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Zinc Finger Protein Prz1 Regulates Ca2+ but Not Cl− Homeostasis in Fission Yeast

2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês

10.1074/jbc.m212900200

1083-351X

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

Endoplasmic Reticulum Stress and Disease

Calcineurin is an important mediator that connects the Ca2+-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 prz1+ 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 Ca2+ 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 Cl−. Instead, theprz1-null cells showed hypersensitivity to Ca2+, consistent with a dramatic decrease in transcription of Pmc1 Ca2+ pump. Interestingly, overexpression of Prz1 did not suppress the Cl− hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Cl− hypersensitivity of calcineurin deletion but not the Ca2+ hypersensitivity of prz1 deletion. In addition, mutations in theits2+/cps1+,its8+, andits10+/cdc7+ 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 is an important mediator that connects the Ca2+-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 prz1+ 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 Ca2+ 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 Cl−. Instead, theprz1-null cells showed hypersensitivity to Ca2+, consistent with a dramatic decrease in transcription of Pmc1 Ca2+ pump. Interestingly, overexpression of Prz1 did not suppress the Cl− hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Cl− hypersensitivity of calcineurin deletion but not the Ca2+ hypersensitivity of prz1 deletion. In addition, mutations in theits2+/cps1+,its8+, andits10+/cdc7+ 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. the nuclear factor of activated T cells mitogen-activated protein kinase immunosuppressant- andtemperature-sensitive Ppb1-responsive zinc finger protein green fluorescent protein digoxigenin serine-rich region phosphatidylinositol 4-phosphate 5 Calcineurin is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase consisting of a catalytic subunit and a regulatory subunit (1Klee C.B. Crouch T.H. Krinks M.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6270-6273Crossref PubMed Scopus (611) Google Scholar). In mammalian cells, calcineurin plays an important role in various Ca2+-mediated processes including T-cell activation (2Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar, 3O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar), cardiac hypertrophy (4Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar), neutrophil chemotaxis (5Hendey B. Klee C.B. Maxfield F.R. Science. 1992; 258: 296-299Crossref PubMed Scopus (173) Google Scholar), apoptosis (6Wang H.G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (967) Google Scholar), angiogenesis (7Graef I.A. Chen F. Chen L. Kuo A. Crabtree G.R. Cell. 2001; 105: 863-875Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), and memory development (8Mansuy I.M. Mayford M. Jacob B. Kandel E.R. Bach M.E. Cell. 1998; 92: 39-49Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). For many of these cellular events, calcineurin exerts its function by regulating the NF-AT1family members. Calcineurin directly dephosphorylates NF-AT transcription factors, causing their activation and trans-location from the cytoplasm to the nucleus (9Crabtree G.R. J. Biol. Chem. 2001; 276: 2313-2316Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Furthermore, calcineurin is specifically inhibited by the immunosuppressants cyclosporin A and FK506 (10Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. In the budding yeast Saccharomyces cerevisiae, calcineurin-deficient strains exhibit normal growth under standard conditions (11Cyert M.S. Kunisawa R. Kaim D. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7376-7380Crossref PubMed Scopus (241) Google Scholar, 12Liu Y. Ishii S. Tokai M. Tsutsumi H. Ohki O. Akada R. Tanaka K. Tsuchiya E. Fukui S. Miyakawa T. Mol. Gen. Genet. 1991; 227: 52-59Crossref PubMed Scopus (143) Google Scholar). However, calcineurin function is required for cell viability under some specific growth conditions. Calcineurin mutants deficient for either the catalytic subunits (CNA1/CNA2) (11Cyert M.S. Kunisawa R. Kaim D. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7376-7380Crossref PubMed Scopus (241) Google Scholar, 12Liu Y. Ishii S. Tokai M. Tsutsumi H. Ohki O. Akada R. Tanaka K. Tsuchiya E. Fukui S. Miyakawa T. Mol. Gen. Genet. 1991; 227: 52-59Crossref PubMed Scopus (143) Google Scholar) or the regulatory subunit (CNB1) (13Kuno T. Tanaka H. Mukai H. Chang C.D. Hiraga K. Miyakawa T. Tanaka C. Biochem. Biophys. Res. Commun. 1991; 180: 1159-1163Crossref PubMed Scopus (84) Google Scholar, 14Cyert M.S. Thorner J. Mol. Cell. Biol. 1992; 12: 3460-3469Crossref PubMed Scopus (207) Google Scholar) die in the presence of high concentrations of different ions including manganese, sodium, lithium, and hydroxyl ions (15Nakamura T. Liu Y. Hirata D. Namba H. Harada S. Hirokawa T. Miyakawa T. EMBO J. 1993; 12: 4063-4071Crossref PubMed Scopus (230) Google Scholar, 16Mendoza I. Rubio F. Rodriguez-Navarro A. Pardo J.M. J. Biol. Chem. 1994; 269: 8792-8796Abstract Full Text PDF PubMed Google Scholar, 17Farcasanu I.C. Hirata D. Tsuchiya E. Nishiyama F. Miyakawa T. Eur. J. Biochem. 1995; 232: 712-717Crossref PubMed Scopus (71) Google Scholar, 18Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Crossref PubMed Scopus (129) Google Scholar). Some of these ion sensitivities are because of a defect in the calcineurin-dependent regulation of several ion transporter genes including PMR1, PMR2, andPMC1 (16Mendoza I. Rubio F. Rodriguez-Navarro A. Pardo J.M. J. Biol. Chem. 1994; 269: 8792-8796Abstract Full Text PDF PubMed Google Scholar, 19Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Crossref PubMed Scopus (383) Google Scholar) whose expressions are regulated through the Crz1/Tcn1 transcription factor (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar, 21Matheos D.P. Kingsbury T.J. Ahsan U.S. Cunningham K.W. Genes Dev. 1997; 11: 3445-3458Crossref PubMed Scopus (280) Google Scholar). The expression ofFKS2, which encodes a β-1,3-glucan synthase, is also regulated by Crz1 through a calcineurin-dependent mechanism (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar). When calcineurin is activated, it dephosphorylates Crz1, causing its rapid trans-location from the cytoplasm to the nucleus (22Stathopoulos-Gerontides A. Guo J.J. Cyert M.S. Genes Dev. 1999; 13: 798-803Crossref PubMed Scopus (202) Google Scholar), 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 (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar). 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 (23Yoshimoto H. Saltsman K. Gasch A.P. Li H.X. Ogawa N. Botstein D. Brown P.O. Cyert M.S. J. Biol. Chem. 2002; 277: 31079-31088Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). 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 terms of relevance to higher systems (24Sugiura R. Sio S.O. Shuntoh H. Kuno T. Cell Mol. Life Sci. 2001; 58: 278-288Crossref PubMed Scopus (56) Google Scholar, 25Sugiura R. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 619-627Crossref PubMed Scopus (47) Google Scholar). S. pombe has a single gene encoding the catalytic subunit of calcineurin,ppb1+ (26Yoshida T. Toda T. Yanagida M. J. Cell Sci. 1994; 107: 1725-1735Crossref PubMed Google Scholar). We have developed a genetic screen for mutants that depend on calcineurin for growth using the immunosuppressant FK506 and have given the designation itsmutants. The analyses of the its mutants revealed that calcineurin is implicated in cytokinesis, septation initiation network, and exocytic pathway in fission yeast (27Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 29Fujita M. Sugiura R. Lu Y. Xu L. Xia Y. Shuntoh H. Kuno T. Genetics. 2002; 161: 971-981Crossref PubMed Google Scholar, 30Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, 31Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar). We have shown that fission yeast calcineurin plays an essential role in maintaining chloride ion homeostasis and acts antagonistically with the Pmk1 MAPK pathway (32Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar, 33Sugiura R. Toda T. Dhut S. Shuntoh H. Kuno T. Nature. 1999; 399: 479-483Crossref PubMed Scopus (72) Google Scholar). 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 theits mutants showing synthetic lethal interaction with calcineurin deletion did not exhibit synthetic lethality with theprz1-null mutation. These results indicate that there are at least two distinct branches of calcineurin signaling pathway. 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 (35Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3148) Google Scholar), have been described previously (34Toda T. Dhut S. Superti-Furga G. Gotoh Y. Nishida E. Sugiura R. Kuno T. Mol. Cell. Biol. 1996; 16: 6752-6764Crossref PubMed Scopus (189) Google Scholar). 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. (35Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3148) Google Scholar). FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Calcineurin and calmodulin were prepared from bovine brain as described previously (36Mukai H. Ito A. Kishima K. Kuno T. Tanaka C. J. Biochem. (Tokyo). 1991; 110: 402-406Crossref PubMed Scopus (15) Google Scholar).Table ISchizosaccharomyces pombe strains used in this studyStrainGenotypeReferenceHM123h−leul-32Our stockHM528h+his2Our stockKP161h−leul-32 ura4-D18 ppbl::ura4+Our stockKP162h−leul-32 ypt3–15Cheng et al. (31Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar)KP165h−leul-32 cps1–12This studyKP167h−leul-32 its3–1Zhang et al. (27Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar)KP533h−leul-32 its8–1Yadaet al. (28Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar)KP553h−leul-32 cdc7-i10Lu et al. (30Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar)KP1003h−leul-32 ura4-D18 przl::ura4+This studyKP1245h+leul-32 ura4–294Lafuente et al. (39Lafuente M.J. Petit T. Gancedo C. FEBS Lett. 1997; 420: 39-42Crossref PubMed Scopus (8) Google Scholar)KP1395h−leul-32 ura4–294 ura4+::nmtl-GFP:przl+This studyKP1464h−leul-32 ura4-D18 przl::ura4+cpsl-i2This studyKP1466h−leul-32 ura4-D18 przl::ura4+cdc7-i10This studyKP1467h−leul-32 ura4-D18 przl::ura4+its8–1This study Open table in a new tab 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 Δ (for example, Δprz1). 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. pombedata base search service (www.sanger.ac.uk). Theprz1+ gene was amplified by PCR with the genomic DNA of S. pombe as a template. The sense primer used for PCR was 5′-CG GGATCCATG GAG CGT CAA AGG TCA GAA GAA GCC AT-3′ (BamHI site and start codon areunderlined), and the antisense primer was 5′-CGGGATCCTCA TTT TTG TTT GCT TGT CGA GGC-3′ (BamHI site and stop codon areunderlined). The amplified product was digested withBamHI, and the resulting fragment was subcloned into Bluescript SK(+). For ectopic expression of proteins, we used the thiamine-repressiblenmt1 promoter at various levels of expression (37Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (931) Google Scholar). Expression was repressed by the addition of 4 μg/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 ofprz1+ was ligated to the C terminus of the GFP carrying the S65T mutation (38Heim R. Cubitt A.B. Tsien R.Y. Nature. 1995; 373: 663-664Crossref PubMed Scopus (1527) Google Scholar). 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 (37Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (931) Google Scholar). 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 theura4+ gene locus of KP1245 (h+leu1–32 ura4–294) (39Lafuente M.J. Petit T. Gancedo C. FEBS Lett. 1997; 420: 39-42Crossref PubMed Scopus (8) Google Scholar). A one-step gene disruption by homologous recombination (40Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar) was performed. Theprz1::ura4+ disruption was constructed as follows. Cloned open reading frame of theprz1+ gene in the Bluescript vector was digested with BamHI and EcoRI, and the resulting fragment containing ∼80%prz1+ gene was subcloned into theBamHI/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). 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 μl 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 mmbenzamidine, 0.1 mm sodium metabisulfite, 0.1 μg/ml chymostatin, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml phosphoramidon, and 0.5 μg/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 μg/5 μl) were subjected to SDS-PAGE and immunoblotted with anti-GFP antibody. Total RNA was isolated by the method of Kohrer and Domdey (41Kohrer K. Domdey H. Methods Enzymol. 1991; 194: 398-405Crossref PubMed Scopus (507) Google Scholar). 20 μg 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 enzyme-linked 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). 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.). A BLAST program search using the peptide sequence of Crz1, the calcineurin-responsive zinc finger transcription factor of S. cerevisiae (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar, 21Matheos D.P. Kingsbury T.J. Ahsan U.S. Cunningham K.W. Genes Dev. 1997; 11: 3445-3458Crossref PubMed Scopus (280) Google Scholar) 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 geneprz1+ (forPpb1-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 domain in many cases (42Perutz M.F. Johnson T. Suzuki M. Finch J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5355-5358Crossref PubMed Scopus (954) Google Scholar). 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. 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 SDS-PAGE 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 mmCaCl2 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 calcineurin-null cells were treated with purified calcineurinin 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 hyperphosphorylated 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. 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 (Ppb1ΔC) increased the steady-state levels of Prz1 mRNA (Fig. 2C). In either wild-type or calcineurin-null cells cultured under standard conditions, GFP-Prz1 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 mmCa2+, GFP-Prz1 remained cytosolic (Fig. 3A). These results are in good agreement with those obtained with GFP-Crz1 in budding yeast (22Stathopoulos-Gerontides A. Guo J.J. Cyert M.S. Genes Dev. 1999; 13: 798-803Crossref PubMed Scopus (202) Google Scholar). 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 cycle-specific 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 (27Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 29Fujita M. Sugiura R. Lu Y. Xu L. Xia Y. Shuntoh H. Kuno T. Genetics. 2002; 161: 971-981Crossref PubMed Google Scholar, 30Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, 31Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar). The addition of calcineurin inhibitor FK506 again completely blocked nuclear accumulation of GFP-Prz1 in these experiments (data not shown).Figure 4Localization of Prz1 to the cytoplasm requires its SRR. Living cells transformed with the vector for the expression of GFP-Prz1 (full-length) or GFP-Prz1ΔSRR (N-terminally truncated) were grown at 30 °C and analyzed as described above. In some cases cells were incubated with 0.5 μm FK506 for 30 min at 30 °C (+ FK506). Arrow indicates a dividing cell whose nucleus shows intense GFP fluorescence. The barindicates 10 μm.View Large Image Figure ViewerDownload (PPT) A GFP-Prz1 fusion lacking the N-terminal 240 residues of Prz1 (GFP-Prz1ΔSRR for lacking serine-richregion) partially complemented the growth defects of aprz1-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 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 (22Stathopoulos-Gerontides A. Guo J.J. Cyert M.S. Genes Dev. 1999; 13: 798-803Crossref PubMed Scopus (202) Google Scholar). 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 0.3m KCl (32Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar). 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 calcineurin-null cells could not grow in the presence of 0.15m CaCl2 or 0.15 mCa(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 ofPMC1, which encodes a Ca2+ pump playing a key role in Ca2+ tolerance (43Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (365) Google Scholar). Consistently, Northern blot analysis revealed that calcineurin (ppb1) deletion andprz1 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 sensitive 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 (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar). Thus, the roles of calcineurin in Ca2+ homeostasis in these two yeasts are suggested to be quite different. 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. (26Yoshida T. Toda T. Yanagida M. J. Cell Sci. 1994; 107: 1725-1735Crossref PubMed Google Scholar), 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. (26Yoshida T. Toda T. Yanagida M. J. Cell Sci. 1994; 107: 1725-1735Crossref PubMed Google Scholar), 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 ofprz1 deletion and that calcineurin acts upstream of the Prz1 transcription factor. As described above, we have isolated mutants that depend on calcineurin for growth using the immunosuppressant FK506 and have given the designation its mutants (27Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 29Fujita M. Sugiura R. Lu Y. Xu L. Xia Y. Shuntoh H. Kuno T. Genetics. 2002; 161: 971-981Crossref PubMed Google Scholar, 30Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, 31Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar). 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 itsmutants with prz1-null cells was performed. As shown in Table II, we found thatprz1-null mutation was synthetically lethal withits3 and its5/ypt3-i5 mutants that encode phosphatidylinositol 4-phosphate 5 (PI(4)P5)-kinase and a small GTPase of the Rab/Ypt family, respectively (27Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 31Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar). On the other hand, mutations in its2+/cps1+,its8+, andits10+/cdc7+ genes (encoding β-glucan synthase, glycosylphosphatidylinositol anchor synthetic enzyme, and protein kinase implicated in septation initiation, respectively) (28Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 30Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, 44Ishiguro J. Saitou A. Duran A. Ribas J.C. J. Bacteriol. 1997; 179: 7653-7662Crossref PubMed Google Scholar) did not show synthetic lethality withprz1-null mutation and double mutants were obtained (TableII). The Δprz1 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.Table IIGenetic interactions between prz1+ or ppb1+ and its (immunosuppressant- and temperature-sensitive) mutantsits2/cps1-i2its3its5/ypt3-i5its8its10/cdc7-i10β-glucan synthase (44)P1(4)P5 kinase (27)Rab family (31)GPI biosynthetic enzyme (28)SIN pathway kinase (30)ΔprzlviablelethallethalviableviableΔppbllethallethallethallethallethalSynthetic lethality in crosses between various mutants was examined by tetrad analysis following sporulation. Open table in a new tab Synthetic lethality in crosses between various mutants was examined by tetrad analysis following sporulation. 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 (32Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar, 33Sugiura R. Toda T. Dhut S. Shuntoh H. Kuno T. Nature. 1999; 399: 479-483Crossref PubMed Scopus (72) Google Scholar). Consistent with our previous results, overexpression ofpmp1+ 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 ofprz1-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 ofprz1+ partially suppressed the CaCl2hypersensitivity 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 (32Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar, 33Sugiura R. Toda T. Dhut S. Shuntoh H. Kuno T. Nature. 1999; 399: 479-483Crossref PubMed Scopus (72) Google Scholar). A model consistent with these data is presented in Fig. 7B. 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 α-factor (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar). 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 calcineurin-regulated gene expression in budding yeast (23Yoshimoto H. Saltsman K. Gasch A.P. Li H.X. Ogawa N. Botstein D. Brown P.O. Cyert M.S. J. Biol. Chem. 2002; 277: 31079-31088Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). On the other hand, with the exception of its hypersensitivity to Ca2+, prz1-null 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 considerably different from that of calcineurin-null cells in fission yeast. 2R. Sugiura, H. Tohda, Y. Giga-Hama, H. Shuntoh, and T. Kuno, unpublished observations. These results strongly suggest that there are at least two branches of calcineurin signaling pathway. Related to this issue is the observation that inS. 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+ (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar), 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 (20Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (385) Google Scholar, 21Matheos D.P. Kingsbury T.J. Ahsan U.S. Cunningham K.W. Genes Dev. 1997; 11: 3445-3458Crossref PubMed Scopus (280) Google Scholar). To further analyze the heterogeneous nature of the calcineurin signaling pathway, we examined the genetic interaction between prz1 deletion andits mutants that are synthetically lethal with calcineurin deletion. As shown in Table II, prz1 deletion is synthetically lethal with mutations in the its3+ andits5+/ypt3+ genes encoding a PI(4)P5 kinase and a Rab family protein (27Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 31Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar), respectively, suggesting that these gene products and Prz1 play an overlapping essential function. In the previous study (32Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar), 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 theits2+/cps1+,its8+, andits10+/cdc7+ genes (encoding β-glucan synthase, glycosylphosphatidylinositol biosynthetic enzyme, and a protein kinase in the SIN pathway, respectively) (28Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 30Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, 44Ishiguro J. Saitou A. Duran A. Ribas J.C. J. Bacteriol. 1997; 179: 7653-7662Crossref PubMed Google Scholar) (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 (34Toda T. Dhut S. Superti-Furga G. Gotoh Y. Nishida E. Sugiura R. Kuno T. Mol. Cell. Biol. 1996; 16: 6752-6764Crossref PubMed Scopus (189) Google Scholar). 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. 3R. Sugiura, H. Shuntoh, and T. Kuno, unpublished observations. 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. 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.