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Mol Biol Cell. Feb 15, 2010; 21(4): 674–685.
PMCID: PMC2820430

The Cell Surface Protein Gene ecm33+ Is a Target of the Two Transcription Factors Atf1 and Mbx1 and Negatively Regulates Pmk1 MAPK Cell Integrity Signaling in Fission Yeast

Daniel J. Lew, Monitoring Editor

Abstract

The highly conserved fission yeast Pmk1 MAPK pathway plays a key role in cell integrity by regulating Atf1, which belongs to the ATF/cAMP-responsive element-binding (CREB) protein family. We identified and characterized ecm33+, which encodes a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. We demonstrated that the gene expression of Ecm33 is regulated by two transcription factors Atf1 and a MADS-box-type transcription factor Mbx1. We identified a putative ATF/CREB-binding site and an RLM1-binding site in the ecm33+ promoter region and monitored the transcriptional activity of Atf1 or Mbx1 in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of the CRE and six tandem repeats of the Rlm1-binding sequence, respectively. These reporter genes reflect the activation of the Pmk1 pathway by various stimuli, thereby enabling the real-time monitoring of the Pmk1 cell integrity pathway. Notably, the Δecm33 cells displayed hyperactivation of the Pmk1 signaling together with hypersensitivity to Ca2+ and an abnormal morphology, which were almost abolished by simultaneous deletion of the components of the Rho2/Pck2/Pmk1 pathway. Our results suggest that Ecm33 is involved in the negative feedback regulation of Pmk1 cell integrity signaling and is linked to cellular Ca2+ signaling.

INTRODUCTION

The mitogen-activated protein kinase (MAPK) pathway is one of the most important intracellular signaling pathways that play a crucial role in cell proliferation, cell differentiation, and cell cycle regulation (Nishida and Gotoh, 1993 blue right-pointing triangle; Marshall, 1994 blue right-pointing triangle; Herskowitz, 1995 blue right-pointing triangle; Levin and Errede, 1995 blue right-pointing triangle). MAPKs deliver extracellular signals from activated receptors to various cellular compartments, especially, the nucleus, where they regulate eukaryotic gene expression at the transcriptional and posttranscriptional levels (Pouyssegur, 2000 blue right-pointing triangle; Sugiura et al., 2003 blue right-pointing triangle; Edmunds and Mahadevan, 2004 blue right-pointing triangle; Satoh et al., 2009 blue right-pointing triangle).

In the budding yeast Saccharomyces cerevisiae, the Slt2/Mpk1 MAPK pathway mediates cell cycle–regulated cell wall synthesis and responds to different signals, including cell cycle regulation, growth temperature, changes in external osmolarity, and mating pheromones (Gustin et al., 1998 blue right-pointing triangle). Signaling proteins involved in the pathway include the GTP-binding protein Rho1, the protein kinase C homologue Pkc1, the MEKK Bck1p/Slk1p, the redundant pair of MAP/ERK kinases (MEKs) Mkk1 and Mkk2, the MAPK Slt2/Mpk1, and the transcription factor targets Rlm1 and SBF (Gustin et al., 1998 blue right-pointing triangle). Moreover, signaling via Mpk1/Slt2-Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis (Jung and Levin, 1999 blue right-pointing triangle; Jung et al., 2002 blue right-pointing triangle).

We have been studying the Pmk1 MAPK signaling pathway in the fission yeast Schizosaccharomyces pombe. The Pmk1 MAPK, a homologue of the mammalian ERK/MAPK plays a central role in cell integrity in fission yeast (Toda et al., 1996 blue right-pointing triangle; Zaitsevskaya-Carter and Cooper, 1997 blue right-pointing triangle). The Pmk1 MAPK pathway is composed of MAPKKK Mkh1 (Sengar et al., 1997 blue right-pointing triangle), MAPKK Pek1 (Sugiura et al., 1999 blue right-pointing triangle), and MAPK Pmk1/Spm1. The Pmk1 MAPK pathway also regulates ion homeostasis and morphogenesis (Satoh et al., 2009 blue right-pointing triangle) and is activated under multiple stresses, including heat shock, hyper- or hypotonic stresses, cell wall damage, or glucose deprivation (Toda et al., 1996 blue right-pointing triangle; Sugiura et al., 1999 blue right-pointing triangle; Madrid et al., 2006 blue right-pointing triangle).

We have previously demonstrated that calcineurin and Pmk1 MAPK play antagonistic roles in Cl homeostasis (Sugiura et al., 1998 blue right-pointing triangle, 2002 blue right-pointing triangle) and genetic screening on the basis of the functional interaction between calcineurin and Pmk1 MAPK has resulted in the isolation of negative regulators of the Pmk1 MAPK pathway, including pmp1+, encoding a dual-specificity MAPK phosphatase (Sugiura et al., 1998 blue right-pointing triangle); pek1+, encoding a MAPK kinase (MAPKK; Sugiura et al., 1999 blue right-pointing triangle); and rnc1+, encoding a novel KH-type RNA-binding protein that stabilizes Pmp1 mRNA (Sugiura et al., 2003 blue right-pointing triangle, 2004 blue right-pointing triangle). Moreover, genetic screening for vic (viable in the presence of immunosuppressant and chloride ion) mutants revealed that the cpp1+ gene, encoding a β subunit of the protein farnesyltransferase, and its target Rho2 GTPase (Ma et al., 2006 blue right-pointing triangle) act as upstream regulators of the Pmk1-signaling pathway.

Most recently, we have identified the Atf1 transcription factor as a downstream target of the Pmk1 MAPK pathway and demonstrated that Atf1 is involved in cell integrity in addition to its well-established role in the stress responses mediated by the Sty1/Spc1 MAPK pathway in fission yeast (Takada et al., 2007 blue right-pointing triangle). Mbx2, an Rlm1 homologue in fission yeast, unlike in budding yeast, displayed only a modest sensitivity to cell wall–damaging agents, suggesting that Mbx2 plays a minor role in this process (Takada et al., 2007 blue right-pointing triangle). Moreover, the intermediate phenotypes of the Δatf1 cells in the cell integrity response suggest that other unidentified target(s) of Pmk1 must play a significant role in the cell integrity pathway in fission yeast.

To identify novel genes involved in cell integrity signaling pathway, we searched for S. pombe homologues of the cell wall biogenesis genes regulated by the Mpk1-Rlm1 pathway in budding yeast. Of these genes, PST1 was particularly interesting because its gene expression was induced upon exposure to various cell wall–damaging agents such as azole and polyene under the control of the Slt2/Rlm1 signaling (Jung and Levin, 1999 blue right-pointing triangle; Agarwal et al., 2003 blue right-pointing triangle). Hence, we speculated that the fission yeast homologue of PST1 might serve as a good candidate for the target of Pmk1 signaling, as well as a good tool for studying the activation mechanism of Pmk1.

In the present study, we focused on ecm33+ (SPAC1705.03c) that encodes a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein, which is similar to ECM33 and PST1 in budding yeast, and characterized the expression mechanism and the role of Ecm33 in the Pmk1-mediated cell integrity signaling. Notably, Ecm33 expression was found to be regulated by two transcription factors, namely, Atf1 and Mbx1, via the putative cAMP-responsive element (CRE) sequence TTACAGTAA and the RLM1-binding sequence GTATATATAG in the promoter region of the ecm33+ gene. Furthermore, we developed reporter systems that allowed the monitoring of the real-time activity of Atf1 and Mbx1 in living cells by constructing a destabilized luciferase reporter gene with a reduced functional half-life fused to three tandem repeats of the CRE (3xCREecm33::luc) or six tandem repeats of the Rlm1-binding sequence (6xRlm1ecm33::luc). These reporter constructs also reflected the Pmk1 activity, thereby enabling the monitoring of the activation of the cell integrity pathway. We also presented evidence that Ecm33 is involved in the Pmk1 signaling by affecting Ca2+ homeostasis in fission yeast.

MATERIALS AND METHODS

Strains, Media, and Genetic and Molecular Biology Methods

S. pombe strains used in this study are listed in Table 1. The complete medium YPD (yeast extract-peptone-dextrose) and the minimal medium EMM (Edinburgh minimal medium) have been described previously (Toda et al., 1996 blue right-pointing triangle). Standard genetic and recombinant DNA methods (Moreno et al., 1991 blue right-pointing triangle) were used, except where specified. FK506 was provided by Astellas Pharma (Osaka, Japan). An S. pombe haploid strain in which the sty1+/spc1+ gene (geneID SPAC24B11.06c) had been deleted was purchased from Bioneer (Daejeon, Korea).

Table 1.
Schizosaccharomyces pombe strains used in this study

Cloning and Knockout of the ecm33+ Gene

The ecm33+ gene was amplified by PCR using the genomic DNA of S. pombe as a template. The sense primer used for PCR was 5′-GAA GAT CTC ATG TTG TTC AAA TCA TTC GCT CTC ACT C-3′ (BglII site and start codon are underlined), and the antisense primer was 5′-GAA GAT CTG CGG CCG CCC ATA GCA AGA GCA GCA ACC AAA AGA G-3′ (BglII and NotI site are underlined). The amplified product was digested with BglII/NotI, and the resulting fragment was subcloned into Bluescript SK(+) to create pBS-ecm33.

To knockout the ecm33+ gene, a one-step gene disruption by homologous recombination was performed (Rothstein, 1983 blue right-pointing triangle). The ecm33+ null mutants were obtained by entire deletion of the corresponding coding sequence and its replacement with the ura4+ cassette by PCR-mediated strategy using plasmid pFA6a-ura4 as the template (Bahler et al., 1998 blue right-pointing triangle).

The ecm33+ Promoter Assay

Firefly luciferase was chosen as a reporter, because the assay is simple to perform and has a high signal-to-noise ratio (Leskinen et al., 2003 blue right-pointing triangle). A 0.5-kb DNA fragment (P0.5, 500/2 base pairs) in the 5′ flanking region of the ecm33+ gene was amplified by PCR primers (forward primer 170, 5′-AA CTG CAG CAA GCT CCT CGT TGG TGT TGT GGCC-3′; reverse primer 126, 5′-CCG CTC GAG ATT GAC TTT AGA CTA TAT AAT GTA GAA ATA TG-3′). Similarly, the 5′-end deletion mutants of P0.5 (P0.45, 450/2 base pairs; P0.4, 402/2 base pairs; P0.37, 369/2 base pairs; and P0.3, 300/2 base pairs) were prepared using the reverse primer 126 and the following forward primers: 228 (5′-AA CTG CAG CAT TGT TTA CAG TAA ACA TTG CAA CG-3′), 229 (5′-AA CTG CAG CCT TTT TAT CTA ACA AGT CAC AAT TC-3′), 192 (5′-AA CTG CAG TTT CCG GGT ATA TAT AGA TGT CTT TTC CGC-3′), and 171 (5′-AA CTG CAG ACA CTC TTT TAC TTC TTT ATT CAT TAC CC-3′). The 3′-end deletion mutants of P0.5 (P0.2, 200/2 base pairs; and P0.1, 100/2 base pairs) were prepared using the forward primer 170 and the following reverse primers: 230 (5′-CCG CTC GAG TTA AAA CTC AAA TGT AGT TCG CTG-3′) and 257 (5′-CCG CTC GAG AAG GGG GAC AAC GAG GTG CGC-3′). The 5′-end and 3′-end deletion mutant of P0.5 (P0.07, 69/2 base pairs) was prepared using the forward primer 192 and the reverse primer 230. The various fragments of the 5′ promoter region of ecm33+ were subcloned into the PstI/XhoI-digested pKB5723 (Deng et al., 2006 blue right-pointing triangle), a multicopy vector that contains the destabilized luciferase gene from pGL3 (R2.2; Promega, Madison, WI).

Cells transformed with these reporter plasmids were cultured at 27°C in EMM to midlog phase. The ecm33+ promoter activity was measured as described by Deng et al. (2006) blue right-pointing triangle, with minor modifications. Briefly, the culture was diluted with fresh medium to OD660 = 0.2, and the cells were grown for 3 h at 27°C. Cells were incubated with 0.5 mM d-luciferin for 10 min at 27°C. Aliquots of the cell culture were pipetted into a 96-well plate, and NaCl was added to a final volume and concentration of 100 μl and 500 mM, respectively. Distilled water, which was used as control, was added to some of the wells. The mixture was incubated at 27°C for 2 h, and light emission levels expressed as relative light units were measured using a luminometer (AB-2300; Atto, Tokyo, Japan) at 12-s intervals.

Live-Cell Monitoring of Pmk1-mediated Transcriptional Activity

A 1.2-kb PstI/XhoI fragment of pKB5721 was replaced with the ecm33+-derived CRE oligonucleotide (sense 259: 5′-GGC TTT TAC AGT AAA TAC ATT ACA GTA AAT ACA CAT TAC AGT AAA TGC AC-3′, antisense 260: 5′-TCG AGT GCA TTT ACT GTA ATG TGT ATT TAC TGT AAT GTA TTT ACT GTA AAA GCC TGCA-3′) that contains three tandem repeats of CRE (TTACAGTAA or TTACTGTAA, underlined), which is the Atf1-binding core identified in the ecm33+ promoter, to yield pKD1934.

The point-mutated CRE reporter (pKD1953) was obtained in the same way as the CRE mutant (Pascual-Ahuir et al., 2001 blue right-pointing triangle) oligonucleotide (sense 269: 5′-GGC TTT TAT TTT AAA TAC ATT ATT TTA AAT ACA CAT TAT TTT AAA TGC AC-3′, antisense 270: 5′-TCG AGT GCA TTT AAA ATA ATG TGT ATT TAA AAT AAT GTA TTT AAA ATA AAA GCC TGCA-3′) that contains the three tandem repeats of the CRE mutant (TTATTTTAA or TTAAAATAA, underlined). Similarly, a 1.2-kb PstI/XhoI fragment of pKB5721 was replaced with the ecm33+-derived RLM1 oligonucleotide (sense 341: 5′-GGC TTG TAT ATA TAG ATA CAG TAT ATA TAG ATA CAC AGT ATA TAT AGA TAC AGT ATA TAT AGA TAC ACA GTA TAT ATA GAT ACA GTA TAT ATA GAT GCAC-3′, antisense 342: 5′-TCG AGT GCA TCT ATA TAT ACT GTA TCT ATA TAT ACT GTG TAT CTA TAT ATA CTG TAT CTA TAT ATA CTG TGT ATC TAT ATA TAC TGT ATC TAT ATA TAC AAG CCT GCA-3′) that contains six tandem repeats of RLM1 (GTATATATAG or CTATATATAC, underlined), which is the Mbx1-binding core identified in the ecm33+ promoter, to yield pKD1936.

The point-mutated RLM1 reporter (pKD1991) was obtained in the same way as the MEF2 mutant (Thai et al., 1998 blue right-pointing triangle) oligonucleotide (sense 367: 5′-GGC TTG TGG GCC CAG ATA CAG TGG GCC CAG ATA CAC AGT GGG CCC AGA TAC AGT GGG CCC AGA TAC ACA GTG GGC CCA GAT ACA GTG GGC CCA GAT GCA C-3′, antisense 368: 5′-TCG AGT GCA TCT GGG CCC ACT GTA TCT GGG CCC ACT GTG TAT CTG GGC CCA CTG TAT CTG GGC CCA CTG TGT ATC TGG GCC CAC TGT ATC TGG GCC CAC AAG CCT GCA-3′) that contains six tandem repeats of the RLM1 mutant (GTGGGCCCAG or CTGGGCCCAC, underlined).

These reporter vectors were used for live-cell monitoring of Pmk1-mediated transcriptional activity in living cells.

Northern Blot Analyses

Total RNA was isolated by the method of Kohrer and Domdey (1991) blue right-pointing triangle. A 20-μg sample of total RNA/lane was subjected to electrophoresis on denaturing formaldehyde 1% agarose gels and transferred to nylon membranes. Hybridization was performed using digoxigenin (DIG)-labeled antisense cRNA probes coding for Ecm33 and Leu1 as previously described (Hirayama et al., 2003 blue right-pointing triangle). The DIG-labeled hybrids were detected by an enzyme-linked immunoassay (ELISA) 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, Indianapolis, IN).

Miscellaneous Methods

Cell extract preparation and immunoblot analysis were performed as previously described (Sio et al., 2005 blue right-pointing triangle).

Ecm33 Monoclonal Antibody

Monoclonal antibody against Ecm33 was raised by using purified Ecm33 from S. pombe. For the first immunization, F344/N rats were subcutaneously injected with Ecm33 (50 μg protein in each rat) dissolved in 500 μl of saline emulsified with an equal volume of complete Freund's adjuvant (Difco, Detroit, MI) at multiple sites. After 3 wk, the rats were subcutaneously and intraperitoneally injected with Ecm33 (total 50 μg protein in each rat) dissolved in 500 μl of saline emulsified with an equal volume of incomplete Freund's adjuvant (Difco). A rat, the serum of which showed strong reactivity with purified Ecm33 expressed in S. pombe and E. coli, received final intraperitoneal and intravenous injections of Ecm33 (total 50 μg protein) without adjuvant. After 3 d, the rat was killed, and spleen cells were fused with P3X63Ag8.653 mouse myeloma cells, as described previously (Ohno et al., 2008 blue right-pointing triangle). Antibody secreted from 1920 hybridoma cultures was screened for reactivity with purified Ecm33 and unrelated proteins using ELISA and immune blotting. One selected hybridoma was cloned twice by the limiting dilution method. A monoclonal antibody designated 2P11 (rat IgG, κ) secreted by cloned hybridoma cells was used as a culture supernatant or as a purified monoclonal antibody as follows. Cloned hybridoma cells were intraperitoneally injected into KSN nude mice (3.0 × 106 cells/mouse) pretreated with 2,6,10,14-tetramethylpentadecane (500 μl/mouse; Pristane; Wako, Osaka, Japan). IgG in the ascites fluid was purified on protein G Sepharose (BD Healthcare, Uppsala, Sweden).

Immunostaining of Whole Cells

Cells were treated with PEMS (100 mM PIPES, pH 6.9, 1 mM EGTA, 1 mM MgSO4, 1 M sorbitol) containing zymolyase 20T (0.3 mg/ml) at 37°C until ~10% of the cells lost their cell walls, as observed under a microscope. Subsequently, the cells were washed with PBS three times and were incubated for 2 h at 4°C with 100 μl of 1% BSA-PBS containing anti-Ecm33 monoclonal antibodies (20 μg/ml). After incubation, the cells were washed three times with PBS and treated with 1:300-diluted FITC-conjugated goat anti-rat immunoglobulin (Sigma, St. Louis, MO) in 100 μl of 1% BSA-PBS in the dark for 1 h at 4°C.

Measurement of Cytosolic Free Ca2+ Concentration Using Aequorin

The intracellular free Ca2+ concentration was determined using a previously described method with minor modifications (Deng et al., 2006 blue right-pointing triangle). In brief, cells containing pREP1-AEQ (apoaequorin) were grown in EMM medium and harvested in the early logarithmic growth phase. Cells were resuspended in fresh EMM containing 10 μM coelenterazine, and the optical density of a 1-ml sample was adjusted to 0.6 at 660 nm. To convert the AEQ to aequorin, the cells were incubated for 4 h at 27°C. The cells were washed three times by centrifugation and resuspension in fresh EMM. The cells were then resuspended in EMM with an optical density of 0.6 at 660 nm for a 1-ml sample, and the cell culture was incubated at 27°C for 30 min before starting the experiment. The light emission levels expressed as relative light units were measured using the luminometer at 12-s intervals.

RESULTS

Identification of the ecm33+ Gene as a Target of Pmk1 and Atf1

To identify novel genes involved in the Pmk1 cell integrity signaling pathway, we searched for S. pombe homologues of the cell wall biogenesis genes regulated by the Mpk1-Rlm1 pathway in budding yeast (Jung and Levin, 1999 blue right-pointing triangle). Here, we focus on the ecm33+ gene (SPAC1705.03c) encoding a putative GPI-anchored cell surface protein (De Groot et al., 2003 blue right-pointing triangle), which is similar to PST1 (30% identity) and ECM33 (28% identity) in budding yeast (Pardo et al., 2004 blue right-pointing triangle).

Northern blot analysis demonstrated that under unstressed conditions, the ecm33+ (SPAC1705.03c) mRNA level was significantly reduced in Δatf1 cells and Δpmk1 cells compared with that in wild-type cells (Figure 1A), suggesting that the expression of ecm33+ is regulated by Pmk1/Atf1 signaling. Here, we characterized the ecm33+ gene as a transcriptional target of Pmk1 and Atf1. The Δecm33 cells, like Δpmk1 cells and Δatf1 cells, were highly sensitive to calcofluor, a cell wall–damaging agent (Figure 1B, +1.4 μg/ml calcofluor). Notably, the sensitivity of Δecm33 cells to calcofluor was higher than that of Δpmk1 cells and Δatf1 cells to this agent (Figure 1B, +1.2 μg/ml calcofluor). The cell integrity defect associated with the Δecm33 cells was further confirmed using β-glucanase, another cell wall–damaging agent. As shown in Figure 1C, the Δecm33 cells showed hypersensitivity to β-glucanase as did Δpmk1 cells. The Δatf1 cells showed intermediate response to β-glucanase compared with the responses of the wild-type cells and Δpmk1 cells (Figure 1C). Disruption of the meu10+ gene (Tougan et al., 2002 blue right-pointing triangle), which also shows significant amino acid similarity to PST1 and ECM33, did not result in cell wall defects (Figure 2E); therefore, we focused on the ecm33+ gene.

Figure 1.
Identification of Ecm33 as a target of Pmk1 and Atf1. (A) Northern blot analysis of total RNA from the wild-type (wt), Δatf1 cells, and Δpmk1 cells. Cells were incubated in YPD medium and collected after culture. Total RNA (20 μg) ...
Figure 2.
Promoter analysis of ecm33+ gene. (A) Deletion analysis of the ecm33+ promoter. Segment from the ecm33+ upstream region indicated at the left was inserted into the multicopy plasmid containing the luciferase reporter gene. The positions of the CRE (*) ...

To examine the expression and regulation of the ecm33+ gene in more detail, we developed a reporter construct containing a 0.5-kb sequence upstream of ATG of the ecm33+ gene fused to the destabilized version of luciferase R2.2 [ecm33 P(0.5)(R2.2)]. As shown in Figure 1D, the ecm33+ promoter analysis using [ecm33 P(0.5)(R2.2)] yielded similar results as obtained by Northern blot analysis under unstressed conditions (Figure 1D, basal). We further investigated whether the expression of the ecm33+ reporter gene was Pmk1- and Atf1-dependent under stress conditions. For this, we examined the effects of various stimuli, which have been reported to activate Pmk1 MAPK (Madrid et al., 2006 blue right-pointing triangle), on the ecm33+ reporter expression. As expected, NaCl (500 mM), CaCl2 (400 mM), KCl (400 mM), calcofluor (2.0 μg/ml), and micafungin (4.0 μg/ml) induced the expression of the ecm33+ reporter gene in wild-type cells (Figure 1D, wt). In contrast, this induction was almost completely abolished in Δpmk1 cells and Δatf1 cells (Figure 1D). Moreover, the overexpression of Pek1DD, the constitutively active version of MAPKK for Pmk1, increased the levels of the Ecm33 reporter gene under unstressed conditions (Figure 1E, wt+Pek1DD OP, basal). Notably, the effect of overexpressing Pek1DD and addition of NaCl (500 mM) seemed to be additive because the reporter response was elevated (Figure 1E, wt+Pek1DD OP, 500 mM NaCl). Knockout of the pmk1+ gene abolished the effects of Pek1DD overexpression as well as of the addition of NaCl (Figure 1E, Δpmk1).

The subcellular localization of Ecm33 was determined using anti-Ecm33 antibody (α-Ecm33) by immunofluorescence microscopy. As shown in Figure 1F, Ecm33 localized to the cell surface. Immunoblotting experiments using anti-Ecm33 antibodies showed that the 43.3-kDa protein was detected in the wild-type cells (wt) and in the cells overproducing the ecm33+ gene (Ecm33 OP), but not in Δecm33 cells, indicating that the antibodies specifically recognized the Ecm33 protein (Figure 1G).

Deletion Analysis of the ecm33+ Promoter

To determine the promoter region involved in the Pmk1-dependent ecm33 expression, the 5′ deletion mutants of the 0.5-kb DNA fragment (P0.5) of the ecm33+ gene promoter were generated and subcloned into the multicopy luciferase vector (Figure 2A). These plasmids were transformed into a wild-type strain, and the promoter assay was performed under basal conditions (Figure 2B, basal, and Table 2). Deletion of the 5′-flanking sequences from −500 to −450 had little effect on the reporter activity (Table 2). Deletion of the region from −450 to −402 reduced the promoter activity by ~54% (Figure 2B, pKD1361). The luciferase reporter construct containing the 0.3-kb sequence upstream of ATG of the ecm33+ gene (P0.3R2.2; Figure 2A, pKD1193) showed almost no detectable promoter activity (Figure 2B, 300–1, basal). Moreover, the luciferase reporter constructs containing the region from −500 to −300 (pKD1952, pKD1361, and pKD1360) displayed an enhanced promoter activity in response to a variety of Pmk1-activating stimuli as shown in Figure 1D, whereas the same stresses failed to induce the promoter activity of (P0.3R2.2; Figure 2B). On the basis of these results, we conclude that the region from −500 to −300 base pairs upstream of ATG of the ecm33+ gene is important for its regulated expression.

Table 2.
Promoter analysis of the ecm33+ gene

A database search (TESS SEARCH) revealed a CRE-like sequence motif TTACAGTAA at position −444 to −436 (Figure 2, A and C, CRE, underlined) and a sequence similar to the RLM1-binding motif GTATATATAG at position −362 to −353 (Figure 2, A and C, RLM1, underlined) of the ecm33+ gene. The presence of putative consensus elements for the binding of the Rlm1-like transcription factor as well as the ATF1/cAMP-responsive element-binding (CREB) family protein in the promoter region of the ecm33+ gene from −500 to −300 upstream of ATG prompted us to examine the involvement of Mbx1-like transcription factors in the regulation of Ecm33 expression. Next, we examined the promoter activity of p0.5(R2.2) in deletion mutant cells of Mbx1-like transcription factor genes, namely, Δmbx1 and Δmbx2, and compared them with the promoter activities of the Δatf1, Δpmk1, or wild-type cells. Notably, disruption of the mbx1+ gene, but not the Rlm1-homologous gene mbx2+, resulted in a significant reduction of the Ecm33 promoter activity compared with that of the wild-type cells (Figure 2D). The relative promoter activity of the Δmbx1 cells was almost equivalent to that of the Δpmk1 cells, but was slightly higher than that of the Δatf1 cells (Figure 2D). Moreover, deletion of Mbx1, but not Mbx2, abrogated the induction of promoter response by various stimuli, which activate the Pmk1 pathway (Figure 2D). Similarly, disruption of the mbx1+ gene, but not the mbx2+ gene, resulted in the hypersensitivity to calcofluor (1.4 μg/ml) as observed in Δpmk1, Δatf1, or Δecn33 cells (Figure 2E). Thus, we concluded that the Mbx1 transcription factor is also involved in the cell integrity pathway by regulating Ecm33 expression.

Real-Time Monitoring of Atf1 Activity in Living Cells

Atf1 activity in living cells was monitored by 3xCREECM33 fused to R2.2 destabilized luciferase [3xCREECM33::luc (R2.2)]. As shown in Figure 3A, wild-type cells harboring the multicopy 3xCREECM33::luc (R2.2) reporter were stimulated by the addition of 500 mM NaCl, a hyperosmotic stress that is reported to stimulate Atf1 activity (Wilkinson et al., 1996 blue right-pointing triangle). Elevated extracellular NaCl caused an extremely rapid increase in the 3xCREECM33::luc (R2.2) reporter response within 3 min, followed by a rapid decrease to reach its lowest value at around 30 min, then again showed a second increase, and finally approached a constant level (Figure 3A, wt). In contrast, the Δatf1 cells harboring the same reporter showed minimal responses to the same stimuli, indicating that multicopy 3xCREECM33::luc (R2.2) reporter appears to be a reliable reporter of Atf1 activity (Figure 3A, Δatf1). To examine whether this CRE site was of functional relevance, we used PCR primers to mutate the −444/−436 element. Compared with the wild-type promoter 3xCREECM33::luc (R2.2) reporter, mutation in the CRE element caused a marked reduction of the promoter activity with (500 mM NaCl) or without (0 mM NaCl) the stimuli [Figure 3A, 3xCREmECM33::luc (R2.2)].

Figure 3.
Real-time monitoring of Atf1 activity in living cells. (A) Wild-type cells or Δatf1 cells harboring the multicopy plasmid [3xCREECM33::luc(R2.2) reporter vector] or the mutant version of the reporter vector [3xCREmECM33::luc(R2.2)] were incubated ...

We also examined whether the 3xCREECM33::luc (R2.2) reporter expression was dependent on two upstream MAPK pathways that phosphorylate and regulate Atf1, namely, the Sty1/Spc1 MAPK and the Pmk1 MAPK pathways. As shown in Figure 3B, the reporter expression of the 3xCREECM33::luc (R2.2) in Δsty1 cells was barely detectable both in the absence and presence of a hyperosmotic stress. In addition, the 3xCREECM33::luc (R2.2) promoter activity was very low in Δpmk1 cells compared with that in the wild-type cells and responded only weakly to the hyperosmotic stress (Figure 3B, Δpmk1). Moreover, when constitutively active MAPKK Pek1DD was overexpressed in the wild-type cells, a significantly higher level of 3xCREECM33::luc (R2.2) reporter response was observed even without any stimulation (Figure 3C, wt-Pek1DD OP), and addition of NaCl to the medium further stimulated the response (Figure 3C, wt-Pek1DD OP, +500 mM NaCl). This induction by Pek1DD and the addition of NaCl was almost abolished in Δpmk1 cells (Figure 3C, Δpmk1), indicating the Pmk1-dependent response of the 3xCREECM33::luc (R2.2) reporter. It should be noted that the relative light units in Δpmk1 cells were ~20% of that of the wild-type cells and were relatively higher than those of the Δatf1 cells and Δsty1 cells. Thus, 3xCREECM33::luc (R2.2) indicates both Sty1 and Pmk1 activation. The biphasic activation of the signal upon NaCl stimulation may reflect the intracellular Ca2+ concentration, i.e., the first sudden increase reflects the Ca2+ influx from the plasma membrane-localized Ca2+ channels, and the second increase indicates a mechanism similar to that of Ca2+-induced Ca2+ release from the intracellular Ca2+ store.

To determine the cross-talk between Pmk1 and Sty1/Spc1 MAPK cascade in the regulation of Ecm33 transcription, we examined whether Pek1DD overexpression can overcome the Δsty1 defect in the transcription from 3xCREECM33. Pek1DD overexpression failed to increase the transcription level of ECM33 from 3xCRE even after NaCl stimulation (Figure 3C, Δsty1). This might be because of instability of the Atf1 protein in the absence of Sty1. Atf1 is a target for the ubiquitin-proteasome system (Lawrence et al., 2009 blue right-pointing triangle), and Sty1 phosphorylation of Atf1 is required for modulating Atf1 stability and is vital for a robust response to certain stresses (Lawrence et al., 2007 blue right-pointing triangle). Therefore, in the absence of the Sty1 protein, the Atf1 protein may be easily degraded and may fail to respond to Pmk1 activation by Pek1DD overexpression.

Real-Time Monitoring of Mbx1 Activity in Living Cells

We next created the reporter construct 6xRLMECM33 fused to R2.2 destabilized luciferase [6xRLMECM33::luc (R2.2)]. As shown in Figure 4A, wild-type cells harboring the multicopy 6xRLMECM33::luc (R2.2) reporter were stimulated by the addition of 500 mM NaCl. Elevated extracellular NaCl also caused a rapid increase in the 6xRLMECM33::luc (R2.2) reporter response within 3 min, followed by a rapid decrease to reach its lowest value at around 30 min, then again showed a second increase, and finally approached a constant level (Figure 4A, wt). In contrast, the Δmbx1 cells harboring the same reporter showed minimal responses to the same stimuli, indicating that multicopy 6xRLMECM33::luc (R2.2) reporter could reflect the Mbx1 activity (Figure 4A, Δmbx1). Disruption of mbx2+ did not affect the promoter response (data not shown). Moreover, mutation at a consensus RLM site dramatically reduced the 6xRLMECM33::luc (R2.2) promoter activity with or without the stimuli (Figure 4A, wt, 6xRLMmECM33).

Figure 4.
Real-time monitoring of Mbx1 activity in living cells. (A) Wild-type cells or Δmbx1 cells harboring the multicopy plasmid [6xRLMECM33::luc(R2.2) reporter vector] or the mutant version of the reporter vector [6xRLMmECM33::luc(R2.2)) were incubated ...

Notably, disruption of the pmk1+ gene reduced the basal promoter activity and almost abolished the induction of the reporter by NaCl addition (Figure 4B, Δpmk1). Sty1 deletion did not significantly affect the rapid response of the 6xRLMECM33::luc (R2.2) reporter, whereas the second increase in the reporter response to NaCl stimulation in Δsty1 cells was distinct from that in the wild-type cells (Figure 4B, Δsty1). Thus, the rapid phase of the 6xRLMECM33::luc (R2.2) is a faithful reporter of Pmk1 pathway activation; Sty1 may also be involved in some aspects of the Mbx1 regulation.

Moreover, the overexpression of the constitutively active Pek1DD induced reporter expression in wild-type cells even in the absence of stimulation (Figure 4C, wt-Pek1DD OP, basal), and addition of NaCl to the medium further increased this response (Figure 4C, wt-Pek1DD OP, +500 mM NaCl). This induction by Pek1DD and its enhancement with the addition of NaCl were almost completely abolished in Δpmk1 cells, thereby indicating a Pmk1-dependent response of the 6xRLMECM33::luc (R2.2) reporter (Figure 4C, Δpmk1). Moreover, the induction by Pek1DD and its enhancement with the addition of NaCl were also abolished in Δmbx1 cells (Figure 4C, Δmbx1). Thus, the Pek1DD-induced transcription from 6xRLM ECM33 observed in wild-type cells depends on the Mbx1 transcription factor.

Role of Ecm33 in Pmk1 Signaling

We previously demonstrated that mutations in the components of the Pmk1 pathway result in the vic phenotype (Ma et al., 2006 blue right-pointing triangle). These components include Pmk1 MAPK, Pek1 MAPKK, Mkh1 MAPKKK, Pck2 protein kinase C, and Rho2. We further examined the functional relationship between Ecm33 and Pmk1 signaling by analyzing whether the disruption of the ecm33+ gene affected the chloride ion hypersensitivity induced by the inhibition of the protein phosphatase calcineurin by using the immunosuppressant FK506, a specific inhibitor of calcineurin (Sugiura et al., 1998 blue right-pointing triangle). The results showed that Δecm33 cells, like wild-type cells, failed to grow in the presence of the immunosuppressant FK506 and 0.12 M MgCl2, whereas Δpmk1 cells grew well under these conditions (Figure 5A, +0.12 M MgCl2 +FK506). Moreover, Δecm33 cells failed to grow in the presence of the immunosuppressant FK506 and 0.08 M MgCl2, wherein wild-type cells grew slowly (Figure 5A, +0.08 M MgCl2 +FK506). Thus, Ecm33 deletion exacerbated the chloride ion hypersensitivity induced by calcineurin inhibition. In our previous study, we showed that the hyperactivation of Pmk1 MAPK by the overexpression of the constitutively active Pek1DD exacerbated the chloride ion hypersensitivity of calcineurin deletion (Sugiura et al., 1999 blue right-pointing triangle). This suggested that Ecm33 deletion, like Pek1DD, induced hyperactivation of Pmk1 signaling. To investigate this possibility, we examined the level of Pmk1 phosphorylation in Δecm33 cells using anti-phospho Pmk1 antibodies that recognize only phosphorylated and hence activated Pmk1 (Sugiura et al., 1999 blue right-pointing triangle). The results revealed that Δecm33 cells showed increased Pmk1 phosphorylation level compared with that of the wild-type cells under normal conditions(Figure 5B, 0 min). Moreover, upon treatment with CaCl2, the phosphorylation of Pmk1 was greatly induced in Δecm33 cells than in wild-type cells (Figure 5B, left panel). Further, the addition of 500 mM NaCl induced a higher-than-normal level of Pmk1 phosphorylation in Δecm33 cells (Figure 5B, right panel). Consistently, the vic-negative phenotype associated with Δecm33 cells was rescued by Pmk1 deletion, because the Δecm33Δpmk1 double mutant cells grew well in the presence of the immunosuppressant FK506 and 0.12 M MgCl2 as did Δpmk1 cells (Figure 5A, Δecm33Δpmk1). In addition, disruption of the rho2+ gene, an upstream activator of the Pmk1 pathway, also rescued the vic-negative phenotype of Δecm33 cells (data not shown). Thus, loss of Ecm33 function induced hyperactivation of the Rho2/Pmk1 cell integrity pathway.

Figure 5.
Ecm33 is involved in Pmk1 MAPK-mediated cell integrity signaling. (A) The ecm33+ gene knockout displayed a vic-negative phenotype. The cells as indicated were spotted onto the plates and then incubated for 4 d at 27°C. (B) Knockout of the ecm33 ...

We next examined the effect of the overexpression of Ecm33 on the chloride ion hypersensitivity of calcineurin deletion (Δppb1). Our previous data showed that overexpression of the dual-specificity phosphatase Pmp1 or the type 2C phosphatases Ptc1 or Ptc3 suppressed the chloride ion hypersensitivity of Δppb1 cells by inhibiting Pmk1 activation (Sugiura et al., 1998 blue right-pointing triangle; Takada et al., 2007 blue right-pointing triangle). If Ecm33 were considered to play a role in the negative regulation of Pmk1 signaling, it would be expected that Ecm33 overexpression would also suppress Δppb1 cells. As expected, Δppb1 cells overexpressing the ecm33+ gene could grow in the presence of 0.12 M MgCl2, whereas those bearing the control vector alone failed to grow (Figure 5C). Moreover, the overexpression of the ecm33+ gene almost abolished the stimulation of Pmk1 phosphorylation both before and after CaCl2 treatment (Figure 5D, Ecm33 OP, left panel). The inhibitory effect of Ecm33 overproduction on Pmk1 phosphorylation was also observed when cells were treated with 500 mM NaCl (Figure 5D, right panel).

Knockout of the pmk1+ Gene Rescued Phenotypes of Δecm33 Cells

Another striking feature of Δecm33 cells is their hypersensitivity to CaCl2. As shown in Figure 6A, Δecm33 cells grew poorly in the media supplemented with 150 mM CaCl2, whereas wild-type cells grew normally. In contrast, Δecm33 cells grew well in the media supplemented with 150 mM MgCl2 or 300 mM NaCl, suggesting that Ca2+ homeostasis is altered in Δecm33 cells (Figure 6A). Moreover, the Ca2+-hypersensitive phenotype observed in Δecm33 cells was rescued by Pmk1 deletion (Figure 6A, Δecm33Δpmk1), suggesting that this phenotype somehow results from Pmk1 hyperactivation in Δecm33 cells. In addition, morphologically, the Δecm33 cells were abnormally enlarged and swollen compared with the wild-type cells (Figure 6B, Δecm33). Notably, simultaneous deletion of Pmk1 almost rescued the morphological abnormality observed in Δecm33 cells (Figure 6B, Δecm33Δpmk1). Interestingly, the above finding that Pmk1 deletion rescued Δecm33 phenotypes clearly contrasts the report in budding yeast where simultaneous deletion of ECM33 and SLT2 results in synthetic lethality (Pardo et al., 2004 blue right-pointing triangle).

Figure 6.
Altered Ca2+ homeostasis in Δecm33 cells. (A) Δecm33 cells exhibited Ca2+ hypersensitivity. Wild-type cells, Δecm33, Δpmk1, or Δecm33Δpmk1 cells were streaked onto the plates as indicated and then incubated ...

Altered Calcium Homeostasis in Δecm33 Cells

To further characterize the Ca2+-related phenotypes associated with Δecm33 cells, we used the 3xCDRE::luc(R2.2) reporter system that was developed to monitor the real-time activity of the Ca2+/calcineurin signaling pathway (Deng et al., 2006 blue right-pointing triangle). We assumed that if Ca2+ homeostasis is compromised in Δecm33 cells, the CDRE reporter response would be altered from that of the wild-type cells. As shown in Figure 6C, the Δecm33 cells showed an enhanced 3xCDRE::luc(R2.2) reporter response in the presence of various concentrations of extracellular CaCl2 (0–200 mM), compared with that of the wild-type cells. Notably, compared with the wild-type cells, Δecm33 cells showed a continuous increase in the 3xCDRE::luc(R2.2) response (Figure 6C, 100 mM CaCl2, 200 mM CaCl2). The enhanced calcineurin activity as evidenced by the CDRE response and Ca2+ hypersensitivity of Δecm33 cells is reminiscent of that observed in Δpmc1 cells, which lack the vacuolar Ca2+-ATPase (Deng et al., 2006 blue right-pointing triangle). On the other hand, the overproduction of Ecm33 (Ecm33 OP) lowered the CDRE response even in the presence of 200 mM CaCl2 (Figure 6D, 100 mM CaCl2, 200 mM CaCl2).

We further examined the effect of Pmk1 deletion on the CDRE reporter response. In the presence of 100 mM CaCl2, the Δpmk1 cells exhibited a slightly lower peak response of the CDRE reporter than the wild-type cells. In the presence of 200 mM CaCl2, the peak response of the Δpmk1 cells was approximately half that of the wild-type cells (Figure 6E, Δpmk1). Importantly, the peak responses of the CDRE reporter in the Δecm33Δpmk1 cells were almost similar to those in the Δpmk1 cells suggesting that the increased Ca2+/calcineurin signaling observed in the Δecm33 mutant is dependent on Pmk1. However, the calcineurin activity remains higher in the Δecm33Δpmk1 cells than in the Δpmk1 cells, after the peak response to CaCl2stimulation has been attainted (Figure 6E, 200 mM CaCl2).

To provide additional information regarding the defective Ca2+ homeostasis in Δecm33 cells, we monitored intracellular Ca2+ levels in wild-type and Δecm33 cells. The Δecm33 cells displayed a higher Ca2+ concentration than that observed in the wild-type cells in terms of the level of peak response (Figure 7). In contrast, intracellular Ca2+ levels in cells overexpressing the ecm33+ gene were significantly lower than those in the cells bearing the control vector alone (Figure 7, Ecm33 OP). Taken together, the results suggest that Ecm33 might exert its effect on Pmk1 signaling by affecting Ca2+ homeostasis in fission yeast.

Figure 7.
Ecm33 regulates Ca2+ influx. The peak response of intracellular Ca2+ monitoring after the addition of CaCl2. Wild-type, Δecm33, and cells overproducing Ecm33 (Ecm33 OP) were transformed with pREP1-AEQ, and their intracellular Ca2+ levels were ...

DISCUSSION

In the present study, the identification of Ecm33 as a novel component of the Pmk1 MAPK cell integrity signaling has led to the discovery that two transcription factors, namely, Atf1 and Mbx1, are involved in the Pmk1-dependent expression of Ecm33. We also developed a reporter system to monitor the real-time activity of these transcription factors and hence the activation of the Pmk1 pathway.

Here, we show that Ecm33 is involved in the cell integrity signaling. First, mutants lacking ecm33+ displayed hypersensitivity to two typical cell wall–damaging agents, calcofluor white and β-glucanase. Second, Δecm33 cells exhibited the vic-negative phenotype and hyperphosphorylation of the Pmk1 MAPK, which is a strong indication of the negative regulation of the Pmk1 signaling (Ma et al., 2006 blue right-pointing triangle). Third, Ecm33 overproduction suppressed calcineurin deletion and inhibited Pmk1 MAPK phosphorylation upon treatment with CaCl2 and NaCl. Fourth, the mRNA levels of ecm33+ were Pmk1/Atf1– as well as Pmk1-Mbx1–dependent. Thus, Ecm33 is a novel component of the Pmk1 MAPK pathway.

In budding yeast, signaling via Mpk1-Rlm1 regulates the expression of several genes implicated in cell wall biogenesis, including PST1 (Jung and Levin, 1999 blue right-pointing triangle; Jung et al., 2002 blue right-pointing triangle). Unexpectedly, the deletion mutants of Mbx2, an Rlm1 homologue in fission yeast, displayed only modest sensitivity to cell wall–damaging agents, such as calcofluor (Figure 2E), suggesting that unlike budding yeast, the Rlm1 homolog only plays a minor role in cell wall integrity in fission yeast. Here, we identified Ecm33, a cell surface GPI-anchor protein homologous to PST1, as a target of Pmk1 and Atf1. Moreover, Mbx1, but not Mbx2, was found to be involved in the regulation of Ecm33 expression (Figure 2D). Mbx1 has been shown to be involved in gene expression in the M-G1 phase as a component of PBF (Pombe cell cycle box binding factor) transcriptional complex (Buck et al., 2004 blue right-pointing triangle). Although Mbx1 genetically and functionally interacts with two forkhead transcription factors Fkh2 and Sep1 together with Plo1, the direct target of Mbx1 and its physiological role have not yet been elucidated; this is because the deletion of mbx1+ has little effect on M–G1 transcription (Papadopoulou et al., 2008 blue right-pointing triangle). In this study, we showed that Mbx1 is involved in cell integrity in fission yeast via the regulation of Ecm33 in a Pmk1-dependent manner. Recently, Papadopoulou et al. (2008) blue right-pointing triangle reported that the Polo kinase Plo1, a key regulator of cell cycle, binds and phosphorylates Mbx1. It would be intriguing to speculate that Plo1 and Pmk1 kinases coordinately regulate cell cycle and/or cell integrity signaling via the phospho-regulation of Mbx1 activity.

Another important finding of this study is the role of Ecm33 in the MAPK cell integrity signaling. The finding that Ecm33 deletion and overproduction affect Ca2+/calcineurin signaling and Ca2+ homeostasis suggested the possibility that some Ca2+-mobilizing mechanism(s) might be involved in the Ecm33-mediated suppression of Pmk1 signaling. Carnero et al. (2000) blue right-pointing triangle reported that ehs1+/yam8+, encoding a homologue of the budding yeast Mid1, is involved in Ca2+ accumulation and cell wall integrity. Interestingly, high extracellular levels of Ca2+ as well as pck2+ overexpression suppressed all the phenotypes of ehs1/yam8 mutants, suggesting that the cell integrity defects of ehs1/yam8 mutant result from inadequate calcium levels in the cell (Carnero et al., 2000 blue right-pointing triangle). Similarly, our recent study showed that Pmk1 is required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca2+ influx and that knockout of pck2+ gene markedly diminished the Yam8/Cch1-dependent stimulation of calcineurin activity, suggesting that Pck2 acts upstream of Pmk1 in this signaling pathway (Deng et al., 2006 blue right-pointing triangle). Thus, exogenous Ca2+ activates the Pck2/Pmk1 signaling, which in turn leads to Yam8/Cch1-mediated Ca2+ influx. The hyperactivation of Pck2/Pmk1 signaling induces lethality associated with strong Ca2+ accumulation (Carnero et al., 2000 blue right-pointing triangle; Deng et al., 2006 blue right-pointing triangle). One way to reverse this effect is by the dephosphorylation and inactivation of Pmk1 via the overexpression of Pmp1 or PP2C phosphatases as evidenced in our previous studies (Sugiura et al., 1998 blue right-pointing triangle; Takada et al., 2007 blue right-pointing triangle). Alternatively, inactivation of the Ca2+-influx machinery, such as Yam8/Cch1 complex, and maintenance of the normal Ca2+ homeostasis within the cell would also rescue cells from the lethal effect.

The molecular mechanism underlying Ecm33-mediated modulation of Pmk1 signaling is currently unknown. Given the plasma membrane localization of Ecm33, we hypothesize that Ecm33, a GPI-anchored protein, like the sensor protein Wsc1 in budding yeast (Philip and Levin, 2001 blue right-pointing triangle), might interact with some component(s) of the plasma membrane localizing MAPK signaling molecules and/or the components of Ca2+-influx machinery to inhibit protein function (Figure 8). Our results showed that Ecm33 deletion and overproduction affects Pmk1 phosphorylation upon treatment with CaCl2 and NaCl, two independent stimuli that activate Pmk1 (Figure 5, B and D). Moreover, Pmk1 deletion markedly suppressed the increased Ca2+/calcineurin signaling observed in Δecm33 cells (Figure 6E). These results favor the possibility that Ecm33 impinges on the Pmk1 MAPK cascade via a Ca2+-independent mechanism and that Pmk1 then regulates Ca2+ influx. However, Δecm33Δpmk1 cells exhibited a higher CDRE reporter activity than the Δpmk1 cells after the peak response (Figure 6E), and Ecm33 overproduction resulted in a lower CDRE response than that in Δpmk1 cells upon in the CaCl2 treatment (Figure 6D). Thus, it does not exclude the possibility that Ecm33 may exert its effect on Ca2+/calcineurin signaling largely via Pmk1 pathway and partly via other Ca2+-influx machineries (Figure 8). Further studies will be required to clarify the precise role of Ecm33 and its involvement in the MAPK signaling.

Figure 8.
A model for the dual regulation of ecm33+ gene expression and its putative involvement in the negative feedback regulation of the Pmk1 MAPK signaling pathway. Regulatory elements in the promoter are schematically represented. Dotted lines denote hitherto ...

In conclusion, to our knowledge, this article provides the first evidence for the involvement of a GPI-anchored cell surface protein in the negative regulation of cell wall integrity Pmk1 MAPK signaling. Furthermore, we also discovered a novel functional link between Ecm33 and cellular Ca2+ signaling. Given the high similarity between the MAPK pathways of fission yeast and the mammals, this study may provide the basis of understanding the regulatory mechanisms underlying MAPK signaling in higher eukaryotes.

ACKNOWLEDGMENTS

We thank Drs. M. Yanagida, and T. Toda and Yeast Resource Centre (YGRC/NBRP; http://yeast.lab.nig.ac.jp/nig) for providing strains and plasmids; Kazue Masuko for able technical assistance; and Susie O. Sio and Yukiko Fujimoto for critical reading of the manuscript. We are grateful to the members of the Laboratory of Molecular Pharmacogenomics for their support. This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas and research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (R.S.). This work was also financially supported by the Antiaging Center Project for Private Universities from Ministry of Education, Culture, Sports, Science, and Technology, 2008–2012. H.T. is a Research Fellow of the Japan Society for the Promotion of Science.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-09-0810) on December 23, 2009.

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