none of the CESR genes seem to have apparent involvement in cellular resistance to the cations. In this study, we isolated sod2+ as a multicopy suppressor of the Li+- and Na+-sensitive phenotypes of the {Delta}spc1 mutant. sod2+ encodes a Na+/H+ antiporter that exports Na+ and Li+ at the plasma membrane, and its expression level is known to correlate with cellular resistance to these cations (22). We found that the sod2+ mRNA level is reduced in the {Delta}spc1 mutant and increased in the wis1DD strain, observations consistent with the idea that sod2+ is a novel transcriptional target of the Spc1 pathway. sod2+ expression showed only a minor reduction in the {Delta}atf1 mutant, and therefore, Atf1 may not be the only transcription factor that mediates sod2+ regulation by Spc1. It was previously reported that the Spc1-Atf1 pathway is responsible for salt-induced expression of cta3+ (17, 38), which encodes a cation-efflux ATPase homologous to the budding yeast Ena1 (16). Although Ena1 is essential for Na+/Li+ resistance of budding yeast cells (19), the {Delta}cta3 mutant is not sensitive to Na+, Li+, and Ca2+ (17, 38; our unpublished results), and the physiological role of Cta3 is unclear.

The involvement of Spc1 SAPK in the cellular response to the cations has been further corroborated by the discovery of its physical interaction with Hal4 kinase. Its budding yeast orthologs, Sat4/Hal4 and Hal5, are essential for cellular resistance to the toxic cations (33), and consistently, we have demonstrated that the S. pombe {Delta}hal4 mutant is hypersensitive to Na+, Li+, and Ca2+. How does Hal4 kinase modulate cellular cation resistance? Studies of budding yeast suggest that Sat4/Hal4 and Hal5 kinases positively regulate cellular potassium uptake, which reduces influx of the toxic cations as follows. The plasma membrane H+-ATPase generates an electrochemical proton gradient across the membrane, negative inside, which drives the secondary transport of nutrients as well as the influx of cations (45). This membrane potential is counteracted by cellular K+ uptake, mainly through the Trk1 and Trk2 transporters; deletion of the TRK1 and TRK2 genes results in the hyperpolarized plasma membrane potential and increased uptake of cations (28), leading to cellular hypersensitivity to the toxic cations (33). Similar phenotypes were observed with the budding yeast sat4 hal5 mutant, and from epistasis analyses, it was proposed that Sat4/Hal4 and Hal5 kinases positively regulate K+ uptake through the Trk transporters (33). Our data also implicate the S. pombe Hal4 kinase in cellular K+ uptake. First, the multicopy suppressor screen of Li+ sensitivity in the {Delta}hal4 mutant isolated the trk1+ potassium transporter gene. Second, the {Delta}hal4 mutant shows a growth defect in K+-limited growth media. Third, like {Delta}trk1, {Delta}hal4 cells show cation-sensitive phenotypes that are complemented by additional potassium in the growth medium. Lastly, we found that {Delta}hal4 cells are hypersensitive also to the cationic antibiotics, such as hygromycin B and G418, as well as tetramethylammonium (our unpublished results), a phenotype indicative of the hyperpolarized plasma membrane (3, 33, 40). Together, these data strongly suggest that the S. pombe Hal4 kinase positively regulates cellular K+ uptake and that the loss of Hal4 activity leads to the cation-sensitive phenotype, probably due to hyperpolarization of the plasma membrane. As proposed in budding yeast, Hal4 may directly or indirectly regulate the Trk transporters. This regulation does not seem to be transcriptional, because the trk1+ mRNA level is not reduced in the {Delta}hal4 mutant (our unpublished results). Interestingly, expression of the truncated Trk1 without the C-terminal 38 residues, but not the full-length protein, can complement the hal4 defects. An intriguing possibility is that this cytoplasmic tail of the Trk1 transporter may be a negative regulatory domain and that the Hal4 function may be to relieve this negative regulation of Trk1. On the other hand, we found that the {Delta}hal4 {Delta}trk1 double mutant is lethal, while the {Delta}trk1 {Delta}trk2 cells are viable. Thus, in contrast to the budding yeast Sat4/Hal4, Hal4 kinase in S. pombe appears to have an additional function independent of the Trk transporters.

Although Spc1 physically interacts with Hal4 and the {Delta}spc1 mutant shows cation-sensitive phenotypes similar to those of {Delta}hal4, Spc1 SAPK does not appear to be important in cellular K+ uptake; {Delta}spc1 cells exhibit no growth defect under K+-limited conditions. In addition, overexpression of the trk1+ gene does not complement the {Delta}spc1 phenotypes. Therefore, Spc1 might modulate cellular resistance to the toxic cations independently of the Hal4 function in K+ homeostasis. Alternatively, the Spc1-Hal4 interaction might become important for cellular K+ homeostasis only under certain conditions. We found that cells expressing the mutant Hal4 without the Spc1-binding domain are hypersensitive to Ca2+, but not to Na+ and Li+, implying that cellular responses to high concentrations of Ca2+ may require additional regulation of Hal4 by Spc1. The molecular mechanisms in which Spc1 regulates the Hal4 function need further investigation. The in vivo phosphorylation state of Hal4 did not appear to be significantly affected by the {Delta}spc1 mutation, and Spc1 failed to phosphorylate Hal4 in vitro (our unpublished results), suggesting that Spc1 may not regulate Hal4 through phosphorylation. Spc1 also does not seem to affect the localization of the Hal4 protein, because cytoplasmic localization of Hal4 is not altered by osmostress that induces nuclear translocation of Spc1 SAPK (our unpublished results). However, the physical interaction may provide necessary coordination between the Spc1 and Hal4 functions for maximum resistance to the toxic cations. It is also possible that the Spc1-Hal4 interaction might play a role during the environmental stress responses mediated by Spc1 SAPK. In bacteria, K+ uptake is part of the cellular response to high-osmolarity stress (26, 60). Thus, it is conceivable that, in addition to transcriptional regulation, Spc1 SAPK may modulate cellular cation homeostasis through interaction with Hal4 kinase under environmental stresses.

In this study, we have identified a novel physiological role of Spc1 SAPK in cellular resistance to the toxic cations. Spc1 regulates expression of a Na+/H+ antiporter and also physically interacts with Hal4 kinase that regulates cellular K+ uptake. During preparation of the manuscript, it was reported that the budding yeast Hog1 SAPK regulates the Na+/H+ antiporter in the plasma membrane through phosphorylation (41), indicating that budding yeast also utilizes SAPK in the cellular cation response. Considering the large evolutional distance between fission and budding yeasts (20), involvement of SAPK in cellular cation homeostasis might be conserved also in other eukaryotes. Further genetic and biochemical studies of these yeast systems will be very useful to characterize this novel SAPK function at a molecular level.


   ACKNOWLEDGMENTS
 
We are grateful to Aminah Ikner for the spc1KM strain and Mina Kikuchi, Thac Tran, Gina Lam, Jennifer Rust, and Patricia Novy for technical assistance. We also thank Tomohiro Matsumoto for a yeast two-hybrid system, Taro Nakamura for an S. pombe genomic library, and Janet Quinn for strains. Valley Stewart and Linda Bisson provided helpful discussions.

L.W. was a recipient of the Jastro-Shields Graduate Research Scholarship Award. This research was supported by a NIH grant (GM59788) awarded to K.S.


   FOOTNOTES
 
* Corresponding author. Mailing address: Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616. Phone: (530) 752-3628. Fax: (530) 752-9014. E-mail: kshiozaki{at}ucdavis.edu. Back

{dagger} Present address: Department of Periodontology, Nihon University School of Dentistry, Chiyoda-ku, Tokyo 101-8310, Japan. Back


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 Materials and Methods
 Results
 Discussion
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Molecular and Cellular Biology, May 2005, p. 3945-3955, Vol. 25, No. 10
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.10.3945-3955.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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