From the Division of Molecular Pharmacology and
Pharmacogenomics, Department of Genome Sciences, and ¶ Division of
Cardiovascular and Respiratory Medicine, Kobe University Graduate
School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan,
ASPEX Division, Research Center, Asahi Glass Co., Ltd. 1150 Hazawa-cho, Kanagawa-ku, Yokohama, 221-8755, Japan, and
** Faculty of Health Science, Kobe University School of
Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 650-0142, Japan
Received for publication, December 18, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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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 Calcineurin is a Ca2+/calmodulin-dependent
serine/threonine protein phosphatase consisting of a catalytic subunit
and a regulatory subunit (1). In mammalian cells, calcineurin plays an
important role in various Ca2+-mediated processes including
T-cell activation (2, 3), cardiac hypertrophy (4), neutrophil
chemotaxis (5), apoptosis (6), angiogenesis (7), and memory development
(8). For many of these cellular events, calcineurin exerts its function by regulating the NF-AT1
family members. Calcineurin directly dephosphorylates NF-AT
transcription factors, causing their activation and trans-location from
the cytoplasm to the nucleus (9). Furthermore, calcineurin is
specifically inhibited by the immunosuppressants cyclosporin A and
FK506 (10), and these drugs have been a powerful tool for identifying
many of the roles of calcineurin.
In the budding yeast Saccharomyces cerevisiae,
calcineurin-deficient strains exhibit normal growth under standard
conditions (11, 12). However, calcineurin function is required for cell viability under some specific growth conditions. Calcineurin mutants deficient for either the catalytic subunits
(CNA1/CNA2) (11, 12) or the regulatory subunit
(CNB1) (13, 14) die in the presence of high concentrations
of different ions including manganese, sodium, lithium, and hydroxyl
ions (15-18). Some of these ion sensitivities are because of a defect
in the calcineurin-dependent regulation of several ion
transporter genes including PMR1, PMR2, and
PMC1 (16, 19) whose expressions are regulated through the
Crz1/Tcn1 transcription factor (20, 21). The expression of
FKS2, which encodes a 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 (24, 25). S. pombe has a single gene encoding the catalytic subunit of calcineurin,
ppb1+ (26). We have developed a genetic screen for
mutants that depend on calcineurin for growth using the
immunosuppressant FK506 and have given the designation its
mutants. The analyses of the its mutants revealed that
calcineurin is implicated in cytokinesis, septation initiation network,
and exocytic pathway in fission yeast (27-31). We have shown that
fission yeast calcineurin plays an essential role in maintaining
chloride ion homeostasis and acts antagonistically with the Pmk1 MAPK
pathway (32, 33). These phenotypes are quite different from those
described above for calcineurin deletion in budding yeast, suggesting
that the upstream or downstream signaling events of calcineurin may be distinct in these two distantly related yeasts.
Here, we cloned the fission yeast prz1+ gene, which
encodes a zinc finger protein highly homologous to Crz1. Consistent
with the hypothesis that Prz1 is the functional homolog of Crz1, Prz1 is dephosphorylated by calcineurin, causing its rapid trans-location from the cytoplasm to the nucleus. However, the prz1-null
cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Cl Strains, Media, and Miscellaneous Procedures--
S.
pombe strains used in this study are
listed in Table I. The complete medium, YPD (1% yeast extract, 2%
polypeptone, 2% glucose), and the minimal medium, Edinbugh
minimal medium (35), have been described previously (34). SPA
mating and sporulation medium contained 10 g/liter glucose, 1 g/liter
KH2PO4, 1 ml/liter 1000× vitamin stock
solution (same as those used for EMM), and 30 g/liter agar. Standard
methods for S. pombe genetics were followed according to
Moreno et al. (35). FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Calcineurin and calmodulin were
prepared from bovine brain as described previously (36).
Gene disruptions are denoted by lowercase letters representing the
disrupted gene followed by two colons and the wild-type gene marker
used for disruption (for example,
prz1::ura4+). Also, gene
disruptions are denoted by an abbreviation of the gene preceded by
Data base searches were performed using the National Center for
Biotechnology Information BLAST network service
(www.ncbi.nlm.nih.gov) and the Sanger Center S. pombe
data base search service (www.sanger.ac.uk).
Cloning and Tagging of the prz1+ Gene--
The
prz1+ gene was amplified by PCR with the genomic DNA
of S. pombe as a template. The sense primer used for PCR was
5'-CG GGA TCC ATG GAG CGT CAA AGG
TCA GAA GAA GCC AT-3' (BamHI site and start codon are
underlined), and the antisense primer was 5'-CG
GGA TCC TCA TTT TTG TTT GCT TGT CGA
GGC-3' (BamHI site and stop codon are
underlined). The amplified product was digested with
BamHI, and the resulting fragment was subcloned into
Bluescript SK(+).
For ectopic expression of proteins, we used the thiamine-repressible
nmt1 promoter at various levels of expression (37). Expression was repressed by the addition of 4 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 of
prz1+ was ligated to the C terminus of the GFP
carrying the S65T mutation (38). GFP-Prz1 fully complements the growth
defects of a prz1-null strain (data not shown). The
GFP-fused gene was subcloned into pREP1, pREP41, or pREP81 vectors to
express the gene at various levels. Maximum expression of the fused
gene was obtained using pREP1, whereas pREP81 contained the most
attenuated version of the nmt1 promoter (37). To obtain the
chromosome-born GFP-Prz1 instead of the plasmid-born GFP-Prz1, the
fused genes with the nmt1 promoter at various levels were
subcloned into the vector containing the ura4+
marker and were integrated into the chromosome at the
ura4+ gene locus of KP1245 (h+
leu1-32 ura4-294) (39).
Deletion of prz1+ Gene--
A one-step gene
disruption by homologous recombination (40) was performed. The
prz1::ura4+ disruption was
constructed as follows. Cloned open reading frame of the
prz1+ gene in the Bluescript vector was digested
with BamHI and EcoRI, and the
resulting fragment containing ~80%
prz1+ gene was subcloned into the
BamHI/EcoRI site of pUC119. A SmaI fragment containing the ura4+ gene then was inserted
into the EcoRV site of the previous construct, causing the
interruption of the open reading frame. The fragment containing
disrupted prz1+ gene was transformed into diploid
cells. Stable integrants were selected on medium lacking uracil, and
disruption of the gene was checked by genomic Southern hybridization
(data not shown).
Cell Extract Preparation and Immunoblot Analysis--
For the
analysis of electrophoretic mobility shift of Prz1, whole-cell extracts
were prepared from cultures of wild-type or calcineurin-null cells
expressing GFP-Prz1 grown at 30 °C to mid-log phase. Cells were
resuspended in 450 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 mM
benzamidine, 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.
Northern Blot Analyses--
Total RNA was isolated by the
method of Kohrer and Domdey (41). 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).
Microscopic Analysis--
Cells were grown to exponential phase
in YPD or EMM medium and shifted to various conditions as indicated in
the figure legends. In some cases, cells were washed with
phosphate-buffered saline, pH 7.0, and then stained with Hoechst 33342 or Calcofluor to visualize the DNA or septum, respectively, before
microscopic observation.
Cells were microscopically examined under an Axioskop microscope (Carl
Zeiss Inc.). Photographs were taken with a SPOT2 digital camera
(Diagnostic Instruments Inc.). Images were processed with the CorelDRAW
software (Corel Corporation Inc.).
Identification of the S. pombe prz1+ Gene--
A BLAST
program search using the peptide sequence of Crz1, the
calcineurin-responsive zinc finger transcription factor of S. cerevisiae (20, 21) against the S. pombe protein data
base at the Sanger Center revealed an open reading frame, SPAC4G8.13c, exhibiting significant similarity to Crz1 (score = 293, p = 6.3e Prz1 is Dephosphorylated by Calcineurin--
We examined whether
Prz1 phosphorylation is modulated by calcineurin. GFP-Prz1 protein
showed a significant alteration in its mobility when analyzed by
immunoblot. GFP-Prz1 protein isolated from calcineurin null or from the
wild-type cells treated with the specific calcineurin inhibitor, FK506,
migrated on 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 mM
CaCl2 migrated with a slightly smaller apparent molecular
mass (Fig. 1C). The change in the ratio of the faster and
slower migrating GFP-Prz1 species suggested that there is a
calcineurin-dependent change in the phosphorylation state
of Prz1. To establish that the mobility change of GFP-Prz1 was indeed
the result of variable degrees in phosphorylation, protein extracts
from calcineurin-null cells were treated with purified calcineurin
in vitro. Treatment of the larger form of GFP-Prz1 with
calcineurin converted it to the smaller form (Fig. 1D). This
change in apparent molecular mass was dependent on Ca2+ ion
and calmodulin (Fig. 1D) and was blocked by the addition of
FK506 (data not shown). These findings confirmed that Prz1 is
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.
High Extracellular Ca2+ and High Temperature Induction
of Prz1 mRNA Is Dependent on Calcineurin Activity--
Fig.
2A showed that Prz1 mRNA
accumulation was induced rapidly in wild-type but not in
calcineurin-null cells grown at 27 °C after the addition of
CaCl2 (30 mM) to the growth medium, indicating that Prz1 expression itself is calcineurin-responsive and that Prz1
controls the activity of its own promoter in response to calcineurin
signaling. We also examined the effect of temperature upshift on the
steady-state levels of Prz1 mRNA. Fig. 2B showed that
the level of Prz1 mRNA is strongly induced by a shift to growth at
42 °C in wild-type but not in calcineurin-null cells, peaking at 20 min after the shift. Pretreatment of the wild-type cells with FK506
completely blocked Prz1 mRNA accumulation induced by both high
extracellular Ca2+ and high temperature, again indicating
that the transient induction observed is calcineurin-responsive (data
not shown). Consistent with this hypothesis, the expression of
constitutively active calcineurin (Ppb1 Activation of Calcineurin Causes the Trans-location of GFP-Prz1
from the Cytoplasm to the Nucleus--
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 mM
Ca2+, GFP-Prz1 remained cytosolic (Fig. 3A).
These results are in good agreement with those obtained with GFP-Crz1
in budding yeast (22). Consistently, wild-type cells expressing the
constitutively active calcineurin showed nuclear localization of
GFP-Prz1 in the absence of exogenous Ca2+ stimulation (Fig.
3A). In addition, cell 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 (27-31). The addition
of calcineurin inhibitor FK506 again completely blocked nuclear
accumulation of GFP-Prz1 in these experiments (data not shown).
A GFP-Prz1 fusion lacking the N-terminal 240 residues of Prz1
(GFP-Prz1 Prz1-null Cells Are Hypersensitive to Ca2+ but Not to
Cl
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 (20). Thus, the roles of calcineurin in
Ca2+ homeostasis in these two yeasts are suggested to be
quite different.
prz1-null and Calcineurin-null Mutants Have Distinct Phenotypes in
Cell Morphology and Mating--
In addition to ion homeostasis,
prz1-null cells showed phenotypes distinct from those of
calcineurin-null cells in cell morphology and mating (Fig.
6). As shown in Fig. 6A,
calcineurin-null cells were enlarged, multiseptated, and branched,
consistent with the previous study by Yoshida et al. (26),
which suggests the involvement of calcineurin in the regulation of the
cell polarity and cytokinesis. On the other hand, prz1-null
cells were indistinguishable from the wild-type cells in
morphology.
Furthermore, unlike calcineurin-null cells, which were sterile as
reported by Yoshida et al. (26), prz1-null cells
were fertile and their mating efficiency and spore morphology were indistinguishable from wild-type cells (Fig. 6B).
prz1-null cells treated with FK506 showed the same
phenotypes as calcineurin-null cells (Fig. 6), indicating that
the effects of eliminating calcineurin is epistatic to the effects of
prz1 deletion and that calcineurin acts upstream of the Prz1
transcription factor.
Some of the Mutants That Show Synthetic Lethality with Calcineurin
Deletion Do Not Show Synthetic Lethality with prz1
Deletion--
As described above, we have isolated mutants that
depend on calcineurin for growth using the immunosuppressant FK506 and
have given the designation its mutants (27-31). These
mutants showed synthetic lethal genetic interaction with calcineurin.
Therefore, their genes encode the functional proteins that may share
essential function with calcineurin. To examine the relationship
between these genes and the prz1+ gene, tetrad
analysis of a diploid derived from a cross between the its
mutants with prz1-null cells was performed. As shown in Table II, we found that
prz1-null mutation was synthetically lethal with
its3 and its5/ypt3-i5 mutants that
encode phosphatidylinositol 4-phosphate 5 (PI(4)P5)-kinase and a small
GTPase of the Rab/Ypt family, respectively (27, 31). On the other hand,
mutations in its2+/cps1+,
its8+, and
its10+/cdc7+ genes (encoding
Prz1 Is Not Involved in Chloride Ion Homeostasis That Is
Antagonistically Regulated by Calcineurin and the Pmk1 MAPK
Pathways--
Our previous study showed that calcineurin acts
antagonistically with the Pmk1 MAPK pathway in Cl 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
Obviously, one branch is the Prz1-dependent branch that
regulates the expression of Pmc1 Ca2+ pump. The
Ca2+ sensitivity of prz1-null cells is
consistent with the markedly reduced level of Pmc1 mRNA, suggesting
a similar regulatory mechanism of Ca2+ homeostasis in these
two distantly related yeasts (20, 21). To further analyze the
heterogeneous nature of the calcineurin signaling pathway, we examined
the genetic interaction between prz1 deletion and
its mutants that are synthetically lethal with calcineurin
deletion. As shown in Table II, prz1 deletion is
synthetically lethal with mutations in the its3+ and
its5+/ypt3+ genes encoding a
PI(4)P5 kinase and a Rab family protein (27, 31), respectively,
suggesting that these gene products and Prz1 play an overlapping
essential function.
In the previous study (32), we showed that overexpression of Pmp1 MAPK
phosphatase could suppress the aberrant cell morphology and
Cl. Instead, the
prz1-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 the
its2+/cps1+,
its8+, and
its10+/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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,3-glucan synthase, is also
regulated by Crz1 through a calcineurin-dependent mechanism
(20). When calcineurin is activated, it dephosphorylates Crz1, causing
its rapid trans-location from the cytoplasm to the nucleus (22),
suggesting similar modes of regulation by calcineurin for its
downstream transcription factors in budding yeast and mammals. A
disruption of CRZ1 gene caused similar phenotypes as those
of calcineurin mutants, and in calcineurin mutants, these phenotypes
are suppressed by CRZ1 overexpression (20). These results
suggest that Crz1 functions downstream of calcineurin to effect most of
the calcineurin-dependent cellular responses in
budding yeast. Furthermore, recent genome-wide analysis of gene
expression regulated by the calcineurin/Crz1 signaling pathway confirm
that Crz1 is the major and possibly the only effector of
calcineurin-regulated gene expression in budding yeast (23).
. Instead, they showed
hypersensitivity to Ca2+. In addition, some of the
its mutants showing synthetic lethal interaction with
calcineurin deletion did not exhibit synthetic lethality with the
prz1-null mutation. These results indicate that there are at
least two distinct branches of calcineurin signaling pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Schizosaccharomyces pombe strains used in this study
(for example,
prz1). Proteins are denoted by Roman
letters, and only the first letter is capitalized (for example, Prz1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
25, identities = 58/125
(46%), positives = 75/125 (60%)). We named the gene
prz1+ (for
Ppb1-responsive zinc finger
protein). As shown in Fig. 1, A and B, the prz1+ gene
encodes a protein of 681 amino acids that contains three C2H2-type zinc
finger motifs at its carboxyl terminus highly homologous to those of
Crz1. However, unlike Crz1, Prz1 does not have a polyglutamine tract,
which acts as a transcriptional activation domain in many cases (42).
Outside of the zinc finger domain, Prz1 also contains a serine-rich
region (SRR, residues 57-219, N score = 8.748 (Prosite)), but it
shows low homology with that of Crz1.
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Fig. 1.
Prz1 is a C2H2-type zinc finger protein and
is a calcineurin substrate. A, schematic structure of
Prz1 protein. Denoted are the SRR and three putative zinc fingers
(ZnF). B, sequence alignment of the three zinc
finger motifs from Prz1 and Crz1 from budding yeast (21). Residues
conserved in two sequences are boxed and
highlighted. Residues that coordinate zinc ions are
indicated by asterisks. C, the
calcineurin-specific inhibitor FK506 induces a Prz1 protein mobility
shift. Cells expressing GFP-Prz1 were incubated in EMM medium
containing 4 µg/ml thiamine at 30 °C with CaCl2 (50 mM) or FK506 (0.5 µg/ml) for 30 min as noted. The
electrophoretic mobility of the GFP-Prz1 protein was investigated by
immunoblotting using a GFP-specific antiserum. D, the
mobility shift of Prz1 caused by calcineurin treatment in
vitro is calmodulin-dependent. GFP-Prz1 was purified
by immunoprecipitation from ppb1 cells expressing
GFP-Prz1, incubated with 1 mM CaCl2 and
calcineurin (lane 1) or with 1 mM
CaCl2, calmodulin, and calcineurin (lane 2)
for 15 min at 37 °C, and analyzed by immunoblotting.
C) increased the steady-state
levels of Prz1 mRNA (Fig. 2C).
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Fig. 2.
Northern blot analysis of Prz1 mRNA
expression. A, induction of Prz1 mRNA by
Ca2+ requires calcineurin activity. Wild-type or
calcineurin-null cells were incubated in YPD medium at 30 °C with 30 mM CaCl2 for 0, 10, 20, or 40 min. Total RNA
(20 µg) were subjected to Northern analysis using a DIG-labeled Prz1
cRNA. B, induction of Prz1 mRNA by heat treatment
requires calcineurin activity. Wild-type or calcineurin-null cells were
cultured to mid-log phase at 27 °C and then incubated at 42 °C
for 0, 10, 20, or 40 min. Total RNA were subjected to Northern analysis
as described above. C, expression of constitutively active
calcineurin induces Prz1 mRNA. Wild-type cells transformed with a
control vector (+ vector) or the vector containing the
constitutively active truncated calcineurin gene (+ ppb1 C) were cultured in EMM medium to mid-log
phase at 30 °C. Total RNA was subjected to Northern analysis as
described above. WT, wild type.
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Fig. 3.
Intracellular localization of GFP-Prz1.
A, translocation of GFP-Prz1 to the nucleus is induced by
Ca2+ addition and requires calcineurin. Wild-type,
calcineurin-null, or wild-type cells transformed with the vector
containing the constitutively active truncated calcineurin gene
(+ ppb1 C) expressing GFP-Prz1 were grown in EMM medium at
30 °C, incubated briefly with Hoechst 33342 to stain DNA, and
analyzed by fluorescence microscopy to observe GFP-Prz1 localization
(GFP-Prz1) or nuclear staining (DNA). In some cases cells were
incubated with 100 mM CaCl2 for 10 min at
30 °C (+ CaCl2). Arrow indicates a
dividing cell whose nucleus shows intense GFP fluorescence. The
bar indicates 10 µm. B, cell cycle-specific
nuclear accumulation of GFP-Prz1. Wild-type cells expressing GFP-Prz1
were grown to mid-log phase at 30 °C in YPD medium. Cells were
stained with Calcofluor, and the percentage of cells showing intense
nuclear fluorescence was measured. At least 500 cells were
counted.
SRR for lacking serine-rich
region) partially complemented the growth defects of a
prz1-null strain (data not shown) and showed nuclear
localization that was not affected by FK506 treatment (Fig.
4, lower panel), indicating
that the serine-rich region of Prz1 is required for
calcineurin-dependent regulation of its localization but is
dispensable for its transcriptional activity. There is no sequence
similarity between the serine-rich region of Prz1 and that of Crz1 or
the NF-ATc with the exception of the richness in serine residue,
although significant sequence similarity between the serine-rich region
of Crz1 and NF-ATc transcription factor has been reported previously
(22).
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Fig. 4.
Localization of Prz1 to the cytoplasm
requires its SRR. Living cells transformed with the vector for the
expression of GFP-Prz1 (full-length) or GFP-Prz1 SRR (N-terminally
truncated) were grown at 30 °C and analyzed as described above. In
some cases cells were incubated with 0.5 µM FK506 for 30 min at 30 °C (+ FK506). Arrow indicates a dividing cell
whose nucleus shows intense GFP fluorescence. The bar
indicates 10 µm.
--
To further investigate the relationship between
Prz1 and calcineurin, we analyzed prz1-null cells for
phenotypes exhibited by calcineurin-null mutants. Calcineurin-null
mutants were hypersensitive to Cl
ion and failed to grow
in the presence of 0.15 M MgCl2 or 0.3 M KCl (32). Contrary to our expectation,
prz1-null cells grew normally in the YPD plate containing
0.2 M MgCl2 where calcineurin-null cells did
not grow (Fig. 5A).
Interestingly, both of the prz1-null cells and
calcineurin-null cells could not grow in the presence of 0.15 M CaCl2 or 0.15 M
Ca(NO3)2 (Fig. 5A). These results
suggest that Prz1 is involved in the regulation of Ca2+ ion
homeostasis but not that of Cl
ion homeostasis. In
budding yeast, Crz1, the homolog of Prz1, is required for
calcineurin-dependent transcriptional regulation of
PMC1, which encodes a Ca2+ pump playing a key
role in Ca2+ tolerance (43). Consistently, Northern blot
analysis revealed that calcineurin (ppb1) deletion and
prz1 deletion resulted in a marked reduction in Pmc1
mRNA levels (Fig. 5B). In fission yeast, the disruption
of pmc1+ gene (SPBC1A4.10c) resulted in severe
hypersensitivity to Ca2+ (data not shown). Thus, the
Ca2+ hypersensitivity of prz1-null cells can be
explained, at least in part, by lowered level of Pmc1.
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Fig. 5.
Prz1 deletion causes hypersensitivity to
Ca2+ and marked reduction of mRNA for vacuolar Pmc1
Ca2+ pump. A, prz1 deletion is
hypersensitive to Ca2+ but not to Cl .
Wild-type (WT), prz1-null (
prz1),
and ppb1-null (
ppb1) cells were streaked onto
YPD plate and incubated for 3 days at 30 °C in the presence or
absence of 0.2 M MgCl2, 0.15 M
CaCl2, 0.3 M KCl, or 0.15 M
Ca(NO3)2. B, Northern blot analysis
of Pmc1 mRNA expression. Wild-type, calcineurin-null
(
ppb1), or prz1-null (
prz1)
cells were incubated in YPD medium at 30 °C with 100 mM
CaCl2 for 15 min. Total RNA (20 µg) were subjected to
Northern analysis using a DIG-labeled Pmc1 cRNA.
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Fig. 6.
Prz1-null and calcineurin-null mutants have
distinct phenotypes in cell morphology and mating. A,
prz1-null cells is normal in size, polarity, and cytokinesis.
prz1-null and calcineurin-null mutants were grown in YPD
medium to mid-log phase at 30 °C, washed with phosphate-buffered
saline, pH 7.0, and stained with Hoechst 33342 and Calcofluor to
visualize the DNA or septum, respectively. B, sporulation in
prz1-null and calcineurin-null mutants. Null mutants and
HM528 strain were crossed on an SPA plate at 30 °C and examined
microscopically after 24 h. Bar, 10 µm.
-glucan synthase, glycosylphosphatidylinositol anchor synthetic
enzyme, and protein kinase implicated in septation initiation,
respectively) (28, 30, 44) did not show synthetic lethality with
prz1-null mutation and double mutants were obtained (Table
II). The
prz1 its8 double mutant grew slower and was more temperature-sensitive than the its8 single mutant (data not
shown), indicating a functional overlapping between Prz1 and the
glycosylphosphatidylinositol anchor synthetic pathway.
Genetic interactions between prz1+ or ppb1+ and its
(immunosuppressant- and temperature-sensitive) mutants
ion
homeostasis and inhibition of Pmk1 MAPK pathway suppressed the
Cl
hypersensitivity of calcineurin-null cells (32, 33).
Consistent with our previous results, overexpression of
pmp1+ gene encoding a MAPK phosphatase for Pmk1
suppressed the Cl
hypersensitivity of calcineurin-null
cells. On the other hand, overexpression of pmp1+
did not affect the Ca2+ hypersensitivity of
prz1-null cells (Fig.
7A). Furthermore,
overexpression of prz1+ did not suppress the
MgCl2 hypersensitivity of calcineurin-null cells. In
addition, overexpression of constitutively active calcineurin did not
suppress the CaCl2 hypersensitivity of prz1-null
cells (Fig. 7A), whereas overexpression of
prz1+ partially suppressed the CaCl2
hypersensitivity of calcineurin-null cells (data not shown). These
results suggest that Prz1 acts downstream of calcineurin and regulates
Ca2+ homeostasis. These results also suggest that Prz1 is
not involved in Cl
homeostasis that is antagonistically
regulated by calcineurin and the Pmk1 MAPK pathways (32, 33). A model
consistent with these data is presented in Fig. 7B.
View larger version (39K):
[in a new window]
Fig. 7.
Prz1 is neither genetically related to the
Pmk1 MAPK pathway nor is involved in the regulation of Cl
homeostasis. A, prz1-null
(
prz1) or ppb1-null (
ppb1) cells
transformed with a control vector (vector) or the multicopy vector
containing pmp1+ (+ pmp1+) and
prz1+ (+ prz1+) or the
constitutively active truncated calcineurin (+ ppb1
C)
gene were streaked onto YPD plate and incubated for 3 days at 30 °C
in the presence of 0.1 M CaCl2 or 0.15 M MgCl2. B, a model of the two
distinct branches of calcineurin signaling pathway. See "Results"
and "Discussion." Ppb1, calcineurin phosphatase;
Pmk1, MAPK phosphatase; Pmp1, MAPK phosphatase
for Pmk1; Pmc1, vacuolar Ca2+ pump;
?, unknown mechanism for Cl
resistance.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor (20). Furthermore, a recent genome-wide analysis of gene
expression regulated by the calcineurin/Crz1 signaling pathway confirms
that Crz1 is the major and possibly the only effector of
calcineurin-regulated gene expression in budding yeast (23). 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.2 These results
strongly suggest that there are at least two branches of calcineurin
signaling pathway. Related to this issue is the observation that in
S. cerevisiae, crz1-null cells and
calcineurin-null cells show opposing phenotypes in the condition of
high Ca2+, i.e. crz1-null cells are
highly Ca2+-sensitive similar to prz1-null
cells, whereas cnb1-null cells are resistant to this ion and
show increased growth on medium containing high levels of
Ca2+ (20), showing that there is also branching in
calcineurin signaling in S. cerevisiae.
hypersensitivity of the calcineurin deletion. Thus,
the second branch of the calcineurin signaling pathway seems to act
antagonistically with the Pmk1 MAPK pathway and regulate various
cellular events, such as morphogenesis and Cl
homeostasis. Furthermore, prz1 deletion is not synthetically lethal with mutations in the
its2+/cps1+,
its8+, and
its10+/cdc7+ genes (encoding
-glucan synthase, glycosylphosphatidylinositol biosynthetic enzyme,
and a protein kinase in the SIN pathway, respectively) (28, 30, 44)
(Table II). Thus, it is suggested that these three genes seem to have
some genetic interactions with the second branch of the calcineurin
signaling pathway. As these genes encode proteins that seem to be
involved in the cell wall synthesis, these genetic data are in good
agreement with the hypothesis that the Pmk1 MAPK pathway is involved in
the regulation of cell wall integrity (34). In our preliminary studies
in which we have been searching for downstream targets of the Pmk1
MAPK, we identified several candidates including certain novel putative transcription factors.3
Studies are in progress to determine whether these factors play some
roles in the Cl
homeostasis and are functionally related
to the calcineurin signaling pathway.
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ACKNOWLEDGEMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
* This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Division of
Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5440; Fax:
81-78-382-5459; E-mail: tkuno@med.kobe-u.ac.jp.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M212900200
2 R. Sugiura, H. Tohda, Y. Giga-Hama, H. Shuntoh, and T. Kuno, unpublished observations.
3 R. Sugiura, H. Shuntoh, and T. Kuno, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NF-AT, the nuclear factor of activated T cells; MAPK, mitogen-activated protein kinase; its, immunosuppressant- and temperature-sensitive; prz, Ppb1-responsive zinc finger protein; GFP, green fluorescent protein; DIG, digoxigenin; SRR, serine-rich region; PI(4)P5, phosphatidylinositol 4-phosphate 5.
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