Zinc Finger Protein Prz1 Regulates Ca2 but Not Cl ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 20, Issue of May 16, pp. 18078 –18084, 2003 Printed in U.S.A.

Zinc Finger Protein Prz1 Regulates Ca2ⴙ but Not Clⴚ Homeostasis in Fission Yeast IDENTIFICATION OF DISTINCT BRANCHES OF CALCINEURIN SIGNALING PATHWAY IN FISSION YEAST* Received for publication, December 18, 2002, and in revised form, January 31, 2003 Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M212900200

Sonoko Hirayama‡§, Reiko Sugiura‡§, Yabin Lu‡, Takuya Maeda‡, Kenji Kawagishi‡, Mistuhiro Yokoyama¶, Hideki Tohda储, Yuko Giga-Hama储, Hisato Shuntoh**, and Takayoshi Kuno‡ ‡‡ From the ‡Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, and ¶Division of Cardiovascular and Respiratory Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan, 储ASPEX Division, Research Center, Asahi Glass Co., Ltd. 1150 Hazawa-cho, Kanagawa-ku, Yokohama, 221-8755, Japan, and **Faculty of Health Science, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 650-0142, Japan

Calcineurin is a Ca2⫹/calmodulin-dependent serine/threonine protein phosphatase consisting of a catalytic subunit and a regulatory subunit (1). In mammalian cells, calcineurin plays an important role in various Ca2⫹-mediated processes including T-cell activation (2, 3), cardiac hypertrophy (4), neutrophil * This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. § Both authors contributed equally to this work. ‡‡ To whom correspondence should be addressed: Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5440; Fax: 81-78-382-5459; E-mail: [email protected].

chemotaxis (5), apoptosis (6), angiogenesis (7), and memory development (8). For many of these cellular events, calcineurin exerts its function by regulating the NF-AT1 family members. Calcineurin directly dephosphorylates NF-AT transcription factors, causing their activation and trans-location from the cytoplasm to the nucleus (9). Furthermore, calcineurin is specifically inhibited by the immunosuppressants cyclosporin A and FK506 (10), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. In the budding yeast Saccharomyces cerevisiae, calcineurindeficient strains exhibit normal growth under standard conditions (11, 12). However, calcineurin function is required for cell viability under some specific growth conditions. Calcineurin mutants deficient for either the catalytic subunits (CNA1/ CNA2) (11, 12) or the regulatory subunit (CNB1) (13, 14) die in the presence of high concentrations of different ions including manganese, sodium, lithium, and hydroxyl ions (15–18). Some of these ion sensitivities are because of a defect in the calcineurin-dependent regulation of several ion transporter genes including PMR1, PMR2, and PMC1 (16, 19) whose expressions are regulated through the Crz1/Tcn1 transcription factor (20, 21). The expression of FKS2, which encodes a ␤-1,3-glucan synthase, is also regulated by Crz1 through a calcineurindependent mechanism (20). When calcineurin is activated, it dephosphorylates Crz1, causing its rapid trans-location from the cytoplasm to the nucleus (22), suggesting similar modes of regulation by calcineurin for its downstream transcription factors in budding yeast and mammals. A disruption of CRZ1 gene caused similar phenotypes as those of calcineurin mutants, and in calcineurin mutants, these phenotypes are suppressed by CRZ1 overexpression (20). These results suggest that Crz1 functions downstream of calcineurin to effect most of the calcineurin-dependent cellular responses in budding yeast. Furthermore, recent genome-wide analysis of gene expression regulated by the calcineurin/Crz1 signaling pathway confirm that Crz1 is the major and possibly the only effector of calcineurin-regulated gene expression in budding yeast (23). We have been studying the calcineurin signaling pathway in fission yeast Schizosaccharomyces pombe because this system is amenable to genetic analysis and has many advantages in 1 The abbreviations used are: NF-AT, the nuclear factor of activated T cells; MAPK, mitogen-activated protein kinase; its, immunosuppressant- and temperature-sensitive; prz, Ppb1-responsive zinc finger protein; GFP, green fluorescent protein; DIG, digoxigenin; SRR, serine-rich region; PI(4)P5, phosphatidylinositol 4-phosphate 5.

18078

This paper is available on line at http://www.jbc.org

Downloaded from http://www.jbc.org/ by guest on December 28, 2015

Calcineurin is an important mediator that connects the Ca2ⴙ-dependent signaling to various cellular responses in a wide variety of cell types and organisms. In budding yeast, activated calcineurin exerts its function mainly by regulating the Crz1p/Tcn1 transcription factor. Here, we cloned the fission yeast prz1ⴙ gene, which encodes a zinc finger transcription factor highly homologous to Crz1/Tcn1. Similar to the results in budding yeast, calcineurin dephosphorylated Prz1 and resulted in the trans-location of Prz1 from the cytoplasm to the nucleus. Prz1 expression was stimulated by high extracellular Ca2ⴙ in a calcineurin-dependent fashion. However, unlike in budding yeast, the prz1-null cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Clⴚ. Instead, the prz1-null cells showed hypersensitivity to Ca2ⴙ, consistent with a dramatic decrease in transcription of Pmc1 Ca2ⴙ pump. Interestingly, overexpression of Prz1 did not suppress the Clⴚ hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Clⴚ hypersensitivity of calcineurin deletion but not the Ca2ⴙ hypersensitivity of prz1 deletion. In addition, mutations in the its2ⴙ/cps1ⴙ, its8ⴙ, and its10ⴙ/cdc7ⴙ genes that showed synthetic lethal genetic interaction with calcineurin deletion did not exhibit synthetic lethality with the prz1 deletion. Our results suggest that calcineurin activates at least two distinct signaling branches, i.e. the Prz1-dependent transcriptional regulation and an unknown mechanism, which functions antagonistically with the Pmk1 MAPK pathway.

Calcineurin and PRZ1 in S. Pombe

18079

TABLE I Schizosaccharomyces pombe strains used in this study Strain

HM123 HM528 KP161 KP162 KP165 KP167 KP533 KP553 KP1003 KP1245 KP1395 KP1464 KP1466 KP1467

Genotype ⫺

h h⫹ h⫺ h⫺ h⫺ h⫺ h⫺ h⫺ h⫺ h⫹ h⫺ h⫺ h⫺ h⫺

leul-32 his2 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32 leul-32

Reference

⫹

ura4-D18 ppbl::ura4 ypt3–15 cps1–12 its3–1 its8–1 cdc7-i10 ura4-D18 przl::ura4⫹ ura4–294 ura4–294 ura4⫹::nmtl-GFP:przl⫹ ura4-D18 przl::ura4⫹ cpsl-i2 ura4-D18 przl::ura4⫹ cdc7-i10 ura4-D18 przl::ura4⫹ its8–1

EXPERIMENTAL PROCEDURES

Strains, Media, and Miscellaneous Procedures—S. pombe strains used in this study are listed in Table I. The complete medium, YPD (1% yeast extract, 2% polypeptone, 2% glucose), and the minimal medium, Edinbugh minimal medium (35), have been described previously (34). SPA mating and sporulation medium contained 10 g/liter glucose, 1 g/liter KH2PO4, 1 ml/liter 1000⫻ vitamin stock solution (same as those used for EMM), and 30 g/liter agar. Standard methods for S. pombe genetics were followed according to Moreno et al. (35). FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Calcineurin and calmodulin were prepared from bovine brain as described previously (36). Gene disruptions are denoted by lowercase letters representing the disrupted gene followed by two colons and the wild-type gene marker used for disruption (for example, prz1::ura4⫹). Also, gene disruptions are denoted by an abbreviation of the gene preceded by ⌬ (for example, ⌬prz1). Proteins are denoted by Roman letters, and only the first letter is capitalized (for example, Prz1). Data base searches were performed using the National Center for Biotechnology Information BLAST network service (www.ncbi.nlm. nih.gov) and the Sanger Center S. pombe data base search service (www.sanger.ac.uk). Cloning and Tagging of the prz1⫹ Gene—The prz1⫹ gene was amplified by PCR with the genomic DNA of S. pombe as a template. The sense

primer used for PCR was 5⬘-CG GGA TCC ATG GAG CGT CAA AGG TCA GAA GAA GCC AT-3⬘ (BamHI site and start codon are underlined), and the antisense primer was 5⬘-CG GGA TCC TCA TTT TTG TTT GCT TGT CGA GGC-3⬘ (BamHI site and stop codon are underlined). The amplified product was digested with BamHI, and the resulting fragment was subcloned into Bluescript SK(⫹). For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter at various levels of expression (37). Expression was repressed by the addition of 4 ␮g/ml thiamine to EMM and was induced by washing and incubating the cells in EMM lacking thiamine. To express GFP-Prz1, the complete open reading frame of prz1⫹ was ligated to the C terminus of the GFP carrying the S65T mutation (38). GFP-Prz1 fully complements the growth defects of a prz1-null strain (data not shown). The GFP-fused gene was subcloned into pREP1, pREP41, or pREP81 vectors to express the gene at various levels. Maximum expression of the fused gene was obtained using pREP1, whereas pREP81 contained the most attenuated version of the nmt1 promoter (37). To obtain the chromosome-born GFP-Prz1 instead of the plasmid-born GFP-Prz1, the fused genes with the nmt1 promoter at various levels were subcloned into the vector containing the ura4⫹ marker and were integrated into the chromosome at the ura4⫹ gene locus of KP1245 (h⫹ leu1–32 ura4 –294) (39). Deletion of prz1⫹ Gene—A one-step gene disruption by homologous recombination (40) was performed. The prz1::ura4⫹ disruption was constructed as follows. Cloned open reading frame of the prz1⫹ gene in the Bluescript vector was digested with BamHI and EcoRI, and the resulting fragment containing ⬃80% prz1⫹ gene was subcloned into the BamHI/EcoRI site of pUC119. A SmaI fragment containing the ura4⫹ gene then was inserted into the EcoRV site of the previous construct, causing the interruption of the open reading frame. The fragment containing disrupted prz1⫹ gene was transformed into diploid cells. Stable integrants were selected on medium lacking uracil, and disruption of the gene was checked by genomic Southern hybridization (data not shown). Cell Extract Preparation and Immunoblot Analysis—For the analysis of electrophoretic mobility shift of Prz1, whole-cell extracts were prepared from cultures of wild-type or calcineurin-null cells expressing GFP-Prz1 grown at 30 °C to mid-log phase. Cells were resuspended in 450 ␮l of ice-cold homogenizing buffer, 50 mM Tris-HCl, pH 7.8, containing 2 mM EDTA, 1 mM dithiothreitol, and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 0.1 mM sodium metabisulfite, 0.1 ␮g/ml chymostatin, 2 ␮g/ml aprotinin, 1 ␮g/ml pepstatin A, 1 ␮g/ml phosphoramidon, and 0.5 ␮g/ml leupeptin). Glass beads (0.2 g) were then added, and cells were broken mechanically by vortexing for 30 s, after which the tubes were placed on ice for 30 s. Vortexing and cooling were repeated five times, after which the glass beads and cellular debris were removed by centrifugation at 15,000 ⫻ g for 5 min. Protein extracts (10⬃20 ␮g/5 ␮l) were subjected to SDS-PAGE and immunoblotted with anti-GFP antibody. Northern Blot Analyses—Total RNA was isolated by the method of Kohrer and Domdey (41). 20 ␮g of total RNA/lane was subjected to electrophoresis on denaturing formaldehyde 1% agarose gels and transferred to nylon membranes. Hybridization was performed using DIG-labeled antisense cRNA probes coding for Prz1 and Pmc1 (SPBC1A4.10c). The DIG-labeled hybrids were detected by an enzymelinked immunoassay using an anti-DIG-alkaline-phosphatase antibody conjugate. The hybrids were visualized by chemiluminescence detection on a light-sensitive film according to the manufacturer’s instructions (Roche Applied Science).


terms of relevance to higher systems (24, 25). S. pombe has a single gene encoding the catalytic subunit of calcineurin, ppb1⫹ (26). We have developed a genetic screen for mutants that depend on calcineurin for growth using the immunosuppressant FK506 and have given the designation its mutants. The analyses of the its mutants revealed that calcineurin is implicated in cytokinesis, septation initiation network, and exocytic pathway in fission yeast (27–31). We have shown that fission yeast calcineurin plays an essential role in maintaining chloride ion homeostasis and acts antagonistically with the Pmk1 MAPK pathway (32, 33). These phenotypes are quite different from those described above for calcineurin deletion in budding yeast, suggesting that the upstream or downstream signaling events of calcineurin may be distinct in these two distantly related yeasts. Here, we cloned the fission yeast prz1⫹ gene, which encodes a zinc finger protein highly homologous to Crz1. Consistent with the hypothesis that Prz1 is the functional homolog of Crz1, Prz1 is dephosphorylated by calcineurin, causing its rapid trans-location from the cytoplasm to the nucleus. However, the prz1-null cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Cl⫺. Instead, they showed hypersensitivity to Ca2⫹. In addition, some of the its mutants showing synthetic lethal interaction with calcineurin deletion did not exhibit synthetic lethality with the prz1-null mutation. These results indicate that there are at least two distinct branches of calcineurin signaling pathway.

Our stock Our stock Our stock Cheng et al. (31) This study Zhang et al. (27) Yada et al. (28) Lu et al. (30) This study Lafuente et al. (39) This study This study This study This study

18080


Microscopic Analysis—Cells were grown to exponential phase in YPD or EMM medium and shifted to various conditions as indicated in the figure legends. In some cases, cells were washed with phosphate-buffered saline, pH 7.0, and then stained with Hoechst 33342 or Calcofluor to visualize the DNA or septum, respectively, before microscopic observation. Cells were microscopically examined under an Axioskop microscope (Carl Zeiss Inc.). Photographs were taken with a SPOT2 digital camera (Diagnostic Instruments Inc.). Images were processed with the CorelDRAW software (Corel Corporation Inc.). RESULTS

Identification of the S. pombe prz1⫹ Gene—A BLAST program search using the peptide sequence of Crz1, the calcineurin-responsive zinc finger transcription factor of S. cerevisiae (20, 21) against the S. pombe protein data base at the Sanger Center revealed an open reading frame, SPAC4G8.13c, exhibiting significant similarity to Crz1 (score ⫽ 293, p ⫽ 6.3e⫺25, identities ⫽ 58/125 (46%), positives ⫽ 75/125 (60%)). We named the gene prz1⫹ (for Ppb1-responsive zinc finger protein). As shown in Fig. 1, A and B, the prz1⫹ gene encodes a protein of 681 amino acids that contains three C2H2-type zinc finger motifs at its carboxyl terminus highly homologous to those of Crz1. However, unlike Crz1, Prz1 does not have a polyglutamine tract, which acts as a transcriptional activation

FIG. 2. Northern blot analysis of Prz1 mRNA expression. A, induction of Prz1 mRNA by Ca2⫹ requires calcineurin activity. Wildtype or calcineurin-null cells were incubated in YPD medium at 30 °C with 30 mM CaCl2 for 0, 10, 20, or 40 min. Total RNA (20 ␮g) were subjected to Northern analysis using a DIG-labeled Prz1 cRNA. B, induction of Prz1 mRNA by heat treatment requires calcineurin activity. Wild-type or calcineurin-null cells were cultured to mid-log phase at 27 °C and then incubated at 42 °C for 0, 10, 20, or 40 min. Total RNA were subjected to Northern analysis as described above. C, expression of constitutively active calcineurin induces Prz1 mRNA. Wild-type cells transformed with a control vector (⫹ vector) or the vector containing the constitutively active truncated calcineurin gene (⫹ ppb1⌬C) were cultured in EMM medium to mid-log phase at 30 °C. Total RNA was subjected to Northern analysis as described above. WT, wild type.

domain in many cases (42). Outside of the zinc finger domain, Prz1 also contains a serine-rich region (SRR, residues 57–219, N score ⫽ 8.748 (Prosite)), but it shows low homology with that of Crz1. Prz1 is Dephosphorylated by Calcineurin—We examined whether Prz1 phosphorylation is modulated by calcineurin. GFP-Prz1 protein showed a significant alteration in its mobility when analyzed by immunoblot. GFP-Prz1 protein isolated from calcineurin null or from the wild-type cells treated with the specific calcineurin inhibitor, FK506, migrated on SDSPAGE gels with a significantly larger apparent molecular mass than that from non-treated wild-type cells, whereas GFP-Prz1 isolated from cells treated with 50 mM CaCl2 migrated with a slightly smaller apparent molecular mass (Fig. 1C). The change in the ratio of the faster and slower migrating GFP-Prz1 species suggested that there is a calcineurin-dependent change in the phosphorylation state of Prz1. To establish that the mobility change of GFP-Prz1 was indeed the result of variable degrees in phosphorylation, protein extracts from calcineurinnull cells were treated with purified calcineurin in vitro. Treatment of the larger form of GFP-Prz1 with calcineurin converted it to the smaller form (Fig. 1D). This change in apparent molecular mass was dependent on Ca2⫹ ion and calmodulin (Fig. 1D) and was blocked by the addition of FK506 (data not shown). These findings confirmed that Prz1 is hyper-


FIG. 1. Prz1 is a C2H2-type zinc finger protein and is a calcineurin substrate. A, schematic structure of Prz1 protein. Denoted are the SRR and three putative zinc fingers (ZnF). B, sequence alignment of the three zinc finger motifs from Prz1 and Crz1 from budding yeast (21). Residues conserved in two sequences are boxed and highlighted. Residues that coordinate zinc ions are indicated by asterisks. C, the calcineurin-specific inhibitor FK506 induces a Prz1 protein mobility shift. Cells expressing GFP-Prz1 were incubated in EMM medium containing 4 ␮g/ml thiamine at 30 °C with CaCl2 (50 mM) or FK506 (0.5 ␮g/ml) for 30 min as noted. The electrophoretic mobility of the GFPPrz1 protein was investigated by immunoblotting using a GFP-specific antiserum. D, the mobility shift of Prz1 caused by calcineurin treatment in vitro is calmodulin-dependent. GFP-Prz1 was purified by immunoprecipitation from ⌬ppb1 cells expressing GFP-Prz1, incubated with 1 mM CaCl2 and calcineurin (lane 1) or with 1 mM CaCl2, calmodulin, and calcineurin (lane 2) for 15 min at 37 °C, and analyzed by immunoblotting.


18081

FIG. 3. Intracellular localization of GFP-Prz1. A, translocation of GFP-Prz1 to the nucleus is induced by Ca2⫹ addition and requires calcineurin. Wild-type, calcineurin-null, or wild-type cells transformed with the vector containing the constitutively active truncated calcineurin gene (⫹ ppb1⌬C) expressing GFP-Prz1 were grown in EMM medium at 30 °C, incubated briefly with Hoechst 33342 to stain DNA, and analyzed by fluorescence microscopy to observe GFP-Prz1 localization (GFP-Prz1) or nuclear staining (DNA). In some cases cells were incubated with 100 mM CaCl2 for 10 min at 30 °C (⫹ CaCl2). Arrow indicates a dividing cell whose nucleus shows intense GFP fluorescence. The bar indicates 10 ␮m. B, cell cycle-specific nuclear accumulation of GFP-Prz1. Wild-type cells expressing GFP-Prz1 were grown to mid-log phase at 30 °C in YPD medium. Cells were stained with Calcofluor, and the percentage of cells showing intense nuclear fluorescence was measured. At least 500 cells were counted.

phosphorylated in cells lacking calcineurin activity and that it serves as a substrate for calcineurin in vitro, suggesting that calcium signals maintain Prz1 protein in a hypophosphorylated state through the activation of calcineurin. High Extracellular Ca2⫹ and High Temperature Induction of Prz1 mRNA Is Dependent on Calcineurin Activity—Fig. 2A showed that Prz1 mRNA accumulation was induced rapidly in wild-type but not in calcineurin-null cells grown at 27 °C after the addition of CaCl2 (30 mM) to the growth medium, indicating that Prz1 expression itself is calcineurin-responsive and that Prz1 controls the activity of its own promoter in response to calcineurin signaling. We also examined the effect of temperature upshift on the steady-state levels of Prz1 mRNA. Fig. 2B showed that the level of Prz1 mRNA is strongly induced by a shift to growth at 42 °C in wild-type but not in calcineurin-null

cells, peaking at 20 min after the shift. Pretreatment of the wild-type cells with FK506 completely blocked Prz1 mRNA accumulation induced by both high extracellular Ca2⫹ and high temperature, again indicating that the transient induction observed is calcineurin-responsive (data not shown). Consistent with this hypothesis, the expression of constitutively active calcineurin (Ppb1⌬C) increased the steady-state levels of Prz1 mRNA (Fig. 2C). Activation of Calcineurin Causes the Trans-location of GFPPrz1 from the Cytoplasm to the Nucleus—In either wild-type or calcineurin-null cells cultured under standard conditions, GFPPrz1 mostly localized to the cytosol (Fig. 3A). In wild-type cells incubated with 100 mM Ca2⫹, GFP-Prz1 trans-located to the nucleus within 10 min. On the other hand, in calcineurin-null cells treated with 100 mM Ca2⫹, GFP-Prz1 remained cytosolic (Fig. 3A). These results are in good agreement with those obtained with GFP-Crz1 in budding yeast (22). Consistently, wild-type cells expressing the constitutively active calcineurin showed nuclear localization of GFP-Prz1 in the absence of exogenous Ca2⫹ stimulation (Fig. 3A). In addition, cell cyclespecific nuclear accumulation of GFP-Prz1 was observed in wild-type cells. As shown in Figs. 3A and 4 (indicated by arrows) and summarized in 3B, GFP-Prz1 accumulated in the nucleus of dividing cell before its septum formation, and this is consistent with the role of calcineurin in cytokinesis and septum initiation (27–31). The addition of calcineurin inhibitor FK506 again completely blocked nuclear accumulation of GFPPrz1 in these experiments (data not shown). A GFP-Prz1 fusion lacking the N-terminal 240 residues of Prz1 (GFP-Prz1⌬SRR for lacking serine-rich region) partially complemented the growth defects of a prz1-null strain (data not shown) and showed nuclear localization that was not affected by FK506 treatment (Fig. 4, lower panel), indicating that the serine-rich region of Prz1 is required for calcineurin-dependent regulation of its localization but is dispensable for its transcriptional activity. There is no sequence similarity between the


FIG. 4. Localization of Prz1 to the cytoplasm requires its SRR. Living cells transformed with the vector for the expression of GFP-Prz1 (full-length) or GFP-Prz1⌬SRR (N-terminally truncated) were grown at 30 °C and analyzed as described above. In some cases cells were incubated with 0.5 ␮M FK506 for 30 min at 30 °C (⫹ FK506). Arrow indicates a dividing cell whose nucleus shows intense GFP fluorescence. The bar indicates 10 ␮m.

18082


2ⴙ

FIG. 5. Prz1 deletion causes hypersensitivity to Ca and marked reduction of mRNA for vacuolar Pmc1 Ca2ⴙ pump. A, 2⫹ ⫺ prz1 deletion is hypersensitive to Ca but not to Cl . Wild-type (WT), prz1-null (⌬prz1), and ppb1-null (⌬ppb1) cells were streaked onto YPD plate and incubated for 3 days at 30 °C in the presence or absence of 0.2 M MgCl2, 0.15 M CaCl2, 0.3 M KCl, or 0.15 M Ca(NO3)2. B, Northern blot analysis of Pmc1 mRNA expression. Wild-type, calcineurin-null (⌬ppb1), or prz1-null (⌬prz1) cells were incubated in YPD medium at 30 °C with 100 mM CaCl2 for 15 min. Total RNA (20 ␮g) were subjected to Northern analysis using a DIG-labeled Pmc1 cRNA.

serine-rich region of Prz1 and that of Crz1 or the NF-ATc with the exception of the richness in serine residue, although significant sequence similarity between the serine-rich region of Crz1 and NF-ATc transcription factor has been reported previously (22). Prz1-null Cells Are Hypersensitive to Ca2⫹ but Not to ⫺ Cl —To further investigate the relationship between Prz1 and calcineurin, we analyzed prz1-null cells for phenotypes exhibited by calcineurin-null mutants. Calcineurin-null mutants were hypersensitive to Cl⫺ ion and failed to grow in the presence of 0.15 M MgCl2 or 0.3 M KCl (32). Contrary to our expectation, prz1-null cells grew normally in the YPD plate containing 0.2 M MgCl2 where calcineurin-null cells did not grow (Fig. 5A). Interestingly, both of the prz1-null cells and calcineurinnull cells could not grow in the presence of 0.15 M CaCl2 or 0.15 M Ca(NO3)2 (Fig. 5A). These results suggest that Prz1 is involved in the regulation of Ca2⫹ ion homeostasis but not that of Cl⫺ ion homeostasis. In budding yeast, Crz1, the homolog of Prz1, is required for calcineurin-dependent transcriptional regulation of PMC1, which encodes a Ca2⫹ pump playing a key role in Ca2⫹ tolerance (43). Consistently, Northern blot analysis revealed that calcineurin (ppb1) deletion and prz1 deletion resulted in a marked reduction in Pmc1 mRNA levels (Fig. 5B). In fission yeast, the disruption of pmc1⫹ gene (SPBC1A4.10c) resulted in severe hypersensitivity to Ca2⫹ (data not shown). Thus, the Ca2⫹ hypersensitivity of prz1-null cells can be explained, at least in part, by lowered level of Pmc1. As noted above, calcineurin-null cells of S. pombe are sensi-

tive to Ca2⫹. In contrast to S. pombe, calcineurin-null cells of S. cerevisiae are resistant to CaCl2 and show increased growth on medium containing high levels of Ca2⫹, whereas the crz1-null cells similar to the prz1-null cells are highly Ca2⫹-sensitive (20). Thus, the roles of calcineurin in Ca2⫹ homeostasis in these two yeasts are suggested to be quite different. prz1-null and Calcineurin-null Mutants Have Distinct Phenotypes in Cell Morphology and Mating—In addition to ion homeostasis, prz1-null cells showed phenotypes distinct from those of calcineurin-null cells in cell morphology and mating (Fig. 6). As shown in Fig. 6A, calcineurin-null cells were enlarged, multiseptated, and branched, consistent with the previous study by Yoshida et al. (26), which suggests the involvement of calcineurin in the regulation of the cell polarity and cytokinesis. On the other hand, prz1-null cells were indistinguishable from the wild-type cells in morphology. Furthermore, unlike calcineurin-null cells, which were sterile as reported by Yoshida et al. (26), prz1-null cells were fertile and their mating efficiency and spore morphology were indistinguishable from wild-type cells (Fig. 6B). prz1-null cells treated with FK506 showed the same phenotypes as calcineurin-null cells (Fig. 6), indicating that the effects of eliminating calcineurin is epistatic to the effects of prz1 deletion and that calcineurin acts upstream of the Prz1 transcription factor. Some of the Mutants That Show Synthetic Lethality with Calcineurin Deletion Do Not Show Synthetic Lethality with prz1 Deletion—As described above, we have isolated mutants that depend on calcineurin for growth using the immunosuppressant FK506 and have given the designation its mutants (27–31). These mutants showed synthetic lethal genetic interaction with calcineurin. Therefore, their genes encode the functional proteins that may share essential function with calcineurin. To examine the relationship between these genes and the prz1⫹ gene, tetrad analysis of a diploid derived from a cross between the its mutants with prz1-null cells was performed. As shown in Table II, we found that prz1-null mutation was synthetically lethal with its3 and its5/ypt3-i5 mutants that encode phosphatidylinositol 4-phosphate 5 (PI(4)P5)-kinase and a


FIG. 6. Prz1-null and calcineurin-null mutants have distinct phenotypes in cell morphology and mating. A, prz1-null cells is normal in size, polarity, and cytokinesis. prz1-null and calcineurin-null mutants were grown in YPD medium to mid-log phase at 30 °C, washed with phosphate-buffered saline, pH 7.0, and stained with Hoechst 33342 and Calcofluor to visualize the DNA or septum, respectively. B, sporulation in prz1-null and calcineurin-null mutants. Null mutants and HM528 strain were crossed on an SPA plate at 30 °C and examined microscopically after 24 h. Bar, 10 ␮m.


18083

TABLE II Genetic interactions between prz1⫹ or ppb1⫹ and its (immunosuppressant- and temperature-sensitive) mutants Synthetic lethality in crosses between various mutants was examined by tetrad analysis following sporulation. its2/cps1-i2

⌬przl ⌬ppbl

its3 ␤-glucan synthase (44)

its5/ypt3-i5 P1(4)P5 kinase (27)

its8 Rab family (31)

its10/cdc7-i10 GPI biosynthetic enzyme (28)

SIN pathway kinase (30)

viable lethal

lethal lethal

lethal lethal

viable lethal

viable lethal

DISCUSSION

We report here the identification and characterization of Prz1, the S. pombe homolog of Crz1, which is a C2H2-type zinc finger protein that binds to the calcineurin-dependent response element and that regulates transcription of various target genes in budding yeast. Similar to its budding yeast homolog, the S. pombe Prz1 is dephosphorylated by calcineurin and its trans-location from the cytoplasm to the nucleus is caused by the activation of calcineurin. However, unlike in budding yeast, prz1-null phenotypes are quite different from those of calcineurin-null cells as shown in the present study. In budding yeast, crz1-null cells showed similar phenotypes as those of calcineurin-null cells, such as hypersensitivity to Mn2⫹ or Li⫹, and survival defect when incubated with ␣-factor (20). Furthermore, a recent genome-wide analysis of gene expression regulated by the calcineurin/Crz1 signaling pathway confirms that Crz1 is the major and possibly the only effector of calcineurinregulated gene expression in budding yeast (23). On the other hand, with the exception of its hypersensitivity to Ca2⫹, prz1null cells showed no typical phenotypes as those observed in calcineurin-null cells, such as aberrant cell morphology, mating defect, or hypersensitivity to Cl⫺. In addition, our preliminary genome-wide analysis using S. pombe DNA microarray suggests that the gene expression pattern of prz1-null cells is

FIG. 7. Prz1 is neither genetically related to the Pmk1 MAPK pathway nor is involved in the regulation of Clⴚ homeostasis. A, prz1-null (⌬prz1) or ppb1-null (⌬ppb1) cells transformed with a control vector (vector) or the multicopy vector containing pmp1⫹ (⫹ pmp1⫹) and prz1⫹ (⫹ prz1⫹) or the constitutively active truncated calcineurin (⫹ ppb1⌬C) gene were streaked onto YPD plate and incubated for 3 days at 30 °C in the presence of 0.1 M CaCl2 or 0.15 M MgCl2. B, a model of the two distinct branches of calcineurin signaling pathway. See “Results” and “Discussion.” Ppb1, calcineurin phosphatase; Pmk1, MAPK phosphatase; Pmp1, MAPK phosphatase for Pmk1; Pmc1, vacuolar Ca2⫹ pump; ?, unknown mechanism for Cl⫺ resistance.

considerably different from that of calcineurin-null cells in fission yeast.2 These results strongly suggest that there are at least two branches of calcineurin signaling pathway. Related to this issue is the observation that in S. cerevisiae, crz1-null cells and calcineurin-null cells show opposing phenotypes in the condition of high Ca2⫹, i.e. crz1-null cells are highly Ca2⫹sensitive similar to prz1-null cells, whereas cnb1-null cells are resistant to this ion and show increased growth on medium containing high levels of Ca2⫹ (20), showing that there is also branching in calcineurin signaling in S. cerevisiae. Obviously, one branch is the Prz1-dependent branch that regulates the expression of Pmc1 Ca2⫹ pump. The Ca2⫹ sensitivity of prz1-null cells is consistent with the markedly reduced level of Pmc1 mRNA, suggesting a similar regulatory mechanism of Ca2⫹ homeostasis in these two distantly related yeasts (20, 21). To further analyze the heterogeneous nature of the calcineurin signaling pathway, we examined the genetic interaction between prz1 deletion and its mutants that are synthetically lethal with calcineurin deletion. As shown in Table II, prz1 deletion is synthetically lethal with mutations in the its3⫹ and its5⫹/ypt3⫹ genes encoding a PI(4)P5 kinase and a Rab 2 R. Sugiura, H. Tohda, Y. Giga-Hama, H. Shuntoh, and T. Kuno, unpublished observations.


small GTPase of the Rab/Ypt family, respectively (27, 31). On the other hand, mutations in its2⫹/cps1⫹, its8⫹, and its10⫹/ cdc7⫹ genes (encoding ␤-glucan synthase, glycosylphosphatidylinositol anchor synthetic enzyme, and protein kinase implicated in septation initiation, respectively) (28, 30, 44) did not show synthetic lethality with prz1-null mutation and double mutants were obtained (Table II). The ⌬prz1 its8 double mutant grew slower and was more temperature-sensitive than the its8 single mutant (data not shown), indicating a functional overlapping between Prz1 and the glycosylphosphatidylinositol anchor synthetic pathway. Prz1 Is Not Involved in Chloride Ion Homeostasis That Is Antagonistically Regulated by Calcineurin and the Pmk1 MAPK Pathways—Our previous study showed that calcineurin acts antagonistically with the Pmk1 MAPK pathway in Cl⫺ ion homeostasis and inhibition of Pmk1 MAPK pathway suppressed the Cl⫺ hypersensitivity of calcineurin-null cells (32, 33). Consistent with our previous results, overexpression of pmp1⫹ gene encoding a MAPK phosphatase for Pmk1 suppressed the Cl⫺ hypersensitivity of calcineurin-null cells. On the other hand, overexpression of pmp1⫹ did not affect the Ca2⫹ hypersensitivity of prz1-null cells (Fig. 7A). Furthermore, overexpression of prz1⫹ did not suppress the MgCl2 hypersensitivity of calcineurin-null cells. In addition, overexpression of constitutively active calcineurin did not suppress the CaCl2 hypersensitivity of prz1-null cells (Fig. 7A), whereas overexpression of prz1⫹ partially suppressed the CaCl2 hypersensitivity of calcineurin-null cells (data not shown). These results suggest that Prz1 acts downstream of calcineurin and regulates Ca2⫹ homeostasis. These results also suggest that Prz1 is not involved in Cl⫺ homeostasis that is antagonistically regulated by calcineurin and the Pmk1 MAPK pathways (32, 33). A model consistent with these data is presented in Fig. 7B.

18084


Acknowledgments—We thank Mitsuhiro Yanagida (Kyoto University, Kyoto, Japan), Takashi Toda and Paul Nurse (Cancer Research UK London Institute, London, United Kingdom) for their generous gift of strains and plasmids, and Susie O. Sio for critical reading of the paper. REFERENCES 1. Klee, C. B., Crouch, T. H., and Krinks, M. H. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6270 – 6273 2. Clipstone, N. A., and Crabtree, G. R. (1992) Nature 357, 695– 697 3. O’Keefe, S. J., Tamura, J., Kincaid, R. L., Tocci, M. J., and O’Neill, E. A. (1992) Nature 357, 692– 694 4. Molkentin, J. D., Lu, J. R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R., and Olson, E. N. (1998) Cell 93, 215–228 5. Hendey, B., Klee, C. B., and Maxfield, F. R. (1992) Science 258, 296 –299 6. Wang, H. G., Pathan, N., Ethell, I. M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T. F., and Reed, J. C. (1999) Science 284, 339 –343 7. Graef, I. A., Chen, F., Chen, L., Kuo, A., and Crabtree, G. R. (2001) Cell 105, 863– 875

3

R. Sugiura, H. Shuntoh, and T. Kuno, unpublished observations.

8. Mansuy, I. M., Mayford, M., Jacob, B., Kandel, E. R., and Bach, M. E. (1998) Cell 92, 39 – 49 9. Crabtree, G. R. (2001) J. Biol. Chem. 276, 2313–2316 10. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Cell 66, 807– 815 11. Cyert, M. S., Kunisawa, R., Kaim, D., and Thorner, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7376 –7380 12. Liu, Y., Ishii, S., Tokai, M., Tsutsumi, H., Ohki, O., Akada, R., Tanaka, K., Tsuchiya, E., Fukui, S., and Miyakawa, T. (1991) Mol. Gen. Genet. 227, 52–59 13. Kuno, T., Tanaka, H., Mukai, H., Chang, C. D., Hiraga, K., Miyakawa, T., and Tanaka, C. (1991) Biochem. Biophys. Res. Commun. 180, 1159 –1163 14. Cyert, M. S., and Thorner, J. (1992) Mol. Cell. Biol. 12, 3460 –3469 15. Nakamura, T., Liu, Y., Hirata, D., Namba, H., Harada, S., Hirokawa, T., and Miyakawa, T. (1993) EMBO J. 12, 4063– 4071 16. Mendoza, I., Rubio, F., Rodriguez-Navarro, A., and Pardo, J. M. (1994) J. Biol. Chem. 269, 8792– 8796 17. Farcasanu, I. C., Hirata, D., Tsuchiya, E., Nishiyama, F., and Miyakawa, T. (1995) Eur. J. Biochem. 232, 712–717 18. Pozos, T. C., Sekler, I., and Cyert, M. S. (1996) Mol. Cell. Biol. 16, 3730 –3741 19. Cunningham, K. W., and Fink, G. R. (1996) Mol. Cell. Biol. 16, 2226 –2237 20. Stathopoulos, A. M., and Cyert, M. S. (1997) Genes Dev. 11, 3432–3444 21. Matheos, D. P., Kingsbury, T. J., Ahsan, U. S., and Cunningham, K. W. (1997) Genes Dev. 11, 3445–3458 22. Stathopoulos-Gerontides, A., Guo, J. J., and Cyert, M. S. (1999) Genes Dev. 13, 798 – 803 23. Yoshimoto, H., Saltsman, K., Gasch, A. P., Li, H. X., Ogawa, N., Botstein, D., Brown, P. O., and Cyert, M. S. (2002) J. Biol. Chem. 277, 31079 –31088 24. Sugiura, R., Sio, S. O., Shuntoh, H., and Kuno, T. (2001) Cell Mol. Life Sci. 58, 278 –288 25. Sugiura, R., Sio, S. O., Shuntoh, H., and Kuno, T. (2002) Genes Cells 7, 619 – 627 26. Yoshida, T., Toda, T., and Yanagida, M. (1994) J. Cell Sci. 107, 1725–1735 27. Zhang, Y., Sugiura, R., Lu, Y., Asami, M., Maeda, T., Itoh, T., Takenawa, T., Shuntoh, H., and Kuno, T. (2000) J. Biol. Chem. 275, 35600 –35606 28. Yada, T., Sugiura, R., Kita, A., Itoh, Y., Lu, Y., Hong, Y., Kinoshita, T., Shuntoh, H., and Kuno, T. (2001) J. Biol. Chem. 276, 13579 –13586 29. Fujita, M., Sugiura, R., Lu, Y., Xu, L., Xia, Y., Shuntoh, H., and Kuno, T. (2002) Genetics 161, 971–981 30. Lu, Y., Sugiura, R., Yada, T., Cheng, H., Sio, S. O., Shuntoh, H., and Kuno, T. (2002) Genes Cells 7, 1009 –1019 31. Cheng, H., Sugiura, R., Wu, W., Fujita, M., Lu, Y., Sio, S. O., Kawai, R., Takegawa, K., Shuntoh, H., and Kuno, T. (2002) Mol. Biol. Cell 13, 2963–2976 32. Sugiura, R., Toda, T., Shuntoh, H., Yanagida, M., and Kuno, T. (1998) EMBO J. 17, 140 –148 33. Sugiura, R., Toda, T., Dhut, S., Shuntoh, H., and Kuno, T. (1999) Nature 399, 479 – 483 34. Toda, T., Dhut, S., Superti-Furga, G., Gotoh, Y., Nishida, E., Sugiura, R., and Kuno, T. (1996) Mol. Cell. Biol. 16, 6752– 6764 35. Moreno, S., Klar, A., and Nurse, P. (1991) Methods Enzymol. 194, 795– 823 36. Mukai, H., Ito, A., Kishima, K., Kuno, T., and Tanaka, C. (1991) J. Biochem. (Tokyo) 110, 402– 406 37. Maundrell, K. (1993) Gene (Amst.) 123, 127–130 38. Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Nature 373, 663– 664 39. Lafuente, M. J., Petit, T., and Gancedo, C. (1997) FEBS Lett. 420, 39 – 42 40. Rothstein, R. J. (1983) Methods Enzymol. 101, 202–211 41. Kohrer, K., and Domdey, H. (1991) Methods Enzymol. 194, 398 – 405 42. Perutz, M. F., Johnson, T., Suzuki, M., and Finch, J. T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5355–5358 43. Cunningham, K. W., and Fink, G. R. (1994) J. Cell Biol. 124, 351–363 44. Ishiguro, J., Saitou, A., Duran, A., and Ribas, J. C. (1997) J. Bacteriol. 179, 7653–7662


family protein (27, 31), respectively, suggesting that these gene products and Prz1 play an overlapping essential function. In the previous study (32), we showed that overexpression of Pmp1 MAPK phosphatase could suppress the aberrant cell morphology and Cl⫺ hypersensitivity of the calcineurin deletion. Thus, the second branch of the calcineurin signaling pathway seems to act antagonistically with the Pmk1 MAPK pathway and regulate various cellular events, such as morphogenesis and Cl⫺ homeostasis. Furthermore, prz1 deletion is not synthetically lethal with mutations in the its2⫹/cps1⫹, its8⫹, and its10⫹/cdc7⫹ genes (encoding ␤-glucan synthase, glycosylphosphatidylinositol biosynthetic enzyme, and a protein kinase in the SIN pathway, respectively) (28, 30, 44) (Table II). Thus, it is suggested that these three genes seem to have some genetic interactions with the second branch of the calcineurin signaling pathway. As these genes encode proteins that seem to be involved in the cell wall synthesis, these genetic data are in good agreement with the hypothesis that the Pmk1 MAPK pathway is involved in the regulation of cell wall integrity (34). In our preliminary studies in which we have been searching for downstream targets of the Pmk1 MAPK, we identified several candidates including certain novel putative transcription factors.3 Studies are in progress to determine whether these factors play some roles in the Cl⫺ homeostasis and are functionally related to the calcineurin signaling pathway.

MECHANISMS OF SIGNAL TRANSDUCTION: Zinc Finger Protein Prz1 Regulates Ca2+ but Not Cl− Homeostasis in Fission Yeast: IDENTIFICATION OF DISTINCT BRANCHES OF CALCINEURIN SIGNALING PATHWAY IN FISSION YEAST

J. Biol. Chem. 2003, 278:18078-18084. doi: 10.1074/jbc.M212900200 originally published online March 13, 2003

Access the most updated version of this article at doi: 10.1074/jbc.M212900200 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 44 references, 24 of which can be accessed free at http://www.jbc.org/content/278/20/18078.full.html#ref-list-1


Sonoko Hirayama, Reiko Sugiura, Yabin Lu, Takuya Maeda, Kenji Kawagishi, Mistuhiro Yokoyama, Hideki Tohda, Yuko Giga-Hama, Hisato Shuntoh and Takayoshi Kuno