1 Thoracic Diseases Research
Unit, The pathogenic
fungus Pneumocystis carinii causes
severe pneumonia in patients with impaired immunity, particularly
patients with acquired immunodeficiency syndrome. The life cycle of
P. carinii is poorly understood, and
the inability to continuously culture P. carinii is a major limitation in understanding its cell
biology. In fungi homologous to P. carinii, pheromone mating factors signal through a
mitogen-activated protein kinase (MAPK) signal transduction cascade,
resulting in mitotic cell cycle arrest and entry into a pathway of
conjugation, cellular differentiation, and proliferation. Using
degenerate PCR and library screening, we have identified a MAPK cDNA in
P. carinii that is highly homologous to fungal MAPKs involved in the pheromone mating signal transduction cascade, and we demonstrate MAPK activity in P. carinii lysates with a specific antiserum derived from
the translated P. carinii MAPK cDNA
sequence.
acquired immunodeficiency syndrome; serine-threonine protein
kinase; signal transduction; molecular cloning
PNEUMOCYSTIS CARINII causes severe
pneumonia in patients with an impaired immune system, particularly in
patients with acquired immunodeficiency syndrome (AIDS), solid or
hematological malignancies, or organ transplantation or patients
requiring chronic immunosuppressive therapy for inflammatory conditions
(14, 27, 31). Despite the availability of a number of effective
medications for prophylaxis and treatment of P. carinii pneumonia, the morbidity and mortality remain
in the range of 15-40%, being higher in patients without AIDS
(14, 31). Since its discovery, investigators have been unsuccessful in attempts to continuously culture P. carinii. A variety of media, cell lines, and
supplements have been tried, with limited success in sustaining growth
for more than a few weeks, and none has been successful in propagating
P. carinii continuously (2, 24).
Currently, the immunosuppressed rat model is the most widely used
source of obtaining P. carinii organisms for study; however, even this
model occasionally yields isolates of P. carinii mixed with other microogranisms or host cells
(2, 24). The inability to consistently grow and propagate
P. carinii poses a serious hindrance
to understanding the cell biology of this important fungal pathogen.
Recent investigations have classified P. carinii with the fungi (6). Molecular analysis of
ribosomal RNA places P. carinii on a
branch within the Ascomycetes, having
homology to Saccharomyces cerevisiae
and Schizosaccharomyces pombe (6).
Microscopically, P. carinii has a complex life cycle composed of two
distinct life forms (4, 11). Trophic forms (trophozoites) are 1-2
µm in size and are the most abundant life cycle form, outnumbering
cysts 10 to 1. Trophic forms attach to the alveolar epithelium through adhesive glycoproteins (13, 15). Cysts, which are 8-10 µm in
size, contain multiple intracystic bodies (up to 8 in number) that
mature and subsequently leave the cyst as trophic forms (16). The
mechanisms regulating growth and differentiation of the life cycle
forms are currently unknown.
In fungi homologous to P. carinii,
differentiation and proliferation are regulated by mitogen-activated
protein kinase (MAPK) signal transduction cascades (1, 7, 19). MAPKs
are serine-threonine protein kinases that phosphorylate substrates in
response to specific signals. A number of mitogenic factors, such as
pheromones and growth factors, and environmental factors, such as
osmotic stress, have been identified as stimuli for activating MAPK
signal transduction cascades (20, 28). Once a stimulus activates a
particular MAPK pathway, the signal is propagated by protein kinases
specific for that pathway. Perhaps the best characterized pathway is
the pheromone mating response cascade in the budding yeast
S. cerevisiae, where haploid cells
conjugate to form diploids by the mutual secretion of peptide mating
factors (or pheromones) by each of the two haploid cell types (1). The
binding of a pheromone peptide to its cell surface receptor activates a
heterotrimeric G protein that initiates a series of sequential protein
phosphorylations that are propagated by protein kinases of a MAPK
signal transduction cascade specific for the mating response (1). The
events that follow binding of the pheromone peptide culminate in an
activated MAPK, which is achieved by phosphorylation of two conserved
threonine and tyrosine residues between kinase subdomains VII and VIII
(9, 17). Once activated, MAPK controls the many cellular events the
organism will undergo in response to the mating signal. One of these
events is a controlled and orderly exit from the mitotic cell cycle,
allowing the cells to synchronize before conjugation (18, 22, 25).
We are investigating the role of MAPK in the regulation of the
P. carinii life cycle. As a first step
in our studies, we have isolated a full-length cDNA from
P. carinii that is highly
homologous to fungal MAPKs involved in the pheromone mating signal
transduction cascade. Additionally, we have identified its chromosomal
location within the P. carinii genome
and demonstrated that this cDNA is expressed as a functional MAPK in
P. carinii organisms.
Materials. All reagents were from
Sigma Chemical (St. Louis, MO) unless otherwise specified. Restriction
endonucleases, Taq polymerase, myelin
basic protein, and H1 histone were from GIBCO-BRL (Life Technologies).
[ Preparation of P. carinii
organisms. All studies described in
this report were approved by the institutional animal care facility. P. carinii pneumonia was induced in
Harlan Sprague-Dawley rats by immunosuppression with dexamethasone, as
reported previously (2, 13, 21). Rats received drinking water
containing dexamethasone (2 mg/l) and were fed a diet containing 8%
protein to intensify the infection. After 1 wk, rats were anesthetized
with ether and inoculated with 1 × 106 P. carinii by intratracheal injection. Six weeks later,
rats were killed by injection with 26% pentobarbital sodium. Lungs from moribund rats were minced in HBSS and homogenized in a Stomacher Tissue Blender (Tekmar) for 10 min. P. carinii were purified from this homogenate by
filtration through a 10-µm filter (Millipore). These preparations
contained ~98% P. carinii
organisms, with the remainder being mostly nonviable host cells.
P. carinii organisms were confirmed by
staining smears of the fluid with Wright-Giemsa, and samples containing
contaminating bacterial or fungal organisms were discarded.
P. carinii enumeration was performed
by counting air-dried samples stained with Diff-Quik, as described (13, 21).
Molecular cloning and chromosomal location of
P. carinii MAPK.
Oligonucleotides were made at the Mayo Clinic Molecular Biology Core
Facility. Genomic DNA was obtained from purified P. carinii organisms as previously described (26). We
designed degenerate PCR primers based on the conserved amino acid
sequences of two fungal MAPKs involved in the pheromone mating cascade,
S. cerevisiae FUS3 and
S. pombe spk1. The codon bias for
P. carinii was used to limit the
amount of degeneracy in the third position of the codon (8). PCR
amplification was performed with 1 µM of each degenerate primer
GAYGVVC,
5'-GG(A/T)GC(A/T)TATGG(A/T)GT(A/T)GT(A/T)TG-3', and
TAVYETM, 5'-G(A/T)GT(A/T)GC(A/T)ACATATTC(A/T)GTCAT-3'
using P. carinii genomic DNA as the
template. An initial 4-min hot start at 94°C was followed by 30 cycles of 94°C for 60 s, 56°C for 60 s, 72°C for 60 s, and
a final 72°C 15-min extension. A single 617-bp amplification
product was identified by electrophoresis on 2% agarose after ethidium
bromide staining and was subcloned into the pCR2.1 vector (Invitrogen).
The amplified product was sequenced by cycle sequencing from both
directions with SP6 and T7 sequencing primers from pCR2.1 and internal
primers from the gene. Nucleotide sequence comparisons to the GenBank
database were done with Basic Local Alignment Search Tool (BLAST)
[National Center for Biotechnology Information (NCBI)].
Separation of P. carinii chromosomes
by contour-clamped homogeneous electrical field electrophoresis (CHEF)
was performed as reported previously (5). The 617-bp PCR amplification
product was labeled with
[ P. carinii MAPK cDNA was cloned from a
rat-derived P. carinii cDNA library in
the bacteriophage Uni-Zap XR. This library was screened using the
617-bp PCR amplicon as a probe using standard methods. Excision of the
phagemid was performed with M13 helper phage following the
manufacturer's instructions. A 1,341-bp insert was identified by
restriction endonuclease digestion with
EcoR I and
Xho I and was completely sequenced on
both strands using cycle sequencing with primers derived from the
plasmid and the gene. Nucleotide sequence comparisons to the GenBank
database were done with BLAST (NCBI).
Generation of P. carinii
MAPK antisera. Analysis of the
translated P. carinii MAPK cDNA
revealed a unique amino acid sequence, SAVSTEDSSS, between kinase
subdomains VII and VIII. The peptide CSAVSTEDSSS was synthesized at the
Mayo Protein Core Facility and coupled to keyhole limpet hemocyanin
(KLH) at the
NH2-terminal cysteine. Rabbits
received a primary subcutaneous injection of the antigen mixed with
complete Freunds adjuvant, followed by intramuscular boosts of the
antigen mixed with incomplete Freunds adjuvant using standard antibody
protocols. Reactivity of the immune sera compared with the preimmune
sera was verified by ELISA using the cognate peptide as a target
antigen. ELISA plates were coated with the target antigen in coupling
buffer (0.1 M NaHCO3, pH 8.3) and
blocked with 1% BSA, and serial dilutions of preimmune or immune serum
were added. Specific binding was detected with a goat anti-rabbit
secondary antibody coupled to horseradish peroxidase using
o-phenylenediamine dihydrochloride as a
substrate, with measurement at 450 nm.
MAPK assays. Immunoprecipitation
kinase assays were performed using the specific P. carinii MAPK antiserum (23). P. carinii was purified from immunosuppressed rats by lung
homogenization and differential filtration as described above.
P. carinii were disrupted in ice-cold
kinase lysis buffer [50 mM Tris · HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 1% (vol/vol) Triton X-100, 5 mM EDTA, 1 mM
EGTA, 200 µM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 0.1 tissue inhibitor units
aprotinin] and vortexed repeatedly over 5 min. The sample was
sonicated for 30 s, and the debris was pelleted by centrifugation at
12,000 g at 4°C for 10 min. The
protein concentration of the lysate was determined
spectrophotometrically using the bicinchoninic acid method (Pierce).
Protein lysate (500 µg) was precleared with a 50% slurry of protein
A-Sepharose at 4°C for 30 min, centrifuged at 12,000 g for 5 min, and then transferred to
clean tubes; immune or nonimmune serum (1:250 dilution) was added; and
the mixture was rocked at 4°C for 4 h. Immune complexes were
precipitated with a 50% slurry of protein A-Sepharose at 4°C for
60 min and collected by centrifugation at 12,000 g for 5 min. The pellet was washed
four times with 750 µl of kinase lysis buffer and two times with
kinase buffer (50 mM HEPES, pH 7.5, 10 mM magnesium acetate, and 1 mM
dithiothreitol). After the final wash, the pellet was resuspended in
50 µl of kinase reaction buffer (50 mM HEPES, pH 7.5, 10 mM magnesium
acetate, 1 mM dithiothreitol, 20 µM cold ATP, 0.3 mg/ml of myelin
basic protein or H1 histone, and 10 µCi [ P. carinii MAPK gene is homologous to
fungal MAPKs involved in differentiation and
proliferation. Cloning of the P. carinii MAPK was performed using a degenerate PCR
cloning strategy and traditional library screening (Fig.
1). The 617-bp PCR amplicon was found to be unique on
GenBank analysis but homologous to fungal MAPKs involved in mating. To
verify that the 617-bp PCR amplicon was part of the P. carinii genome, it was hybridized to
P. carinii chromosomes separated by
CHEF. The probe hybridized to a single chromosomal band
(Fig. 2). With the use of the 617-bp PCR amplicon as a
probe, a 1,341-bp full-length cDNA was obtained by screening a
P. carinii cDNA library in the
bacteriophage Uni-Zap XR (Fig. 1).
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP and
[
-32P]dATP were
obtained from ICN Pharmaceuticals.
-32P]dATP using
the random-primer method (Rediprime System; Amersham). After
prehybridization for 30 min (ExpressHyb solution; Clontech), the CHEF
nitrocellulose membrane was incubated with the probe (1.5 × 106
counts · min
1 · ml
1)
at 60°C for 1 h, washed one time at 37°C for 40 min in 2×
saline-sodium citrate (SSC) buffer containing 0.05% SDS,
washed one time at 50°C for 40 min in 2× SSC buffer
containing 0.1% SDS, and examined by autoradiography.
-32P]ATP) and
incubated at 30°C for 30 min. The reaction was stopped with the
addition of Laemmli buffer, boiled for 5 min, and centrifuged at 12,000 g for 5 min, and the supernatant was
resolved by 12% SDS-PAGE. The gel was fixed in 40% methanol-10%
acetic acid for 30 min, washed in distilled water for 15 min, and
exposed to autoradiography film at
70°C. To control for host
cells that may pass through the filtration process, protein lysates
from healthy rat lungs were prepared and filtered in an identical
manner as described above and were immunoprecipitated with the immune
or nonimmune serum (1:250 dilution) using the identical methods
described.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (89K):
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Fig. 1.
Nucleotide and predicted amino acid sequence of
Pneumocystis carinii
mitogen-activated protein kinase (MAPK). The 5'- and
3'-untranslated regions are in lowercase letters. The single open
reading frame encodes a 351-amino acid protein with predicted molecular
mass of 40,571. The T and
Y amino acids are the conserved
putative phosphorylation sites for MAPK activation. (The sequence data
are available from GenBank accession number AF043941.)
View larger version (87K):
[in a new window]
Fig. 2.
Chromosomal location of the P. carinii
MAPK gene. The P. carinii MAPK gene is
present on a single chromosome within the P. carinii genome. The P. carinii MAPK probe hybridized to a single
P. carinii chromosome. M, DNA ladder.
Lanes 1-10: samples of
P. carinii DNA resolved by
contour-clamped homogeneous electrical field electrophoresis (CHEF).
A: ethidium bromide-stained CHEF gel
shows separated samples of P. carinii
chromosomes. B: hybridization blot of
32P-labeled MAPK probe to
separated samples of P. carinii
chromosomes transferred to nitrocellulose.
Analysis of the P. carinii MAPK cDNA against the GenBank database revealed this gene to be unique. Homology on the amino acid level (BLASTX) revealed 80% identity to Fusarium solani and Magnaporthe grisea MAPKs, 78% identity to Candida albicans Cek1, 63% to S. pombe Spk1, 64% to S. cerevisiae FUS3 and KSS1, 58% to Homo sapiens ERK1 and ERK2, and 57% to Rattus norvegicus ERK1 (Fig. 3). The single open reading frame encodes a 351-amino acid protein with predicted molecular weight of 40,571. The putative regulatory phosphorylation sites necessary for P. carinii MAPK activity are present in amino acids Thr(T)-176 and Tyr(Y)-178, and the putative active site is present in amino acid Asp(D)-140 (Fig. 1). The kinase domains HRDLKPSN [containing the active site Asp(D)-140] and TEYVATRWRAPE [containing the phosphorylation sites Thr(T)-176 and Tyr(Y)-178] are 100% conserved between P. carinii and the other MAPKs listed in Fig. 3 with the exception of S. cerevisiae Kss1 (Fig. 3).
|
Demonstration of characteristic MAPK activity from
P. carinii. To phosphorylate
substrates, MAPK needs to be activated by phosphorylation on two
conserved threonine and tyrosine amino acids; this occurs in response
to a specific signal propagated by the protein kinase cascade. To
determine whether P. carinii organisms
possess an activated MAPK capable of kinase activity, P. carinii protein extracts were
immunoprecipitated with the specific P. carinii MAPK antiserum and reacted with
[-32P]ATP and
myelin basic protein or H1 histone as substrates. An immune serum was
generated in rabbits against a unique amino acid region in the
P. carinii MAPK between kinase domains
VII and VIII. The specificity of this immune serum was verified by
ELISA against the target antigen (Fig.
4A). Next, kinase
activity was detected in P. carinii
lysates immunoprecipitated with this specific P. carinii MAPK antiserum but not with the nonimmune serum
(Fig. 4B). Because we used the
immunosuppressed rat model to obtain P. carinii organisms, control experiments were performed
to account for the possibility of host cells that might come through
the purification process of separating P. carinii from host lung. These failed to demonstrate
significant protein kinase activity, thus confirming that the observed
kinase activity was specifically derived from P. carinii.
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DISCUSSION |
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MAPKs are highly conserved serine-threonine protein kinases involved in regulating cellular differentiation and proliferation. The fungal MAPK active in the pheromone mating cascade is essential for the transition from mitotic growth into a pathway of conjugation and cellular differentiation (1, 7, 19). MAPK phosphorylates and thereby activates proteins required for conjugation, cell fusion, and for the direct inhibition of Cdc2 kinase activity, resulting in mitotic cell cycle arrest (18, 22, 25). Loss of fungal MAPK activity results in sterility, with the inability to enter meiosis and proliferate (7). Given its essential role in an organism's life cycle, MAPK is regulated at many levels. Dual-activating phosphorylations by the upstream kinase MEK occur on conserved threonine and tyrosine amino acid residues in the TEY sequence between kinase subdomains VII and VIII, and both phosphorylations are required for enzymatic function (9, 17).
The P. carinii MAPK is most closely homologous to fungal MAPKs involved in differentiation and proliferation, which suggests that P. carinii may use similar pathways. Amino acid sequence comparisons revealed homology to two phytopathogenic filamentous fungi of the ascomycetes family, M. grisea and F. solani (80% identity on the amino acid level), to the opportunistic yeast C. albicans (78% identity), and to S. pombe spk1 and S. cerevisiae FUS3 and KSS1.
M. grisea is a phytopathogenic fungus that causes rice blast disease. The M. grisea MAPK Pmk1 controls differentiation of a specialized invasion structure, the appressorium, which is required for the fungus to infect the host rice plant. M. grisea strains containing mutations in Pmk1 fail to form appressoria and thus cannot grow invasively in rice plants. Pmk1 has also been found to complement the mating defect in S. cerevisiae Fus3/Kss1 double-mutant yeast (30). F. solani, which has historically been recognized as a phytopathogen, has recently been identified as a cause of invasive human infections in immunocompromised patients with hematological malignancies (3, 10). An F. solani MAPK has recently been identified that is homologous to MAPKs involved in growth regulation, but further investigation is needed to determine its function within this fungus (12). The P. carinii MAPK shares homology to the C. albicans MAPK Cek1, which was cloned as a gene that interfered with the pheromone-mediated cell cycle arrest of S. cerevisiae and conferred a phenotype similar to the S. cerevisiae MAPK Kss1 (29) and to S. cerevisiae Fus3/Kss1 and S. pombe Spk1 MAPKs, which are involved in regulating the pheromone mating signal transduction cascade in these yeasts.
In summary, we have identified an MAPK from P. carinii that is homologous to fungal MAPKs that have important roles in regulating growth, differentiation, and proliferation pathways. Chromosomal analysis reveals that the P. carinii MAPK is present on a single chromosome within the P. carinii genome. In addition, we are able to demonstrate MAPK activity from P. carinii lysates using a specific antiserum derived from the translated P. carinii MAPK cDNA sequence, suggesting that this gene is expressed as a functional protein kinase in P. carinii organisms. We believe that these observations represent an essential first step in identifying the regulatory pathways of growth and proliferation of P. carinii. Further investigations of the regulation and activity of the P. carinii MAPK should lead to an enhanced understanding of the complex life cycle of this pulmonary pathogen.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Cushion, University of Cincinnati College of Medicine, for providing the contour-clamped homogeneous electrical field electrophoresis blot of Pneumocystis carinii chromosomes and photograph of the ethidium bromide-stained gel. The following reagent was obtained through the acquired immunodeficiency syndrome (AIDS) Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH): P. carinii cDNA library from Biotechnology General.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-55934-03, AI-34336-05, and HL-57125-01 to A. H. Limper. C. F. Thomas, Jr., is a 1996 Glaxo Wellcome Pulmonary Fellowship Award Recipient.
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. §1734 solely to indicate this fact.
Address for reprint requests: C. F. Thomas, Thoracic Diseases Research Unit, E18B Mayo Bldg., 200 First St. S.W., Mayo Clinic and Foundation, Rochester, MN 55905.
Received 4 February 1998; accepted in final form 1 April 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bardwell, L.,
J. G. Cook,
C. J. Inouye,
and
J. Thorner.
Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae.
Dev. Biol.
166:
363-379,
1994[Medline].
2.
Bartlett, M. S.,
J. A. Fishman,
S. A. Queener,
M. M. Durkin,
M. A. Jay,
and
J. W. Smith.
New rat model of Pneumocystis carinii pneumonia.
J. Clin. Microbiol.
26:
1100-1102,
1988[Medline].
3.
Boutata, E. I.,
and
E. J. Anaissie.
Fusarium, a significant emerging pathogen in patients with hematologic malignancy: ten years experience at a cancer center and implications for management.
Blood
90:
999-1008,
1997
4.
Campbell, W. G.
Ultrastructure of Pneumocystis in human lung.
Arch. Pathol.
93:
312-330,
1972[Medline].
5.
Cushion, M. T.,
J. Zhang,
M. Kaselis,
D. Giuntoli,
S. L. Stringer,
and
J. R. Stringer.
Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats.
J. Clin. Microbiol.
31:
1217-1223,
1993[Abstract].
6.
Edman, J. C.,
J. A. Kovacs,
H. Masur,
D. V. Santi,
H. J. Elwood,
and
M. L. Sogin.
Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi.
Nature
334:
519-522,
1988[Medline].
7.
Elion, E. A.,
P. L. Grisafi,
and
G. R. Fink.
FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation.
Cell
60:
649-664,
1990[Medline].
8.
Fletcher, L. D.,
L. C. Berger,
S. A. Peel,
R. S. Baric,
R. R. Tidwell,
and
C. C. Dykstra.
Isolation and identification of six Pneumocystis carinii genes utilizing codon bias.
Gene
129:
167-174,
1993[Medline].
9.
Gartner, A.,
K. Nasmyth,
and
G. Ammerer.
Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1.
Genes Dev.
6:
1280-1292,
1992[Abstract].
10.
Hennequin, C.,
V. Lavarde,
J. L. Poirot,
M. Rabodonirina,
A. Datry,
S. Aractingi,
J. Dupouy-Camet,
D. Caillot,
F. Grange,
L. Kures,
O. Morin,
B. Lebeau,
S. Bretagne,
C. Guigen,
D. Basset,
and
R. Grillot.
Invasive fusarium infections: a retrospective survey of 31 cases.
J. Med. Vet. Mycol.
35:
107-114,
1997[Medline].
11.
Itatani, C. A.,
and
J. Marshall.
Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage.
J. Parasitol.
74:
700-712,
1988[Medline].
12.
Li, D.,
L. Rogers,
and
P. E. Kolattukudy.
Cloning and expression of cDNA encoding a mitogen-activated protein kinase from a phytopathogenic filamentous fungus.
Gene
195:
161-166,
1997[Medline].
13.
Limper, A. H.,
and
W. J. Martin.
Pneumocystis carinii: inhibition of lung cell growth mediated by parasite attachment.
J. Clin. Invest.
85:
391-396,
1990[Medline].
14.
Limper, A. H.,
K. P. Offord,
T. F. Smith,
and
W. J. Martin.
Pneumocystis carinii pneumonia: differences in lung parasite number and inflammation in patients with and without AIDS.
Am. Rev. Respir. Dis.
140:
1204-1209,
1989[Medline].
15.
Limper, A. H.,
J. E. Standing,
O. A. Hoffman,
M. Castro,
and
L. W. Neese.
Vitronectin binds to Pneumocystis carinii and mediates organism attachment to cultured lung epithelial cells.
Infect. Immun.
61:
4302-4309,
1993[Abstract].
16.
Limper, A. H.,
C. F. Thomas,
R. A. Anders,
and
E. B. Leof.
Interactions of parasite and host epithelial cell cycle regulation during Pneumocystis carinii pneumonia.
J. Lab. Clin. Med.
130:
132-138,
1997[Medline].
17.
Ma, D.,
J. G. Cook,
and
J. Thorner.
Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.
Mol. Biol. Cell
6:
889-909,
1995[Abstract].
18.
McKinney, J. D.,
and
F. R. Cross.
FAR1 and the G1 phase specificity of cell cycle arrest by mating factor in Saccharomyces cerevisiae.
Mol. Cell. Biol.
15:
2509-2516,
1995[Abstract].
19.
Nielsen, O.
Signal transduction during mating and meiosis in S. pombe.
Trends Cell Biol.
3:
60-65,
1993.
20.
Nishida, E.,
and
Y. Gotoh.
The MAP kinase cascade is essential for diverse signal transduction pathways.
Trends Biochem. Sci.
18:
128-131,
1993[Medline].
21.
O'Riordan, D. M.,
J. E. Standing,
K. Y. Kwon,
D. Chang,
E. C. Crouch,
and
A. H. Limper.
Surfactant protein D interacts with Pneumocystis carinii and mediates organism adherence to macrophages.
J. Clin. Invest.
95:
2699-2710,
1995[Medline].
22.
Peter, M.,
and
I. Herskowitz.
Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1.
Science
265:
1228-1231,
1994[Medline].
23.
Reuter, C. W. M.,
A. D. Catling,
and
M. J. Weber.
Immune complex kinase assays for mitogen-activated protein kinase and MEK.
Methods Enzymol.
255:
245-256,
1995[Medline].
24.
Sloand, E.,
B. Laughon,
M. Armstrong,
M. S. Bartlett,
W. Blumenfeld,
M. Cushion,
A. Kalica,
J. A. Kovacs,
W. J. Martin,
and
E. Pitt.
The challenge of Pneumocystis carinii culture.
J. Eukaryot. Microbiol.
40:
188-195,
1993[Medline].
25.
Stern, B.,
and
P. Nurse.
Fission yeast pheromone blocks S-phase by inhibiting the G1 cyclin B-p34cdc2 kinase.
EMBO J.
16:
534-544,
1997
26.
Thomas, C. F.,
R. A. Anders,
M. P. Gustafson,
E. B. Leof,
and
A. H. Limper.
Pneumocystis carinii contains a functional cell-division-cycle Cdc2 homologue.
Am. J. Respir. Cell Mol. Biol.
18:
297-306,
1998
27.
Walzer, P. D.,
M. LaBine,
T. J. Redington,
and
M. T. Cushion.
Predisposing factors in Pneumocystis carinii pneumonia: effects of tetracycline, protein malnutrition, and corticosteroids on hosts.
Infect. Immun.
46:
747-753,
1984[Medline].
28.
Waskiewicz, A. J.,
and
J. A. Cooper.
Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast.
Curr. Opin. Cell Biol.
7:
798-805,
1995[Medline].
29.
Whiteway, M.,
D. Dignard,
and
D. Y. Thomas.
Dominant negative selection of heterologous genes: isolation of Candida albicans genes that interfere with Saccharomyces cerevisiae mating factor-induced cell cycle arrest.
Proc. Natl. Acad. Sci. USA
89:
9410-9414,
1992[Abstract].
30.
Xu, J. R.,
and
J. E. Hamer.
MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea.
Genes Dev.
10:
2696-2706,
1996[Abstract].
31.
Yale, S. H.,
and
A. H. Limper.
Pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated disorders and prior corticosteroid therapy.
Mayo Clin. Proc.
71:
5-13,
1996[Medline].