From the Department of Biochemistry and Molecular
Biology, § Thoracic Diseases Research Unit and Division of
Pulmonary, Critical Care, and Internal Medicine, and the ¶ Section
of Hematology Research, Mayo Clinic, Rochester, Minnesota 55905
Received for publication, August 28, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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Pneumocystis carinii is an
opportunistic fungal pathogen phylogenetically related to the fission
yeast Schizosaccharomyces pombe. P. carinii causes
severe pneumonia in immunocompromised patients with AIDS and
malignancies. Although the life cycle of P. carinii remains
poorly characterized, morphologic studies of infected lung tissue
indicate that P. carinii alternates between numerous small
trophic forms and fewer large cystic forms. To understand further the
molecular mechanisms that regulate progression of the cell cycle of
P. carinii, we have sought to identify and characterize
genes in P. carinii that are important regulators of
eukaryotic cell cycle progression. In this study, we have isolated a
cDNA from P. carinii that exhibits significant
homology, but unique functional characteristics, to the mitotic
phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was
shown to rescue growth of the temperature-sensitive S. pombe
cdc25-22 strain and thus provides an additional tool to
investigate the unique P. carinii life cycle. Although
P. carinii Cdc25 could also restore the DNA damage
checkpoint in cdc25-22 cells, it was unable to restore
fully the DNA replication checkpoint. The dissociation of checkpoint
control at the level of Cdc25 indicates that Cdc25 may be under
distinct regulatory control in mediating checkpoint signaling.
Despite advances in prophylaxis and treatment, Pneumocystis
carinii remains an important cause of life-threatening pneumonia in patients with impaired immunity. Patients with AIDS, organ transplantation, and those receiving chemotherapy are particularly vulnerable to P. carinii pneumonia. The case fatality rate
of P. carinii pneumonia ranges from ~10 to 40%, being
substantially higher in infected immunocompromised patients without
AIDS (1-3). Unfortunately, a considerable number of patients are
intolerant of the currently available agents used to prevent and treat
this infection, such as sulfamethoxazole and pentamidine. The
development of new chemotherapeutic agents to treat P. carinii pneumonia has been hampered by a limited understanding of
the P. carinii life cycle.
Morphological and ultrastructural studies of infected lung tissues
indicate that P. carinii alternates between numerous
diminutive trophic forms and fewer larger cyst forms (4). Trophic forms are known to bind preferentially type I alveolar epithelial cells and
eventually develop into mature cysts characterized by a thickened cell
wall (5, 6). The mature cyst, containing eight intracystic bodies,
ruptures releasing these bodies as haploid trophic forms (4). Although
the proliferation of P. carinii in the lung is thus enhanced
by the attachment of trophic forms to lung epithelium, only recently
have there been initial reports of limited success with in
vitro cultivation of P. carinii in the absence of
feeder cells (7-9). However, long term in vitro propagation
P. carinii has perennially remained a key obstacle in the
investigations of the life cycle of the organism.
Based on DNA sequence analysis, P. carinii has been
classified as a member of the fungi and is phylogenetically related to the fission yeast Schizosaccharomyces pombe (10-12). The
life cycle and the cell cycle of S. pombe have been well
characterized and utilized to identify cell division cycle
(cdc)1 genes in
other organisms with obligate roles in cell proliferation (13-15).
Despite the fact that these cell division cycle genes are well
conserved, it is known that diverse eukaryotic organisms differentially
regulate their cell cycle machinery in response to environmental
stimuli and other internal signaling pathways (16).
To understand better the molecular mechanisms that regulate progression
of the cell cycle of P. carinii, we have sought to identify
and characterize genes in P. carinii that are important regulators of cell cycle progression in related organisms. Accordingly, we began our investigations by demonstrating that P. carinii
utilizes a cyclin-dependent kinase, Cdc2, over its life
cycle (17). Activated Cdc2, in association with cyclin B (cdc13),
phosphorylates a variety of targets, such as histones and nuclear
laminins, to initiate mitosis (18, 19). The kinase activity of Cdc2 is
regulated by both inhibitory and stimulatory phosphorylations (20). One important positive regulator is the Cdc25 phosphatase that activates Cdc2 by removing the inhibitory phosphoryl group on Tyr15
of Cdc2 (21, 22). In fission yeast, Cdc25 protein levels and activity
rise in the G2 phase of the cell cycle (23). Therefore, at
the G2/M border Cdc2 is able to phosphorylate and activate Cdc25, initiating an autofeedback loop resulting in a rapid entry into
mitosis (24, 25). In addition to mitotic control, Cdc25 has roles in
regulating meiosis and imposing a checkpoint arrest in the DNA repair
and DNA replication processes (26-31). Thus, Cdc25 represents an
integral component of eukaryotic cell cycle regulation.
Current models state that the phosphorylation of the Tyr15
residue of Cdc2 is a key component of maintaining the S/M and
G2/M checkpoints (32, 33). DNA damage or unreplicated DNA
activates the appropriate checkpoint pathway that propagates signals
that lead to the removal of Cdc25 from the nucleus and/or inactivation of Cdc25 activity, thereby maintaining the
Tyr15-phosphorylated state of Cdc2 (26, 31, 34-37). In
this study, we have identified and characterized a cDNA and
corresponding genomic clone from P. carinii that is
structurally and functionally homologous to the essential Cdc25 mitotic
regulator. In the context of a heterologous fungal system, this homolog
rescues growth in Cdc25 temperature-sensitive mutants and restores the
DNA damage checkpoint. However, it is unable to restore fully the DNA
replication checkpoint following hydroxyurea treatment. These findings
(i) provide evidence for distinct molecular mechanisms regulating G2/M and S/M checkpoints previously thought to be under
similar control, and (ii) identify a key regulatory molecule involved in P. carinii life cycle progression.
Materials--
Nitrocellulose membranes containing separated
P. carinii chromosomes were kindly provided by Dr. Melanie
T. Cushion, University of Cincinnati College of Medicine (38). A
P. carinii cDNA library was obtained from the National
Institutes of Health AIDS Research and Reference Reagent program. The
temperature-sensitive S. pombe cdc25-22 mutant strain and
the pREP41 vector were the kind gifts of Dr. Kathleen Gould, Vanderbilt
University. Dr. Barbara Painter generously provided Ciprofloxacin
(Miles Pharmaceuticals, Inc., West Haven, CT). All other reagents were
from Sigma unless stated otherwise.
P. carinii Isolation--
All animal studies were approved by
the Mayo Institutional Animal Care and Utilization Committee. Harlan
Sprague-Dawley rats were immunosuppressed with dexamethasone and
subsequently challenged with P. carinii sp. f. (special
form) carinii via transtracheal injection to induce
P. carinii pneumonia (39, 40). P. carinii were
purified by differential filtration through 10-µm filters as
we have previously reported (17).
Cloning of the P. carinii Cdc25 Mitotic
Phosphatase--
P. carinii genomic DNA was prepared (17,
41, 42), and degenerate oligonucleotide primers were synthesized to the
conserved CH2A and CH2B domains of Cdc25 family members. The following
primers were used for amplification, 5'-ATW ATW GAT TGT CGS TTG-3' and 5'-WGG ATA ATA SAA AAA WGG ATA-3'. PCR was performed as follows: 94 °C for 2 min, followed by 30 cycles of 94 °C for 1 min,
55 °C for 1 min, and 72 °C for 2 min, and a final extension at
72 °C for 5 min. A single 280-bp fragment was amplified, subcloned into the pGEM-Teasy vector (Promega), and sequenced. The amplicon was
radiolabeled with [ Amplification of Cdc25 from P. carinii, Rat, and S. pombe--
Total RNA was isolated from uninfected rat lung, P. carinii-infected rat lung, isolated P. carinii, and
S. pombe via the TRIZOL® method (Life
Technologies, Inc.). Total RNA from each sample was used to make
cDNA with the AdvantageTM RT-for-PCR Kit
(CLONTECH Laboratories, Inc., Palo Alto, CA). Cdc25
primers for polymerase chain reaction amplification were used as
follows: P. carinii-specific primers at nucleotide 108 from
the beginning of the coding region (5'-GGGAAGAAATATACTGATAAGAATAATAAATTCCGG-3)' and at nucleotide 748 (5'-GCAATATCAGTATCAGTATCATTCTTTATCTG-3'); rat-specific primers at nucleotide 39 (5'-CTCTTCACTTGCAGCCCCACTC-3') and nucleotide 691 (5'-CATCATTCTTCTGATTCTCTCCATCCAG-3'); and S. pombe-specific primers at nucleotide 295 (5'-CTTGTACTGTAGAAACCCCTCTCGC-3') and at
nucleotide 899 (5'-GAATTATCATTGCAAGCACCAAAATCACTG-3'). PCR components except primers were added and split into three tubes for
each template. Specific primers were then added. Amplification conditions were the same in all reactions as discussed previously, and
the products were electrophoresed on a 2% agarose TBE (100 mM Tris, 90 mM boric acid, 1 mM
EDTA) gel.
Production of Cdc25 Proteins in Bacteria and p-Nitrophenyl
Phosphate Assays--
The full-length cDNAs for the P. carinii Cdc25 homolog, an inactive mutant (C432A), and the
S. pombe cdc25 were subcloned into the pGEX-2T vector
(Amersham Pharmacia Biotech) and transformed into competent
Escherichia coli BL21 cells. The bacteria were grown to an
A600 of 0.5 at 27 °C in LB containing
100 µg/ml ampicillin. Isopropylthio- Complementation of a S. pombe cdc25 Temperature-sensitive Mutant
Using P. carinii cdc25--
An NdeI-BamHI
fragment encoding the entire 1614-bp open reading frame encoding the
P. carinii Cdc25 homolog was subcloned into
NdeI-BamHI-digested pREP41 (containing a leu2
marker). The resulting construct contained the P. carinii
gene in sense orientation under control of the thiamine-repressible
nmt promoter. A 7-kb SalI genomic clone of the
S. pombe Cdc25 was subcloned into SalI-digested pREP41 in an analogous fashion. The constructs, including pREP-C432A and pREP alone, were transformed into the S. pombe
h+ cdc25-22 leu1-32 ura4-218 ade6-M210 strain as
described (44) and selected on leucine- and thiamine-deficient media.
Leucine-positive clones that grew at the permissive temperature
(25 °C) were then tested for growth at the restrictive temperature
(35 °C). The expression of the P. carinii Cdc25 was
repressed by the addition of 25 µM thiamine.
DNA Damage and DNA Replication Checkpoint Analysis with
PcCdc25-complemented cdc25-22 cells--
For the DNA replication
checkpoint studies, 12 mM hydroxyurea was added to
asynchronous yeast cultures (1 × 106/ml) and fixed
with 3% formaldehyde at selected time points and then stained with
DAPI (45). Cells were analyzed by fluorescence microscopy and counted
for cells containing mitotic cuts. For the DNA damage checkpoint
studies, cells were synchronized in G1 in media lacking
nitrogen, released, and irradiated with 200 Gy from a 137Cs
source. Following fixation and DAPI staining, images were taken on an
Olympus AX-70 fluorescence microscope and analyzed using the Metamorph
software program (Universal Imaging Corp., West Chester, PA).
Cloning of the P. carinii Cdc25 Mitotic Phosphatase--
A
degenerative PCR approach was taken to determine whether P. carinii contained a Cdc25 homolog. Because the active site of Cdc25 family members is highly conserved across species, degenerate primers were designed from the CH2A and CH2B domains flanking the
catalytic site. To optimize the efficiency of the degenerate primers,
codon bias was used to reflect the adenine/thymine-rich (>65%)
P. carinii DNA (46). A 280-base pair product was amplified from P. carinii genomic DNA. Sequencing the product revealed
that the 280-base pair fragment was unique but homologous to
cdc25 genes from other organisms (data not shown). To
confirm the P. carinii origin of the amplicon, it was
hybridized to rat-derived P. carinii chromosomes separated
by contour-clamped homogenous field electrophoresis (CHEF). Under high
stringency, the amplicon hybridized to a single chromosome from
P. carinii and to a 2.3-kb P. carinii RNA
transcript (Fig. 1 and data not shown).
Subsequent screening of a P. carinii cDNA library
identified a 2.1-kb fragment that was isolated and characterized. Based
on Northern data, this cDNA clone appears to contain the entire
coding region and most of the surrounding regulatory regions. The
genomic clone was isolated by PCR amplification from freshly isolated
P. carinii genomic DNA. Comparison of the cDNA and
genomic sequences indicated that the coding region is interrupted by
five introns that span 40-50 base pairs in length and contain the
GT/AG intron/exon junction sequence (GenBankTM accession
numbers AF098935 and AF098936, respectively). Fig.
2 shows the predicted amino acid
alignment of P. carinii Cdc25 with Cdc25 proteins from other
eukaryotic species. Consistent with other Cdc25 family members, PcCdc25
exhibits a highly conserved C terminus containing the catalytic site
and a variable N terminus. The PcCdc25 protein was found to be most
closely related to fission yeast, being 61.2% homologous and 40.3%
identical to the S. pombe Cdc25. The PcCdc25 homolog
contained the HCXXXXXR consensus sequence (amino acids
431-438) and the conserved DCR motif at amino acids 389-391 unique to
Cdc25 phosphatases (47). Although the homolog does not contain an LIGD
motif found in higher eukaryotes, it has three putative Cdc2 consensus
phosphorylation sites on the N terminus at amino acids 91-93,
112-114, and 141-143 (47). These results demonstrate that the
isolated cDNA is from P. carinii and exhibits
significant homology to other Cdc25 species.
The P. carinii cdc25 Transcript Is Specific for P. carinii--
One of the major concerns in cloning genes from
Pneumocystis is host cell or other microbial contamination
during the isolation of the organism. Whereas the CHEF blot provides
substantial evidence for P. carinii origin, these
preparations likely contain minor amounts of host material. As such, to
ensure specificity, a PCR-based approach was taken to determine whether
specific primers designed from one cdc25 gene would
amplify a cdc25 gene from another organism. Primers were
designed near the 5' end of the putative P. carinii cdc25+ homolog, rat cdc25+, and
S. pombe cdc25+ genes,
reflecting the region of the proteins that are the least conserved (see
"Experimental Procedures"). RT-PCR was performed with RNA isolated
from normal rat lung, P. carinii-infected rat lung, isolated
P. carinii, and S. pombe. The P. carinii
cdc25+ primers amplified products from only P. carinii-infected lungs and isolated P. carinii (Fig.
3A). Rat
cdc25+ primers only generated a product from rat
lung and P. carinii-infected rat lung, whereas S. pombe cdc25+ primers specifically produced
a transcript from S. pombe cDNA. By having verified the
origin of the cDNA, we wished to determine the gene expression
pattern in the two major life forms of P. carinii. From
Northern analysis, the P. carinii cdc25+
transcript was expressed in both the trophic and cyst form, with a
moderate increase in expression in the cysts (Fig. 3B).
These results, in addition to the CHEF hybridization and nucleotide and
amino acid sequence differences, clearly demonstrate the Cdc25 gene
product is from P. carinii and is expressed in both life cycle forms.
PcCdc25 Exhibits in Vitro Phosphatase Activity--
Cdc25 proteins
are dual-specific phosphatases known to catalyze the transition from
various cell cycle checkpoints (25, 28, 48, 49). To determine whether
the P. carinii Cdc25 homolog had similar enzymatic activity
as that seen for other Cdc25 proteins, we expressed the protein fused
to a glutathione S-transferase (GST) domain at the N
terminus in E. coli. Phosphatase activity was measured by
the ability to dephosphorylate p-nitrophenol phosphate and
compared with GST fusion proteins of human Cdc25C and S. pombe Cdc25. As shown in Fig. 4, the
kinetic parameters of the partially purified GST-PcCdc25, GST-SpCcdc25,
and the GST-HuCdc25C were similar, with Km values of
17, 26, and 35 mM, and Vmax values
of 6, 3, and 31 nmol·min
To ensure that the phosphatase activity observed in Fig. 4 reflected an
enzymatic activity of GST-PcCdc25 and not some bacterial contaminant, a
Cys432 P. carinii Cdc25 Rescues cdc25-22 Temperature-sensitive Mutants
Thereby Restoring Growth--
The absence of defined genetics and
culture systems makes it difficult to do genetic manipulations in
P. carinii. As such, it is presently not possible to examine
directly the role of the P. carinii Cdc25 homolog in
P. carinii proliferation. However, to overcome that
limitation, we determined whether the P. carinii Cdc25
cDNA would complement a temperature-sensitive Cdc25 S. pombe mutant (cdc25-22). This strain grows normally at
a permissive temperature of 25 °C, but at the restrictive
temperature of 35 °C the thermo-labile endogenous Cdc25 is no longer
active and the strain arrests at the G2/M phase border
(48). The PcCdc25 cDNA, the inactive PcCdc25(C432A) mutant, and the
SpCdc25 genomic clone were subcloned downstream of a
thiamine-repressible nmt promoter (50) and transformed into
cdc25-22 cells. Individual clones containing the pREP
expression vector were then selected on leucine-deficient minimal media
plates and transformants shifted to the restrictive temperature to
measure their ability to restore growth. As shown in Fig.
5, although the pREP vector alone and the
PcCdc25(C432A) mutant did not restore growth at the restrictive temperature of 35 °C, complementation was observed in the S. pombe clone containing the PcCdc25 homolog (Fig. 5C).
Moreover, clones grown in the presence of thiamine, which represses
expression of the PcCdc25 cDNA, were unable to grow (Fig.
5D). The growth of pREP-SpCdc25 in the presence of thiamine
(Fig. 5D, panel 2) reflects the intact promoter activity
upstream of the genomic S. pombe Cdc25 sequence. Therefore,
in the context of a heterologous fungal system, the P. carinii Cdc25 homolog is able to initiate mitosis and support
fungal growth.
PcCdc25 Restores the DNA Damage Checkpoint but Not the DNA
Replication Checkpoint in cde25-22 Cells--
Checkpoint pathways in
eukaryotic cells ensure that genomic integrity is maintained in
response to environmental and genotoxic stress (51). In fission yeast,
the entry into mitosis is blocked when DNA synthesis is incomplete or
when DNA is damaged by such agents as ionizing radiation (52). The
G2/M checkpoint is imposed by a signal transduction system
that ultimately leads to the sequestration of the Cdc25 protein into
the cytoplasm (mediated by Rad24, a 14-3-3 protein) and/or inactivation
of Cdc25 activity. These events prevent the phosphatase from activating
Cdc2 (34-36, 49). S. pombe Cdc25 has three canonical
14-3-3-binding sites, Arg-Ser-Leu-Ser99-Cys-Thr,
Arg-Arg-Thr-Gln-Ser359-Met-Phe, and
Arg-Ser-Arg-Ser192-Ser-Gly, that are phosphorylated by Chk1
and Cds1 in response to DNA damage and incomplete DNA replication (26,
36, 49). In contrast to S. pombe Cdc25, we observed that the
P. carinii Cdc25 contained only one similar consensus
14-3-3-binding site at serine 314 (Arg-Arg-Thr-Gln-Ser314-Leu-Tyr). As such, it was unclear
whether a complete checkpoint response could be provided by PcCdc25. To
that end, we subjected PcCdc25-complemented cdc25-22 cells
to ionizing radiation (IR) to determine whether the P. carinii Cdc25 restored the DNA damage checkpoint. The DNA damage
checkpoint was measured by determining the number of cells passing
mitosis following irradiation. G1-synchronized cells were
exposed to 200 Gy of ionizing radiation and harvested over the next
12 h. As shown in Fig.
6A, SpCdc25 and
PcCdc25-complemented yeast show the characteristic
G2-arrested elongated phenotype following exposure to IR.
When the number of septated cells (i.e. cells that have past
mitosis and thus nonarrested) were quantified, SpCdc25-complemented
cultures showed a significant decrease in the number of septated cells
starting at 4 h and recovering around 12 h (Fig.
6B). A similar result held true for the PcCdc25-complemented yeast in that a delay into mitosis was observed. Although the kinetics
of the delay were somewhat slower, the results demonstrate that in
response to ionizing radiation, PcCdc25 complemented yeast arrest
similar to that observed with the endogenous S. pombe Cdc25 protein. Thus, the DNA damage checkpoint is intact in cells harboring PcCdc25.
Since the DNA damage and DNA replication checkpoints are proposed to
signal through Cdc25 by Chk1 and Cds1 phosphorylation, we determined
whether PcCdc25-complemented clones would similarly maintain a
checkpoint arrest in response to inhibitors of DNA replication. DNA
synthesis was inhibited by treating cultures with hydroxyurea (HU), an
agent that inhibits the function of ribonucleotide reductase. Yeast
that cannot maintain the DNA replication checkpoint enter mitosis with
incompletely replicated DNA. This results in an unequal distribution of
DNA as cells pass through mitosis and septate, therefore generating
cells that contain little or no DNA (referred to as mitotic "cut"
cells). As shown in Fig. 7A,
SpCdc25-complemented yeast grown in 12 mM HU maintained the replication checkpoint and generated very few cells exhibiting the
mitotic cut phenotype. However, unlike HU-treated SpCdc25-complemented yeast, a significant fraction of the PcCdc25-complemented cells grown
in 12 mM HU bypassed the S/M checkpoint and entered into mitosis. Quantification of mitotic cut cells showed that
SpCdc25-complemented cells grown in HU maintained the DNA replication
checkpoint in that only 1% of cells exhibited the mitotic cut
phenotype at 6 h (Fig. 7B). This is contrasted by the
PcCdc25-complemented cells grown in HU where the number of cut cells
increased to 8.0% at 8 h. This result is similar to the 9-fold
induction of mitotic cut cells observed with SpCdc25-S3 yeast that have
a diminished replication checkpoint response due to serine mutations in
the three 14-3-3-binding sites (36). We therefore conclude that PcCdc25
does not restore the DNA replication checkpoint. Although the
SpCdc25-S3 mutation is unable to complement either the DNA replication
or DNA damage checkpoints (34, 36), we demonstrate that the PcCdc25
protein differentially responds to these genotoxic assaults. The data
indicate that the pathways that eukaryotic organisms employ in response
to DNA damage and incomplete DNA synthesis can be dissociated at the
level of Cdc25 regulation.
Our laboratory has previously characterized roles for P. carinii Cdc2 and its cognate partner Cdc13 in cell cycle
regulation. In this report, we have continued our approach to define
key mediators of this system in P. carinii. A 2.1-kb
cDNA and corresponding genomic clone has been isolated from
P. carinii that shows greatest homology to the S. pombe cdc25 gene (Figs. 1 and 2). The predicted amino acid sequence contains a consensus HCXXXXXR motif in
the active site and many other consensus sites found in the Cdc25 family (47). We show that the cdc25+ transcript
is specific for P. carinii and is expressed in both the
trophic and cyst life cycle forms (Fig. 3). The Cdc25 homolog contains
in vitro phosphatase activity and demonstrates similar kinetic parameters to other Cdc25 homologs (Fig. 4). Since molecular genetics in P. carinii are currently not feasible, we
examined the function of PcCdc25 in a heterologous fungal system with a temperature-sensitive deficiency of endogenous Cdc25. The results demonstrate that the PcCdc25 cDNA rescues the defect and supports growth of the cdc25-22 strain (Fig. 5). Therefore, in
fission yeast, PcCdc25 provides the necessary function(s) for complete cell cycle progression. Interestingly, yeast cells containing the
P. carinii Cdc25 homolog demonstrated differential responses to the G2/M and S/M checkpoints. Although the
PcCdc25-complemented cdc25-22 yeast maintained the
G2/M checkpoint in response to damaged DNA by IR (Fig. 6),
the S/M DNA replication checkpoint, as tested with HU, was impaired
(Fig. 7). Thus, PcCdc25 is capable of dissociating the signals
regulating normal proliferation and checkpoint control.
Although the P. carinii life cycle has been well
characterized morphologically, the molecular events regulating life
cycle progression have only recently begun to be defined (17, 53-55). It is evident that whereas cell division cycle molecules have key roles
in the proliferation of P. carinii, the unique requirements of P. carinii for life cycle progression suggest other
regulatory mechanisms. For instance, P. carinii attachment
to type I pneumocytes promotes proliferation, whereas Candida
albicans, Saccharomyces cerevisiae, and S. pombe propagate independent of binding to a substrate.
Furthermore, P. carinii progresses through a cyst
form that is critical for survival and necessary for life cycle
progression (54, 56). As such, when P. carinii-infected rats
are treated with In response to environmental stimuli, signal transduction pathways are
activated that eventually impact on the cell cycle machinery. Cdc25 is
a key regulator of several cellular processes including regulating
entry into mitosis, meiotic phase transitions, and maintaining
G2/M and S/M checkpoints in the response to DNA damage and
incomplete DNA replication (26-31). The protein complexes that sense
DNA damage or stalled DNA replication forks transmit signals that
ultimately lead to the inactivation of Cdc2 (32, 33, 37). One such
mechanism includes the phosphorylation of Cdc25 by Chk1 and Cds1
kinases. Phosphorylation of Cdc25 results in the inactivation and/or
sequestration of Cdc25 to the cytoplasm thereby maintaining Cdc2 in the
inactive Tyr15-phosphorylated state (26, 31, 34-36).
The observation that PcCdc25 restored the DNA damage (Fig. 6) but not
the DNA replication checkpoint (Fig. 7) indicates a dissociation of
checkpoint pathways at the level of Cdc25 regulation. In response to
IR, PcCdc25-complemented yeast exhibited a decrease in the number of
cells passing mitosis and an increase of cells in G2
(elongated phenotype). Although the DNA damage checkpoint is dampened
in fission yeast harboring a S99A mutation in Cdc25 (34), PcCdc25
restored the checkpoint even though it lacks the appropriate context of
this regulatory site found in S. pombe Cdc25. Although
PcCdc25 can restore the checkpoint response to IR (Fig. 6), following
treatment with HU, PcCdc25-complemented yeast exhibited an 8-fold
increase in mitotic cut cells when compared with untreated cells. The
impaired S/M checkpoint elicited by PcCdc25 in response to inhibitors
of DNA replication is consistent with that observed in yeast containing
a Cdc25 protein in which the 14-3-3-binding/Chk1 and Cds1
phosphorylation sites have been mutated (SpCdc25-S3) (31). Although
Chk1 appears to be the major kinase involved in the response to DNA
damage and Cds1 in the incomplete DNA replication checkpoint, they both
appear to phosphorylate Cdc25 at the same residues (Ser99,
Ser192, and Ser359) in fission yeast (34).
The definitive role of the three Chk1/Cds1 phosphorylation sites
(Ser99, Ser192, and Ser359) has yet
to be clarified. Whereas Zeng et al. (36) analyzed the
HU-induced S/M checkpoint with a Cdc25 containing Ser It is surprising that PcCdc25 would only restore one checkpoint pathway
since cell cycle proteins are highly conserved and can function
normally in heterologous systems. Since PcCdc25 complements growth in
fission yeast, the host machinery must recognize sites of control in
the primary sequence and secondary structure of PcCdc25. Furthermore,
this differential control cannot easily be explained by the fact that
PcCdc25 is overexpressed in yeast (due to the nmt promoter) in that
cdc25-22 yeast containing overexpressed SpCdc25 maintain
both checkpoints. The data provide initial evidence for distinct
domains on Cdc25 family members or other unknown checkpoint regulatory
sites that differentially regulate the response to IR and HU. Further
studies will identify the sequence(s) controlling this differential
response in checkpoint control and determine the manner which Chk1,
Cds1 and/or Rad 24 interact with PcCdc25.
Our current studies continue to be focused on the role of the
P. carinii Cdc25 homolog in regulating the cyst/trophic form transitions. The data presented (Fig. 3B) show that PcCdc25
is expressed in both life forms. We hypothesize that PcCdc25 will have
a vital role in regulating organism replication and life cycle
transitions. Furthermore, cdc25 gene expression in
the trophic forms provides evidence for the model in which trophic
forms might undergo their own mitotic cycle. Although these studies are
limited by the ability to manipulate genetically the organism, the
further development of an axenic culture will be necessary to address this problem. It is anticipated that further studies of the P. carinii Cdc25 homolog in the context of life cycle progression will provide new insights to understanding P. carinii
pathogenesis and Cdc25 biology in checkpoint control.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP (Amersham Pharmacia
Biotech) with the RadPrime DNA Labeling System (Life Technologies,
Inc.) and hybridized to a nitrocellulose membrane containing P. carinii chromosomes. The P. carinii chromosomes were
separated by contour-clamped homogenous field electrophoresis (CHEF)
and transferred to nitrocellulose (38). After 30 min of
prehybridization in ExpressHyb (CLONTECH
Laboratories, Inc., Palo Alto, CA), the CHEF blot was incubated with
probe (1 × 106 cpm/ml) at 68 °C for 60 min, washed
four times at room temperature in 2× SSC, 0.05% SDS buffer, washed
twice at 50 °C for 40 min in 0.1× SSC, 0.1% SDS, and visualized by
autoradiography. In addition, the probe was used to screen a
rat-derived P. carinii cDNA library under similar
conditions. Clones were isolated after plaques were purified to
homogeneity. A 2.1-kb clone was purified and sequenced (GenBankTM accession number AF098935). The genomic sequence
(GenBankTM accession number AF098936) was determined by PCR
amplification using freshly isolated P. carinii genomic DNA
as the template. Primers made from the 5' end and the 3' end of the
cDNA used for amplification are as follows:
5'-CATATATGGATACTTCACCTCTTG-3' and 5'-CGGACGTTACTCACATC-TTTTTGCAG-3'. PCRs were carried out as
previously mentioned.
-galactosidase was added for
4 h at a final concentration of 1 mM for BL21 cells
harboring P. carinii Cdc25 and C432A expression plasmids and
0.2 mM for BL21 cells containing the S. pombe
Cdc25 expression plasmid. The pelleted cells were suspended in
phosphate-buffered saline containing 10 µg/ml leupeptin, pepstatin,
aprotinin, and phenylmethylsulfonyl fluoride. A final concentration of
100 µg/ml of lysozyme and 1% Triton was added, incubated on ice for
15 min, lysed by mild sonication, and centrifuged to remove insoluble debris. The GST fusion proteins were purified from the soluble fraction
using glutathione-Sepharose beads and eluted with 10 mM
reduced glutathione, 50 mM Tris, pH 8.0. The inactivated
P. carinii Cdc25 was created by mutating the
Cys432 residue in the active site to an Ala residue with
the Stratagene Quikchange Site-directed Mutagenesis Kit®.
The following primers were used: C432A-5'
(5'-GTTTGATTATTTTTCATGCTGAATATAGTTCACATCGTGC-GCCA-3') and C432A-3'
(5'-GGCGCACGATGTGAACTATATTCAGCATG-AAAAATAATCAAAC-3') with
the mutated codon underlined. DNA sequencing verified the presence of the C432A mutation and the absence of other mutations. Reactions for p-nitrophenyl phosphate assays were performed
as described by Dunphy and Kumagai (43). For GST-PcCdc25
purification, the eluted protein was loaded onto a Superose 12 (Amersham Pharmacia Biotech) gel filtration column, and fractions were
collected and visualized by Coomassie-stained SDS-polyacrylamide gel
electrophoresis. Fractions containing the fusion protein were
subsequently loaded onto a Mono Q (Amersham Pharmacia Biotech) anion
exchange column. Two buffers consisting of 25 mM Tris, pH
8.2, 1 mM dithiothreitol with and without 1 M
NaCl were used to create a 40-ml gradient to elute the protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PcCdc25 PCR product hybridizes to P. carinii chromosomes. Lanes 1-4, four
preparations of P. carinii chromosomes separated by CHEF.
A, ethidium bromide-stained agarose gel of P. carinii chromosomes. B, autoradiograph of the
corresponding nitrocellulose blot hybridized with the 280-bp product
amplified from P. carinii genomic DNA.
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Fig. 2.
Comparison of the predicted amino acid
sequence from PcCdc25 with other Cdc25 family members. Sequences
from the P. carinii Cdc25 cDNA (accession number
AF098935), S. pombe Cdc25 (accession number
M13158), S. cerevisiae Cd25 homolog, MIH1 (accession number
J04846), human Cdc25C (accession number 4502706), and rat Cdc25B
(accession number D16237) were obtained from the GenBankTM
data base. Alignments were performed with the Baylor College of
Medicine Boxshade program. Black boxes indicate
conserved identical amino acids, and gray boxes indicate
homologous amino acids.
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Fig. 3.
Pc cdc25+ transcript is specific
for P. carinii. A, RT-PCR was performed with
primers made near the 5' end of P. carinii
cdc25+, rat cdc25+, and S. pombe
cdc25+. Templates are as follows: U, uninfected
rat lung; I, P. carinii-infected rat lung;
Pc, isolated P. carinii; Sp, S. pombe. The cDNA templates were made from 1 µg of total RNA
primed with oligo(dT) primers. The same template was used for each of
the different primer sets, and the appropriate size fragment was
amplified from each condition. P. carinii
cdc25+, 640 bp; rat cdc25+, 652 bp; and
S. pombe cdc25+, 604 bp. B, Northern
blot analysis of 5 µg of total RNA isolated from the trophic
(Troph) and cyst forms of P. carinii. The
ethidium bromide-stained gel (EtBr) shows the ribosomal
subunits of P. carinii. The blot was hybridized with the
radiolabeled 280-bp amplicon.
1·mg
1,
respectively. Similar kinetics have been reported for Xenopus laevis Cdc25 with a Km of 50 mM and
Vmax of 56 nmol·min
1·mg
1
(21).
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Fig. 4.
PcCdc25 exhibits phosphatase activity.
The velocities of PcCdc25 ( ), PcCdc25-C432A catalytic site
mutant (
), SpCdc25 (
), and HuCdc25C (inset) were
compared in a standard V versus [S] plot with
p-nitrophenol phosphate as the substrate.
Vmax and Km values were
obtained from fitting the data to the Michaelis-Menten equation. The
data represent the mean and ± S.E. of samples from two different
GST preparations each analyzed in duplicate.
Ala mutation was introduced in the predicted
catalytic domain of GST-PcCdc25. This mutation abolishes phosphatase
activity by deletion of the active site nucleophile (43). As shown in
Fig. 4, the introduced mutation abolished P. carinii Cdc25
activity. In addition, the GST-PcCdc25 fusion protein was purified to
>80% purity using a gel filtration and ion exchange (Mono Q)
chromatography (data not shown). The resulting purification resulted in
a 7-fold increase in the specific activity of the protein. Moreover,
the Km of the Mono Q-purified enzyme (average of two
preparations) remained very close to the partially purified protein (15 versus 17 mM, respectively), but the
Vmax increased from 6 to 15 nmol·min
1·mg
1.
These data not only documented that the phosphatase activity was due to
the added fusion protein, but strongly suggested that GST-PcCdc25 has a
similar active site to other Cdc25 proteins.
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Fig. 5.
PcCdc25 rescues the S. pombe
temperature-sensitive Cdc25 mutant. S. pombe
cdc25-22 cells were transfected with the thiamine-repressible vector
alone (1), pREP-SpCdc25 (2), pREP-PcCdc25
(3), or pREP-PcCdc25-C432A (4). An equal number
of cells were plated on all conditions. A, transformants
grown at the permissive temperature of 25 °C. B,
transformants grown at the permissive temperature with the addition of
25 µM thiamine. C, transformants grown at the
restrictive temperature of 35 °C. D, transformants grown
at the restrictive temperature with the addition of 25 µM
thiamine.
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Fig. 6.
The DNA damage checkpoint is restored in
PcCdc25-complemented cdc25-22 cells. A,
photomicrographs of the cdc25-22:SpCdc25 cells exposed to
200 Gy of radiation, unexposed cdc25-22:PcCdc25 cells, and
cdc25-22:PcCdc25 cells exposed to 200 Gy of radiation. After
G1 release, cells were exposed to IR versus
unexposed, fixed after 8 h, and stained with DAPI for fluorescence
microscopy analysis. Each panel was taken at × 1000 magnification, and the white bars represent 10 microns. The
arrows indicate septated cells that represent dividing,
nonarrested cells. B, quantitative analysis measuring the
number of septated cells following radiation exposure or no treatment.
At indicated time points, the cells were fixed and mounted. The
graph on the right represents PcCdc25-complemented clones,
and the graph on the left represents
SpCc25-complemented clones. The % septated cells were determined in
unexposed cultures ( ) and IR-exposed cultures (
). Numbers
were derived from two clones analyzed in four separate experiments with
at least 100 cells counted per time point.
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Fig. 7.
PcCdc25 does not restore the DNA replication
checkpoint. A, photomicrographs of the cdc25-22:SpCdc25
cells grown in the presence of 12 mM hydroxyurea,
untreated, and HU-treated cdc25-22:PcCdc25 cells.
Asynchronous cultures were grown in either the presence or absence of
12 mM hydroxyurea, fixed after 8 h, and stained with
DAPI. The arrows indicate yeast that exhibit the mitotic cut
phenotype representing nonarrested cells. B, quantitative
analysis measuring the number of mitotic cut cells in the presence of
HU. The white bar represents PcCdc25-complemented clones,
and the black bar represents SpCdc25 clones. At the
indicated time points, the cells were fixed and mounted for
fluorescence microscopy. The % mitotic cut cells were quantified from
cells from HU-treated cdc25-22:PcCdc25 and
cdc25-22:SpCdc25. Four separate experiments were performed
from two clones from each group with at least 100 cells counted at each
time point.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucan synthesis inhibitors to prevent cyst
formation, the infection is eliminated (56). This is in contrast to
budding and fission yeast haploid forms that utilize a mitotic cycle
independent of forming an ascus, unless environmental conditions are
unfavorable (57). Although these differences need to be considered as
the P. carinii life cycle is investigated, a common feature
found in all eukaryotic proliferation is a central regulatory role for cell division cycle gene products (16). Therefore, it is very likely
that these molecules are intimately involved in similarly coordinating P. carinii life cycle progression.
Ala mutations
of the three sites (SpCdc25-S3), they did not dissect the role of each
site individually in maintaining the arrest nor was the response to IR
examined. Although a SpCdc25-S99A mutant generated by Furnari et
al. (34) was found to impair both the S/M and G2
checkpoints, the role of the other two mutations was not determined.
Moreover, results from both studies suggest that there may be other
Cdc25 sites weakly phosphorylated by Cds1 or Chk1. It remains unclear
whether these additional sites have functional significance. Finally,
the findings that (i) these Cdc25 mutations do not completely abolish
the checkpoints and (ii) Mik1 and Wee1 kinases phosphorylate and
inhibit Cdc2 activity in response to DNA damage and unreplicated DNA
(36, 58, 59) indicate that multiple mechanisms cooperate with Cdc25 to
maintain genomic integrity.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Melanie T. Cushion for providing the CHEF ethidium bromide-stained gel photograph and nitrocellulose blot; Dr. Kathy Gould for the S. pombe cdc25-22 mutants, the pREP41 vector, and the S. pombe cdc25+ genomic clone; and Dr. Larry Karnitz for critical reading of the manuscript. We also thank Joseph Standing for technical assistance in the generation of P. carinii used in these studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 HL55934, RO1 HL 57125, and RO1 HL 62150.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF098935 and AF098936.
To whom correspondence should be addressed: Guggenheim 642C,
Mayo Clinic and Foundation, Rochester, MN 55905. Tel.: 507-284-5717; Fax: 507-284-4521; E-mail: leof.edward@mayo.edu.
Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M007814200
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ABBREVIATIONS |
---|
The abbreviations used are: cdc, cell division cycle; bp, base pair; CHEF, contour clamped homogeneous field electrophoresis; DAPI, 4,6-diamidino-2-phenylindole; GST, glutathione S-transferase; Gy, gray; HU, hydroxyurea; IR, ionizing radiation; PCR polymerase chain reaction, RT-PCR, reverse transcriptase polymerase chain reaction; kb, kilobase pair.
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