Potential Involvement of Extracellular
Signal-regulated Kinase 1 and 2 in Encystation of a Primitive
Eukaryote, Giardia lamblia
STAGE-SPECIFIC ACTIVATION AND INTRACELLULAR LOCALIZATION*
John G.
Ellis IV,
Monica
Davila, and
Ratna
Chakrabarti
From the Department of Molecular Biology and Microbiology,
University of Central Florida, Orlando, Florida 32826-2362
Received for publication, September 10, 2002, and in revised form, October 22, 2002
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ABSTRACT |
Mitogen-activated protein kinase (MAPK) pathways
are major signaling systems by which eukaryotic cells convert
environmental cues to intracellular events such as proliferation and
differentiation. We have identified Giardia lamblia
homologues of two members of the MAPK family ERK1 and ERK2. Functional
characterization of giardial ERK1 and ERK2 revealed that both kinases
were expressed in trophozoites and encysting cells as 44- and 41-kDa
polypeptides, respectively, and were catalytically active. Analysis of
the kinetic parameters of the recombinant proteins showed that ERK2 is
~5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the
native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in
trophozoites showed ERK1 staining mostly in the median body and in the
outer edges of the adhesive disc and ERK2 staining in the nuclei and in
the caudal flagella. Our study also showed a noticeable change in the
subcellular distribution of ERK2 during encystation, which became more
punctate and mostly cytoplasmic, but no significant change in the ERK1
localization at any time during encystation. Interestingly, both ERK1
and ERK2 enzymes exhibited a significantly reduced kinase activity
during encystation reaching a minimum at 24 h, except for an
initial ~2.5-fold increase in the ERK1 activity at 2 h, which
resumed back to the normal levels at 48 h despite no apparent
change in the expression level of either one of these kinases in
encysting cells. A reduced concentration of the phosphorylated ERK1 and
ERK2 was also evident in these cells at 24 h. Our study suggests a
functional distinction between ERK1 and ERK2 and that these kinases may
play a critical role in trophozoite differentiation into cysts.
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INTRODUCTION |
Giardia lamblia, an evolutionary primitive eukaryotic
protozoan parasite and an intestinal pathogen of humans and animals, is
one of the major causes of water-borne diseases worldwide (1). This
flagellated protozoan undergoes complex life cycle stages while inside
the host. Exposure to the highly acidic condition in stomach and
proteases in the upper small intestine triggers excystation of
trophozoites from the ingested cysts. Newly emerged trophozoites swim
freely in the intestinal fluid and colonize the upper small intestine
to replicate (2, 3). As enterocytes migrate to the tip of the villus
and get sloughed off into the intestinal lumen, the attached
trophozoites either reattach to new enterocytes to remain in the
intestine or differentiate into infective cysts. Although the life
cycle of this primitive eukaryote and physiological signals that
regulate induction of excystation and encystation have been studied
extensively, the molecular mechanisms by which trophozoites sense and
respond quickly to the environmental signals in the intestine to
initiate encystation remain largely unknown.
Encystation is an adaptive process to cope with the depletion of
nutrients, specifically cholesterol, in the presence of high bile
concentration in the lower small intestine (4, 5). It is accomplished
in three steps as follows: (i) induction of the encystation-specific
gene expression, specifically those that are necessary for the
synthesis and processing of cyst wall proteins (CWP)1 (6-9); (ii) synthesis
and intracellular transport of cyst wall proteins through newly
developed secretory organelles such as Golgi apparatus (10) and
encystation-specific vesicles (11); and (iii) assembly of the
extracellular cyst wall (12, 13). Encysting trophozoites undergo
distinct morphological changes such as disappearance of the median
bodies and disorganization of the adhesive disc and flagella. The
attached trophozoites gradually round up and detach. They lose mobility
and become refractile as the cyst wall is assembled. The vacuoles that
lie beneath the trophozoite membrane become the periplasmic space
separating the parasite from the cyst wall (14). Unlike differentiating
mammalian cells, giardial differentiation into cysts initiates in the
cells arrested at G2 (15). During encystation two rounds of
DNA replication without an intervening cell division occur, and nuclear
division precedes chromosomal duplication (15). The dramatic changes in
the cytoskeleton reorganization and gene expression during encystation
suggest the existence of an orchestrated signaling mechanism. We
speculate that the activation or inactivation of microtubule-associated
proteins and transcription factors by kinases might play an important
role in differentiation of trophozoites into cysts in response to
cholesterol starvation.
A variety of critical cellular functions in eukaryotic cells are
mediated through the MAP kinase pathway in response to extracellular stimuli (reviewed in Ref. 16). The mammalian MAP kinases p44 and p42,
also known as ERK1 and ERK2, are serine/threonine kinases and are the
two best-studied members of the MAP kinase family. Full activation of
ERK1 and ERK2 requires dual phosphorylation at the serine and tyrosine
residues at the TXY motif by MEK1/2 (17, 18). Upon
activation, both ERK1 and ERK2 translocate to the nucleus and
phosphorylate a variety of proteins including transcription factors,
such as AP-1 (19), Elk1 (20), Ets1 (21), and SREBP (22), that directly
or indirectly stimulate cell proliferation or differentiation.
A differential expression and/or activity of various MAP kinase
homologues has been observed in parasitic protozoa. The fluctuation of
expression or activity of MAP kinases has also been implicated for
proliferation, differentiation, or development in protozoan parasites.
Studies have shown that the activity of KFR1, a MAP kinase homologue of
Trypanosoma brucei, is higher in the differentiating bloodstream form than the procyclic form (23). LMAPK, a MAPK homologue
of Leishmania major, is expressed in promastigotes and amastigotes, but the activity is detected only in amastigotes. Similarly, expression of PfMAP2, a MAPK homologue identified in Plasmodium falciparum, has been detected only in gametocytes
of malaria parasites (24). Here we report functional characterization of giardial homologues of ERK1 and ERK2. Our study provides evidence of
differential subcellular localization and activation of giardial ERK1
and ERK2 in trophozoites and in encysting cells. Our results indicate,
for the first time, that giardial ERK1 and ERK2 possess functional
differences, when they are thought to have overlapping functions, and
that ERK1 and ERK2 may be regulated differentially.
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EXPERIMENTAL PROCEDURES |
Materials--
Unless specified, all materials were obtained
from Sigma, Fisher, and Invitrogen.
Giardia Cell Culture and Differentiation--
G.
lamblia (WB clone C6, ATCC 50803) were maintained in TYI-S-33
medium with 10% bovine serum and bile (25). Encystation was induced
essentially as described by Kane et al. (4). Briefly, encystation was induced by growing trophozoites for one culture cycle
in TYI-S-33. Media were poured off along with unattached trophozoites
and replaced with encystation medium containing 10 mg/ml bovine bile at
pH 7.8 and incubated at 37 °C for the time specified. Encystation
medium was replaced with TYI-S-33 medium at 24 h, and incubation
was continued for 48 h. Encysting cells were harvested at
different time points by chilling and centrifugation and subsequently
used for protein extraction.
PCR Amplification--
A 2.0-kb DNA fragment of ERK1 containing
the entire open reading frame and a 530-bp fragment of ERK2 were
obtained by PCR amplification of Giardia genomic DNA using
primers (forward, 5'-CCCTAATTAAGCCAGTGACATA-3', and reverse,
5'-AAGCCCGTAGCCCCAGTTC-3' for ERK1 and forward,
5'-GCGGGCATGCTGATTGTGTTAG-3', and reverse, 5'-ATAGACGACGATGATAAGAG for
ERK2) designed using sequences available in the
Giardia Genome Sequencing Data base (27, www.mbl.edu/Giardia/). The amplified fragments were sequenced, and the
ERK2 fragment was used to screen a Giardia
ZapII cDNA library.
Screening of a cDNA Library--
The full-length cDNA of
the G. lamblia homologue of ERK2 was obtained by screening a
Giardia
ZAPII cDNA library using a
[32P]dATP-labeled ERK2 probe amplified using a
strand-specific primer as described previously (28). Cloned fragments
were sequenced using a capillary electrophoresis-based automated DNA
sequencer (ABI Prism, 310 Genetic analyzer; ABI, Foster City, CA). Raw
output was analyzed using ABI Prism sequence analysis software. The
sequence identity was compared with the NCBI data base using PSI-BLAST. Multiple sequence alignment was done using MegAlign program from DNAstar (Madison, WI) software. Molecular three-dimensional models of
gERK1 and gERK2 were generated using Geno3D on-line model analysis service, and pdb1erk and pdb2erk as templates, respectively (29).
Southern Blot and Northern Blot--
Genomic DNA from
Giardia was isolated using a standard protocol as described
by Maniatis and co-workers (30). Total RNA was extracted using an RNA
isolation kit (Stratagene) according to the supplier's protocol.
32P-Labeled Giardia ERK1 (gERK1) and ERK2
(gERK2) cDNA probes were used for Southern hybridization as
described previously (31). For Northern blots, strand-specific probes
were used (28).
Expression and Purification of Recombinant Protein--
The open
reading frames of gERK1 and gERK2 were cloned into T7 polymerase-driven
bacterial expression vectors pET41 (Novagen) and pET30Ek/LIC (Novagen),
respectively, according to the manufacturer's protocol. Two
primers (forward, 5'-AGTCCCATGGGAATGCCGCTGGCC-3', and reverse,
5'-AGTGCGGCCGCTTACATCCACACAGA-3') were designed to amplify gERK1 ORF
(1.158 kb) containing NcoI and NotI sites for cloning into pET41. The ORF of gERK2 (1.086 kb) was cloned into pET30
by ligation-independent cloning method (27) using primers (forward
5'-GACGACGACAAGATGTCTGACGACGAAT-3' and reverse
5'-GAGGAGAAGCCCGGTTCACTCGTCTTCCTT-3'). Expression constructs were
used to transform BL21(DE3) Escherichia coli cells.
Transformed cells were induced with either 1.0 mM isopropyl-1-thio-
-D-galactopyranoside (ERK1) or 0.5 mM (ERK2) isopropyl-1-thio-
-D-galactopyranoside at 25 °C to
overexpress recombinant gERK1 (75 kDa) and gERK2 (47 kDa) as
glutathione S-transferase-tagged and His-tagged fusion
proteins, respectively. Expressed proteins were purified through cobalt
affinity column (TALON superflow resin, Clontech
Laboratories) and used for in vitro kinase assays and
generation of rabbit polyclonal antibodies through a commercial vendor
(Cocalico, Reamstown, PA). The His tag (pET30 ERK2) and glutathione
S-transferase tag (pET41 ERK1) sequences added 6 and 31 kDa
of molecular masses to the fusion proteins, respectively.
Western Blot and Immunoprecipitation--
Trophozoites or
encysting cells at different time points were harvested, and crude cell
extracts (50 µg) in Tris-HCl (100 mM, pH 7.0) were used
for immunodetection of endogenous dephosphorylated ERK1 and ERK2 using
anti-gERK1 and anti-gERK2 polyclonal antibodies. Phosphorylated ERK1
and ERK2 in the crude extracts with or without treatment with
phosphatase (2000 units for 30 min at 30 °C) were detected by an
anti-phospho-ERK polyclonal antibody that recognized both ERK1 and ERK2
(Cell Signaling Technologies). Expression of CWP1 in encysting cells
was detected by immunoblot analysis using anti-CWP1 monoclonal antibody
(kindly provided by Dr. Henry Stibbs, Waterborne Inc.). Positive
signals were detected using a chemiluminescence kit (Pierce) and an
anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary
antibody. For immunoprecipitation (28), trophozoites or encysting cells
were lysed in TAN buffer (10 mM Tris acetate, pH 8.0, 1%
Nonidet P-40, 100 mM NaCl, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 2 µg/ml aprotinin). Crude cell extracts were
treated with rabbit serum followed by protein A-agarose beads to
eliminate nonspecific binding of proteins to antibody. The protein
A-agarose beads were collected by centrifugation, and the supernatants
were incubated with the appropriate antibody. Protein-antibody
complexes were captured by protein A-agarose beads, washed, and
collected by centrifugation. Protein A-agarose beads bound to the
immune complex were then resuspended in 100 mM Tris, pH
7.0, and used for subsequent experiments.
Kinase Assay--
Purified recombinant gERK1 and gERK2 (5 µg)
or semi-compact protein A-agarose beads pellets of immunoprecipitated
gERK1 and gERK2 (25 µl) were used in a 50-µl reaction containing
100 mM Tris-HCl, pH 7.5, 600 µM ATP, 1 mM MgCl2 (ERK2) or 10 mM
MgCl2 (ERK1), 6 nM [
-32P]ATP,
and 100 µM of myelin basic protein (MBP). The reaction mixture was incubated at 30 °C for 10 (ERK2) or 30 min (ERK1), and
30 µl (recombinant protein only) were spotted on phosphocellulose filter discs after addition of 5 µl of EDTA (100 mM) to
stop the reaction. Filter discs were washed with 1%
H3PO4, and bound radioactivity was detected in
a liquid scintillation counter (Beckman Instruments). The remaining 20 µl of the reaction mix (recombinant protein) or the 50 µl of the
reaction mix (immunoprecipitated protein) was combined with 4× Laemmli
sample buffer and resolved in SDS-PAGE (12-14%). Phosphorylation of
MBP was detected by autoradiography and PhosphorImager (Amersham
Biosciences) analysis.
Immunofluorescence Analysis--
Trophozoites (350-700
cells/ml) were cultured on 12-mm glass coverslips placed in 24-well
tissue culture dishes and allowed to grow for 65-70 h in an AnaeroPack
jar (Mitshubishi Gas Chemical Co.) at 37 °C. In some experiments,
trophozoites were exposed to encystation medium, and cells were fixed
after the specified time. Trophozoites or encysting cells attached on
coverslips were fixed in 100% chilled methanol (
20 °C) for 10 min
at
20 °C and further permeabilized with 0.5% Triton X-100 in PBS
for 10 min at room temperature. Permeabilized cells were blocked in
blocking buffer (5% goat serum and 1% glycerol in PBS) and incubated
with appropriate rabbit polyclonal antibody (ERK1 1:3000 and ERK2
1:6000 in blocking buffer). At the end of incubation, cells were washed with PBS and incubated with an Alexa 488-conjugated anti-rabbit (Molecular Probes, Eugene, CA) secondary antibody diluted in blocking buffer (1:100). Cells were washed in PBS and counterstained with DAPI
(0.1 µM). Cells were washed in PBS and post-fixed with
4% paraformaldehyde (Electron Microscopy Sciences), rinsed with PBS and mounted on Gelmount (Biomeda Corp.). Localization of the target proteins was detected in a Delta Vision Image Restoration Microscope using SoftWoRX image analysis software. DIC images of the
localization were captured in a Zeiss LSM 510 laser scanning confocal microscope.
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RESULTS |
Sequence Characterization and Expression Analysis of gERK1 and
gERK2--
We have cloned giardial homologues of ERK1 (ORF 1.15 kb,
386 amino acids) by direct PCR amplification of the genomic DNA and ERK2 (ORF 1.086 kb, 362 amino acids) by screening a
ZAPII
Giardia cDNA library. Upon screening the library using a
530-bp fragment as a probe, a 1.15-kb cDNA clone of ERK2 was
obtained (GenBankTM accession number AY149274 for ERK1 and
accession number AY149275 for ERK2). Multiple sequence analysis of the
translated amino acid sequence of the ORF showed that
Giardia ERK1 had 51% identity with Dictyostelium
discoideum ERK1, 49% identity with Chlamydomonas reinhardtii MAPK, 48% identity with Arabidopsis
thaliana MAPK, 42% identity with Schizosaccharomyces
pombe SPK1, and 43% identity with human ERK1 with a probability
score between e-100 and 1e-76. The deduced amino acid sequence
of Giardia ERK2 ORF exhibited 66% identity with D. discoideum ERK2, 59% identity with L. mexicana MAPK2,
57% identity with P. falciparumMAPK1, and 50% identity with human ERK8 with a probability score between e-131 and 2e-94. The gERK2 cDNA clone contains a short 9-bp 5'- and a 56-bp
3'-untranslated sequence that are typical for Giardia
transcripts. Multiple sequence alignment of the amino acid sequence of
the ERK1/2 catalytic domain revealed that all 11 subdomains,
characteristic of serine/threonine kinases, are present in both gERK1
and gERK2 (Figs. 1A and
2A). This includes the consensus motif
GXGXXGXV
(Gly26-Val33 gERK1 and
Gly25-Val32 gERK2) in subdomain I for
anchoring non-transferable phosphates of ATP, the invariant Lys
(Lys-48 ERK1 and Lys-47ERK2) and the
surrounding region (Val45-Ser51, ERK1,
Val44-Ser50 ERK2) in subdomain II for binding
and orienting ATP for maximum enzymatic activity, and the glutamic acid
(Glu66 ERK1, Glu65 ERK2) in the subdomain III
involved in formation of a salt bridge with Lys48 and
Lys47. The catalytic loops in both ERK1 and ERK2 are nearly
100% homologous with the HRDLKXXN motif
(His143-Asn150 ERK1 and
His138-Asn145 ERK2). The conserved TXY motif
for dual phosphorylation (Thr191-Tyr193 ERK1
and Thr180-Tyr182 ERK2) for the activation of
the enzyme is also present. Five other amino acids, Phe54
(PheF53 gERK2), Arg62 (Arg61
gERK2), His121 (His116 gERK2),
Lys134 (Lys129 gERK2), and Cys157
(Cys152 gERK2), considered to be the signature residues for
MAPK as they are not shared by any other kinases including the CDKs
(32), are also present in gERK1 and gERK2. Molecular three-dimensional modeling of gERK1 and gERK2 revealed the presence of additional
-helices (Fig. 1, B and C, and Fig.
2, B and C) in
giardial ERK1/2 when compared with rat extracellular signal-regulated
kinase (pdb1erk) and phospho-ERK2 (pdb2erk) indicating significant
structural differences between mammalian ERK1/2 and giardial ERK1/2.
Functional implication of these additional
-helices is unclear at
this moment.

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Fig. 1.
Multiple sequence alignment and
structural analysis of gERK1. A, Gl,
G. lamblia; Sp, S. pombe;
Hs, Homo sapiens; Dd, D. discoideum. The solid bars above the
sequences indicate the G loop, the invariant Lys48
(black arrow) and surrounding region, the catalytic loop,
the TXY motif, and APE site, respectively. Black
arrows indicate the conserved Phe54,
Arg62, Glu66, His121,
Lys134, and Cys157, respectively. The
black double-headed arrows indicate the 10-amino acid
insertion. B, three-dimensional model of gERK1.
C, three-dimensional model of rat ERK. White
arrows indicate additional -helices.
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Fig. 2.
Multiple sequence alignment and structural
analysis of gERK2. A, Gl, G. lamblia; Dd, D. discoideum;
Hs, H. sapiens; Lm, L. mexicana. The solid bars above the sequences
indicate the G loop, the invariant Lys47 (black
arrow) and surrounding region, the catalytic loop, the
TXY motif, and APE site, respectively. Black
arrows indicate the conserved Phe53,
Arg61, Glu65, His116,
Lys129, and Cys152 respectively. The
black diamond shows serine instead of alanine in the APE
site. B, three-dimensional model of gERK2. C,
three-dimensional model of rat ERK2. White arrow indicates
additional -helix.
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Analysis of genomic organization by Southern blot and search of
Giardia genome sequence data base suggest that both gERK1 and gERK2 are single copy genes (data not shown). Northern blot analysis of gERK1 and gERK2 revealed that both are expressed as a
single transcript of 1.3 kb for ERK1 and 1.2 kb for ERK2, respectively, in trophozoites (data not shown). Analysis of the protein products by
Western blotting of cell extracts indicated that the expression of the
44-kDa gERK1 or the 41-kDa gERK2 did not fluctuate during growth and
encystation (Figs. 8 and 9). The size of the native gERK1 and gERK2 was
in agreement with the deduced amino acid sequences from the ORFs.
Giardial ERK1 and ERK2 Were Catalytically Active--
For
functional characterization of gERK1 and gERK2, the catalytic activity
of the recombinant ERK1 and ERK2 was determined next using MBP as the
substrate. In vitro kinase assays revealed that both
recombinant ERK1 and ERK2 were catalytically active (Fig.
3) and phosphorylated MBP with different
efficiency (specific activities: 3.7 ± 0.3 nmol of
phosphate/µmol of ERK1/s and 20.1 ± 0.06 nmol of
phosphate/µmol of ERK2/s). Recombinant ERK2 was ~5 times more
efficient than ERK1 in incorporating phosphates into MBP and was
capable of autophosphorylation (Fig. 3, E and C).
No autophosphorylation was noted for gERK1, which suggests that gERK1
possibly can maintain a basal level of activity without being activated
by phosphorylation (Fig. 3A). This is apparently reflected
into the lower efficiency of phosphorylation of MBP by the recombinant
gERK1, and therefore, the activity of the recombinant gERK1 may not
represent the full phosphorylating potential of the endogenous gERK1.
At the standard reaction conditions using 5 µg of the enzyme and
varying concentrations of MBP, the apparent Km
values for the MBP were in the micromolar range (31.8 ± 8.5 µM ERK1 and 33.1 ± 7.3 µM ERK2) (Fig.
3, B, D and E), which are close to that reported
for the mouse ERK2 (33). Both ERK1 and ERK2 preferred ATP to GTP as the
phosphate donor (ERK1,
Mg2+ATPKm
265.5 ± 36.0 µM; ERK2,
Mg2+ATPKm
32.1 ± 2.5 µM); however, ERK2 was 8 times more efficient in utilizing ATP than ERK1. Giardial ERK1 and ERK2 also favored Mg2+ than Mn2+ as the divalent cation
(ERK1, MgKm, 1.53 ± 0.4 mM; ERK2, MgKm, 0.265 ± 0.001 µM) (Fig. 3E).

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Fig. 3.
Phosphorylation of MBP by gERK1 and gERK2.
A and C, upper panel, Coomassie
Blue-stained SDS-PAGE showing increasing concentration of MBP in the
kinase assay of ERK1 and ERK2, respectively. Lower panel,
autoradiogram showing phosphorylated MBP and ERK2 (C) and no
phosphorylation in the MBP only lane (A). B and
D, non-linear plots of the phosphorylated MBP from filter
binding kinase assay of ERK1 and ERK2. Each point represents the mean
of three separate experiments. D, specific activity and
apparent Km for MBP, ATP, and MgCl2 for
ERK1 and ERK2.
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Although the kinetic parameters for the recombinant gERK2 were
comparable with that for the mammalian ERK2, the catalytic efficiency
of the recombinant gERK1 was relatively poor. To determine the
catalytic activities of the endogenous ERK1 and ERK2, we
immunoprecipitated ERK1 and ERK2 from the trophozoite extracts using
anti-gERK1 and -gERK2 rabbit polyclonal antibodies. The specificity of
the anti-ERK1 and -ERK2 antibodies was determined previously by Western
blot analysis of the purified recombinant enzymes and crude trophozoite extracts (Fig. 4 and
5), which indicated a single peptide band of 75 kDa for the recombinant ERK1 and a 44-kDa polypeptide band for
the endogenous ERK1 recognized by the antibody at a dilution of 1:4000
(Fig. 4, A and B). Similarly, anti-ERK2 antibody
recognized a single major polypeptide band of 47 kDa for the
recombinant ERK2 and a 41-kDa band for the endogenous ERK2 at a
dilution of 1:30,000 (Fig. 5, A and B). Neither
ERK1 nor ERK2 antibodies cross-reacted with the recombinant gERK2 or
gERK1, respectively (data not shown). Immunoprecipitated ERK1 and ERK2
were subjected to in vitro kinase assays using MBP as the
substrate. Results presented in Figs. 4 (C and D)
and 5 (C and D) indicated that both endogenous
ERK1 and ERK2 were catalytically active and capable of phosphorylating MBP.

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Fig. 4.
Immunoblot analysis and kinase assay of
immunoprecipitated ERK1. A, Western blot analysis of
the recombinant ERK1 (2 µg) showing a single ~75-kDa band using
anti-gERK1 antibody (1:2500). B, Western blot analysis of
the native ERK1 in the crude trophozoite extract (50 µg) showing a
single polypeptide of 44 kDa at different dilutions of the antibody
(lane 1, 1:500; lane 2, 1:1000; lane
3, 1:2000, lane 4, 1:3000; lane 5, 1:4000;
lane 6, preimmune serum). C, panel 1,
Coomassie-stained SDS-PAGE of the kinase assay using immunoprecipitated
extract showing MBP and ERK1 polypeptides. Panel 2, autoradiogram of the kinase assay showing phosphorylated MBP.
D, Western blot of the immunoprecipitated extract showing
the ERK1 polypeptide.
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Fig. 5.
Immunoblot analysis and kinase assay of
immunoprecipitated ERK2. A, Western blot analysis of
the recombinant ERK2 (2 µg) showing a single ~47-kDa band using
anti-gERK2 antibody (1:6000). B, Western blot analysis of
the native ERK1 in the crude trophozoite extract (50 µg) showing a
single polypeptide of 41 kDa at different dilutions of the antibody
(lane 1, 1:5,000; lane 2, 1:10,000; lane
3, 1:15,000, lane 4, 1:20,000; lane 5,
1:25000; lane 6, 1:30,000; lane 7, preimmune
serum). C, panel 1, Coomassie-stained SDS-PAGE of
the kinase assay using immunoprecipitated extract showing MBP and ERK2
polypeptides. Panel 2, autoradiogram of the kinase assay
showing phosphorylated MBP. D, Western blot of the
immunoprecipitated extract showing the ERK2 polypeptide.
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Giardial ERK1 and ERK2 Showed Differential Subcellular
Localization--
To assess the functional role of giardial ERK1 and
ERK2 and whether they have overlapping functions, we monitored
intracellular distribution of ERK1 and ERK2 by immunofluorescence
analysis using primary antisera specific for gERK1 and gERK2 and
deconvolution restoration microscopy. Signals obtained for the ERK1
antibody indicated localization of ERK1 mostly in the flagellar basal
bodies and in the median body. Localization of ERK1 was also seen in the adhesive disc, the ventral groove, and along the caudal flagella (Fig. 6A). On the other hand,
signals for the ERK2 antibody were mostly at the cell membrane, at the
anterior and caudal flagella, and in the nuclei (Fig.
7A). In support of the studies
indicating that mammalian ERK1 and ERK2 are two microtubule-associated
proteins (34), the overall pattern of gERK1 and gERK2 localization to the specific intracellular structures is suggestive of their possible association with cytoskeletal structures.

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Fig. 6.
Immunofluorescence analysis of gERK1.
Immunofluorescence microscopy was employed to investigate whether there
was any change in the cellular localization of gERK1 during
encystation. Trophozoites were seeded onto coverslips and grown in
growth medium for 24 h before encystation was induced with
encystment medium. At the indicated time points the cells were fixed
and permeabilized. Distribution of gERK1 was detected using anti-gERK1
polyclonal antiserum (1:3,000) and anti-rabbit ALEXA Fluor-488
(green). Nuclei were stained with DAPI (blue).
A, trophozoites, BB, basal bodies; MB,
median body; AD, adhesive disc. B-D, encysting
cells at 2, 24, and 48 h. VG, ventral groove.
Inset, DIC and DIC plus fluorescence merge images of the
localization.
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Fig. 7.
Immunofluorescence analysis of gERK2.
Immunofluorescence microscopy was used to investigate whether there was
any change in the cellular localization of gERK2 during encystation.
Trophozoites were seeded onto coverslips and treated the same way as
mentioned in Fig. 6. Cells at the indicated time points were fixed and
permeabilized. Localization of gERK2 was detected using anti-gERK2
polyclonal antiserum (1:6000) and anti-rabbit ALEXA Fluor-488
(green). Nuclei were stained with DAPI (blue).
A, trophozoites; AF, anterior flagella;
CF, caudal flagella; N, nucleus. B,
C, and D, encysting cells at 2, 24, and 48 h. V, vesicles. Inset, DIC and DIC plus
fluorescence merge images of the localization.
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Altered Localization, Phosphorylation, and Catalytic Activity of
ERK1 and ERK2 during Encystation--
Giardia trophozoites undergo
remarkable structural changes during encystation that are associated
with altered intracellular events, such as disappearance of the median
body, cell cycle arrest, appearance of encystation-specific vesicles,
and increased synthesis and transport of cyst wall proteins. Because
ERK1 and ERK2 from higher eukaryotes are known to be involved in a
variety of cellular functions associated with cell proliferation and
cellular differentiation (35, 36), we set out to analyze intracellular
localization and catalytic activity of ERK1 and ERK2 at different
stages of encystation by immunofluorescence analysis and in
vitro kinase assays of the immunoprecipitated samples.
Trophozoites were treated with the encystment media and harvested at 2, 6, 10, 24, and 48 h. A distinct change in the localization of ERK2
in the encysting cells was seen as early as 2 h of encystation, in
which the antibody showed a punctate but even staining throughout the
cytoplasm. No staining in the nuclei or in the flagella by the antibody
was noted at 2, 24, and 48 h following exposure to the encystment media (Fig. 7, B-D). At 24 h, localization of ERK2 was
seen mostly toward the posterior end of the cell and surrounding the
large vesicle-like structures (Fig. 7C), which started to
appear around 10 h during encystation (data not shown). At 48 h, more cells with vesicles were present, and similar staining of ERK2
was seen in the cytoplasm and at the perivesicular region. On the other hand, a more subtle change in the localization of ERK1 was noted during
encystation, which includes lack of staining in the caudal flagella at
2 h onward and in the median body presumably because of the
disappearance of the flagella and the median body at the early stage of
encystation (Fig. 6, B-D). Also, accumulation of ERK1 in
the ventral groove was evident in encysting cells at 24 h. No
alteration in the staining of ERK1 in the adhesive disc and in the
flagellar basal bodies was noted in any stages of encystation. The
observation that ERK1 and ERK2 are targeted to different locations in
trophozoites and that localization of both proteins changes during
encystation suggests a possible functional difference between gERK1 and
gERK2 and important roles of ERK1 and ERK2 during giardial differentiation.
To understand the roles played by gERK1 and gERK2 during encystation,
we next monitored the catalytic activity of ERK1 and ERK2 in encysting
cells, as it is well known that signals for both cell proliferation and
differentiation are mediated through activation or inactivation of ERK1
and ERK2 (36, 37). Although immunoblot analysis indicated no
significant change in the expression of either ERK1 or ERK2 in
encysting cells at different stages of encystation (Figs.
8A, 9A, and
10C, panel a), altered activities of both kinases
were apparent when phosphorylation of MBP by the immunoprecipitated
enzymes was used to monitor the catalytic activity. At 2 h of
encystation, a ~2.5-fold increase in the activity of ERK1 was noted
which gradually declined during 6 and 10 h reaching the lowest
point at 24 h. At 48 h, the activity of ERK1 resumed back to
the level of uninduced cells (Fig. 8, B-D). Interestingly, the activity of ERK2 dropped significantly at 2 h and remained at
that level until 10 h. At 24 h, the activity of ERK2 in
encysting cells reached the minimum level but recovered back to the
uninduced level at 48 h, thus exhibiting a 5-8-fold increase in
the activity within 24 h (Fig. 9,
B-D). Also, it is noteworthy that the catalytic activity of
both ERK1 and ERK2 in encysting cells was minimum at 24 h when the
maximum number of cysts appeared in the culture medium.

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Fig. 8.
In vitro kinase assay with
immunoprecipitated gERK1. Trophozoites were allowed to encyst for
the times indicated, and gERK1 was immunoprecipitated from the crude
cell extracts. Immunoprecipitated ERK1 was used for in vitro
kinase assays. A, immunoblot analysis of the ERK1 in the
crude extract prior to immunoprecipitation showing the level of ERK1
expression. B, autoradiogram showing phosphorylation of MBP
by the immunoprecipitated extracts. C, Coomassie
Blue-stained SDS-PAGE of the kinase assay showing equal amounts of
added MBP. D, densitometric analysis of the phosphorylated
MBP by the immunoprecipitated ERK1 at different times of encystation.
Data represent mean ± S.D. of three separate experiments.
|
|

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Fig. 9.
In vitro kinase assay with
immunoprecipitated gERK2. Trophozoites were allowed to encyst for
the times indicated, and gERK2 was immunoprecipitated from the crude
cell extracts. Immunoprecipitated ERK2 was used for in vitro
kinase assays. A, immunoblot analysis of the ERK2 in the
crude extract prior to immunoprecipitation showing the level of ERK2
expression. B, autoradiogram showing phosphorylation of MBP
by the immunoprecipitated extracts. C, Coomassie
Blue-stained SDS-PAGE of the kinase assay showing equal amounts of
added MBP. D, densitometric analysis of the phosphorylated
MBP by the immunoprecipitated ERK2 at different times of encystation.
Data represent mean ± S.D. of three separate experiments.
|
|
Because the activity of ERK1 and ERK2 depends on the activating
phosphorylation of these two proteins, we set out to assess the
phosphorylation status of ERK1 and ERK2 at different stages of
encystation using a phospho-ERK antibody generated against a 20-amino
acids peptide antigen around the TXY motif of human p44
ERK1, which showed 65 and 52% identity with the same region of the
giardial ERK1 and 2, respectively. Immunoblot analysis revealed that
the concentration of phospho-ERK1 in encysting cells remained the same
as that of the untreated trophozoites until 10 h except for a
marginal increase at 2 h (Fig.
10C, panel b). A
significant decrease in the phospho-ERK1 concentration was noted in the
encysting cells at 24 h, which was possibly reflected into the
decreased kinase activity at that stage. The concentration of
phospho-ERK2 in encysting cells showed a slight decrease in 6 and
10 h but a dramatic drop at 24 h confirming the
minimum kinase activity as observed at that stage of encystation.
Although at a much lower intensity, the band corresponding to
phospho-ERK2 was detectable at 48 h representing an increased
phosphorylation of ERK2, while concentration of the phosphorylated ERK1
remained low at 48 h. The expression of cyst wall protein 1 (CWP1)
was used as a marker for the encystation process, which showed an increased expression of CWP1 at 10 and 24 h, as expected (Fig. 10C, panel c). Recognition of the phospho-gERK1 and -2 by
the anti-phospho-ERK antibody was validated by the treatment of the
crude trophozoite extracts with
-phosphatase, which resulted in
disappearance of the polypeptide bands corresponding to the
phospho-gERK1 and -2 (Fig. 10A). Furthermore, phospho-ERK
antibody did not recognize the recombinant gERK1 and -2 (Fig.
10B).

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Fig. 10.
Assessment of the concentration of
phospho-ERK1/ERK2 during encystation. A,
detection of the phospho-ERK1 and -2 and dephosphorylated ERK1 and -2 in crude trophozoite extracts (30 µg) with and without treatment with
phosphatase to determine the specificity of the phospho-ERK
antibody. B, Western blot analysis of the recombinant ERK1
and -2 using anti-phospho-ERK and anti-gERK1 and-gERK2 antibodies to
confirm that phospho-ERK antibody does not recognize dephosphorylated
ERK1 and 2. C, immunoblot analysis of the dephosphorylated
ERK1 and ERK2 using anti-ERK1 (1:4,000) and ERK2 (1:30,000) antibodies
together (panel a) and phosphorylated ERK1 and ERK2 using an
anti-phospho ERK antibody (1:100) (panel b) in the crude
extracts (50 µg) from encysting cells at different stages of
encystation. Expression of CWP1 protein (panel c) using
anti-CWP1 antibody (1:1 × 106) was used as a marker
for encystation.
|
|
 |
DISCUSSION |
ERK1 and ERK2 are two highly conserved ubiquitously expressed
enzymes in a variety of organisms and play a pivotal role in regulating
complex cellular processes in response to environmental cues. To
understand the functional role of MAPK in growth and differentiation of
Giardia trophozoites, we characterized giardial homologues
of ERK1 and ERK2. Sequence analysis indicated that over 50% identity
exists between gERK1/gERK2, and the orthologues from other model
organisms and the Giardia homologues contain all subdomains
that are conserved in other eukaryotes. However, a 10-amino acid
insertion of unknown significance is present in ERK1 at the activation
domain before the TXY motif, which is not shared by other
ERK1 or ERK2 from different species (Figs. 1A and
2A). Computer-based three-dimensional modeling indicated
distinct differences in the structures of giardial ERK1/2 when compared with the mammalian ERK1/2, although the overall structures were the
same (Fig. 1, B and C, and Fig. 2, B
and C). Both recombinant and native ERK1 and ERK2 were
catalytically active and phosphorylated myelin basic protein, a widely
used substrate for ERK1 and ERK2 in assay systems. Thus, the structural
similarity and substrate specificity confirm that the cloned ORFs are
of gERK1 and gERK2. In addition, search of the large scale
Giardia genome sequencing data base (www.mbl.edu/Giardia/)
revealed the presence of putative homologues of giardial MEK and MEKK
(data not shown) suggesting the possibility of the presence of the
three-kinase module of MAPK pathway in Giardia. The presence
of ERK1 and ERK2 genes in Giardia, a primitive eukaryote
that is believed to be the earliest diverging member of the eukaryotic
line of descent, raises the enticing possibility that they might play
an important role in giardial growth and differentiation.
Analysis of the kinetic parameters of the recombinant ERK1 and ERK2
indicated that ERK2 was more efficient in phosphorylating MBP than
ERK1, although the immunoprecipitated ERK1 phosphorylated MBP with
apparently similar efficiency. It is possible that the full activation
of gERK1 requires the activating phosphorylation at the TXY
motif by MEK, which could be partially achieved by the recombinant ERK2
through autophosphorylation. Both gERK1 and gERK2 required low amounts
of Mg2+ as the divalent cation. MnCl2 and a
higher concentration of MgCl2 completely inhibited
phosphorylation by both kinases (data not shown).
Because ERK1 and ERK2 phosphorylate a variety of proteins,
intracellular localization of these kinases defines their functional specificity. Accordingly, the precise distribution of ERK1 in the basal
bodies, the median body, and in the adhesive discs may signify a
possible interaction of ERK1 with proteins associated with these
structures. Basal bodies of the flagellated cells are rod-like
structures that correspond to the centrosomes of the higher eukaryotic
cells and are associated with origins of the flagellar axonemes. In
flagellated cells, basal bodies are involved in flagellar motility;
therefore, localization of ERK1 into basal bodies presumably reflect
its potential association with microtubule-associated proteins. This
assumption is based on the findings that the association of mammalian
ERK1 and ERK2 with the microtubule-binding protein tubulin played an
important role in the spindle assembly checkpoint (38), the mammalian
ERK interacts with the spindle microtubule motor CENP-E during mitosis
(39), and that the active ERK associates with microtubules and
regulates microtubule stability in fibroblasts and breast epithelial
cells (40). It is possible that gERK1 may associate with tubulins in
the median body and adhesive discs for the maintenance of microtubule
stability. Staining with the gERK2 antibody showed a distinctly
different pattern of gERK2 localization, in particular to the cell
membrane, anterior and caudal flagella, and presumably to the nuclei,
which suggests a possible interaction of gERK2 with
microtubule-associated proteins in cytoskeletal structures different
from what was observed with gERK1. It is well documented that mammalian
ERK1 and -2 translocate to the nucleus upon activation where they
phosphorylate specific transcription factors (16, 41). So far no
information is available on the specific transcription factors in
Giardia that are phosphorylated by ERK. Detailed studies are
required to understand the function of the subset of ERK1 and ERK2
targeted to various cytoskeletal structures and to the nuclei in
Giardia.
Analysis of the distribution of ERK1 and ERK2 in encysting cells
revealed an evident relocation of ERK2 in the cytoplasm. Redistribution
of ERK2 was noticeable as early as 2 h of encystation, which did
not change significantly in the latter part of encystation, except for
its localization around the perivesicular region of the large
vesicle-like structures that were present in the encysting cells at 24 and 48 h. On the other hand, localization of ERK1 did not alter
significantly except for the high intensity staining by the antibody in
the ventral groove area at 24 h. The ventral groove is a shallow
region, which extends from the adhesive disc to the caudal region of
the body and is the site of origin of the ventral flagellar pair. It is
believed that the ventral groove generates the force to develop a
negative pressure inside the adhesive disc for the attachment of the
parasite (14, 42). The rationale for the accumulation of a subset of
ERK1 in this area is unclear. The disappearance of ERK1 staining in the
median body at 24 and 48 h was due to disorganization of the
median body during encystation. Interestingly, a parallel fluctuation
in the kinase activity of the endogenous ERK2 and ERK1 was noted during encystation. A significantly reduced activity of ERK2 was maintained up
to 24 h as long as the cells were exposed to the encystation medium. Interestingly, upon replacement of the encystation medium with
the regular growth medium, a 5-8-fold increase in the activity was
noted within 24 h. The activity of ERK1 instead was biphasic with
an initial ~2.5-fold increase followed by a gradual decline until
24 h. Upon replacement of the regular medium at 24 h, the activity was restored to the level of uninduced cells. It is possible that the initial increase in the ERK1 activity may be required for the
initiation but not for the maintenance of encystation. Also a
parallel decrease in the concentration of phospho-ERK1 and -ERK2 at
24 h suggests that the activating phosphorylation of ERK1 and ERK2
might be necessary for the complete activation of giardial ERK1 and
ERK2 as observed in mammalian ERK1/2.
The results obtained from this study also suggest that the high bile
concentration in the encystation medium, which may induce cholesterol
starvation, may be a factor that indirectly prevents ERK1 and ERK2
activation. However, it is interesting that the exposure to a high bile
concentration and thereby low cholesterol concentration generated
signals that may have different effects on the activation of ERK1 and
ERK2. This finding also suggests the possibility that the function of
giardial ERK1 and ERK2 may be regulated through different mechanisms.
Differential phosphorylation of ERK1 and ERK2 by the
PCPH oncogene has been reported in 293T cells (43).
It was proposed that in these cells, ERK1 activation might have been
mediated through a Ras/MEK-independent pathway (43). Although
Giardia homologues of MEK and MEKK have been identified, it
is not known which signaling pathway(s) activates ERK1 and ERK2 in
trophozoites and in encysting cells. In depth studies are needed to
elucidate the regulation of activation of giardial ERK1/ERK2 during encystation.
The first step of encystation is the induction of expression of CWP
proteins, which is triggered by the depletion of cholesterol. It has
been shown that in higher eukaryotes, cholesterol homeostasis is
maintained by the transcription activation of various genes through
activation of transcription factors, SREBP1a and SREBP2, that bind to
the sterol-response element (SRE) (44). The promoter of CWP1 and CWP2
contains degenerated SRE, and it has been hypothesized that CWP
expression might be induced by interaction with the SRE-binding proteins (SREBP) (44). Importantly, recent studies (45) have shown that
mammalian SREBP1a and SREBP2 are activated by ERK1 and ERK2, and their
activation may be linked to the MAPK cascade. It is possible that the
activation of CWP gene transcription during encystation is mediated
through SREBP, which is in turn activated by ERK1. Recently, the
observation that Giardia trophozoites express a cell-surface
cholesterol-sensing receptor, receptor-Ck, and SREBP (26),
suggests that a sterol-regulated signaling pathway might be present in
Giardia. So far, no information on the function of giardial
homologues of SREBP is available. Further study in this area will shed
light on the involvement of ERK1 in transcription of CWP genes.
Based on our observations, we speculate that inactivation of ERK2 was
necessary for the formation of cysts, whereas an increased activity of
ERK1 was a prerequisite for the encystation process to begin.
Phosphorylation status of ERK1 and ERK2 correlated best with the
activity profile at 24 h supporting the conjecture of inactivation
of ERK1 and ERK2 during encystation. However, despite the fact that the
activity of ERK1 and ERK2 was back to the original level in cells at
48 h upon maintaining them in TYI-S-33 media for the last 24 h, no significant change in the localization of ERK1 or ERK2 was noted
in encysting cells except for the lack of accumulation of ERK1 in the
ventral groove. Functional significance of the differential
distribution and activity of ERK1 and ERK2 during encystation
remains to be determined.
 |
FOOTNOTES |
*
This work was supported by the Office of Research and
College of Health and Public Affairs, University of Central Florida.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.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Microbiology, University of Central Florida, Biomolecular Research Annex, 12722 Research Pkwy., Orlando, FL 32826-2362. Tel.:
407-384-2187; Fax: 407-384-2062; E-mail:
rchak@pegasus.cc.ucf.edu.
Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M209274200
 |
ABBREVIATIONS |
The abbreviations used are:
CWP, cyst wall
proteins;
MAPK, mitogen-activated protein kinase;
ERK, extracellular
signal-regulated kinase;
MBP, myelin basic protein;
ORF, open reading
frame;
DAPI, 4,6-diamidino-2-phenylindole;
PBS, phosphate-buffered
saline;
SRE, sterol-response element;
SREBP, SRE-binding protein;
DIC, differential interference contrast.
 |
REFERENCES |
1.
|
Marshall, M. M.,
Naumovitz, D.,
Ortega, Y.,
and Sterling, C. R.
(1997)
Clin. Microbiol. Rev.
10,
67-85[Abstract]
|
2.
|
Thompson, R. C.,
Reynoldson, J. A.,
and Mendis, A. H.
(1993)
Adv. Parasitol.
32,
71-160[Medline]
[Order article via Infotrieve]
|
3.
|
Gillin, F. D.,
Reiner, D. S.,
and McCaffery, J. M.
(1996)
Annu. Rev. Microbiol.
50,
679-705[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Kane, A. V.,
Ward, H. D.,
Keusch, G. T.,
and Pereira, M. E. A.
(1991)
J. Parasitol.
77,
974-981[Medline]
[Order article via Infotrieve]
|
5.
|
Lujan, H. D.,
Mowatt, M. R.,
Byrd, L. G.,
and Nash, T. E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7628-7633[Abstract/Free Full Text]
|
6.
|
Lujan, H. D.,
Mowatt, M. R.,
and Nash, T. E.
(1998)
Parasitol. Today
14,
446-450[CrossRef]
|
7.
|
Lujan, H. D.,
Mowatt, M. R.,
Conrad, J. T.,
Bowers, B.,
and Nash, T. E.
(1995)
J. Biol. Chem.
270,
29307-29313[Abstract/Free Full Text]
|
8.
|
Jarroll, E. L.,
Macechko, P. T.,
Steinle, P. A.,
Bulik, D.,
Karr, C. D.,
van Keulen, H.,
Paget, T. A.,
Gerwig, G.,
Kamerling, J.,
Vliegenthart, J.,
and Erlandsen, S.
(2001)
J. Eukaryotic Microbiol.
48,
22-26[Medline]
[Order article via Infotrieve]
|
9.
|
Mowatt, M. R.,
Lujan, H. D.,
Coten, D. B.,
Bowers, B.,
Yee, J.,
Nash, T. E.,
and Stibbs, H. H.
(1995)
Mol. Microbiol.
15,
955-963[Medline]
[Order article via Infotrieve]
|
10.
|
Lujan, H. D.,
Marotta, A.,
Mowatt, M. R.,
Sciaky, N.,
Lippincott-Schwartz, J.,
and Nash, T. E.
(1995)
J. Biol. Chem.
270,
4612-4618[Abstract/Free Full Text]
|
11.
|
Reiner, D. S.,
McCaffery, M.,
and Gillin, F. D.
(1990)
Eur. J. Cell Biol.
53,
142-153[Medline]
[Order article via Infotrieve]
|
12.
|
Erlandsen, S. L.,
Macechko, P. T.,
Van Keulen, H.,
and Jarroll, E.
(1996)
J. Eukaryotic Microbiol.
43,
416-429[Medline]
[Order article via Infotrieve]
|
13.
|
Hehl, A. B.,
Marti, M.,
and Kohler, P.
(2000)
Mol. Biol. Cell
11,
1789-1800[Abstract/Free Full Text]
|
14.
|
Adam, R. D.
(2001)
Clin. Microbiol. Rev.
14,
447-475[Abstract/Free Full Text]
|
15.
|
Bernander, R.,
Palm, J. E.,
and Svard, S. G.
(2001)
Cell. Microbiol.
3,
55-62[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Widmann, C.,
Gibson, S.,
Jarpe, M. B.,
and Johnson, G. L.
(1999)
Physiol. Rev.
79,
143-180[Abstract/Free Full Text]
|
17.
|
Canagarajah, B. J.,
Khokhlatchev, A.,
Cobb, M. H.,
and Goldsmith, E. J.
(1997)
Cell
90,
859-869[Medline]
[Order article via Infotrieve]
|
18.
|
Gartner, A.,
Nasmith, K.,
and Ammerer, G.
(1992)
Genes Dev.
6,
1280-1292[Abstract]
|
19.
|
Whitmarsh, A. J.,
and Davis, R. J.
(1996)
J. Mol. Med.
74,
589-607[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Tian, J.,
and Karin, M.
(1999)
J. Biol. Chem.
274,
15173-15780[Abstract/Free Full Text]
|
21.
|
Paumelle, R.,
Tulashe, D.,
Kherrouche, Z.,
Plaza, S.,
Leroy, C.,
Reveneau, S.,
Vandenbunder, B.,
and Fafeur, V.
(2002)
Oncogene
21,
2309-2319[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Roth, G.,
Kotzka, J.,
Kremer, L.,
Lehr, S.,
Lohaus, C.,
Meyer, H. E.,
Krone, W.,
and Muller-Wieland, D.
(2000)
J. Biol. Chem.
275,
33302-33307[Abstract/Free Full Text]
|
23.
|
Hua, S.-B.,
and Wang, C. C.
(1997)
J. Biol. Chem.
272,
10797-10803[Abstract/Free Full Text]
|
24.
|
Ruben, L.,
Kelly, J. M.,
and Chakrabarti, D.
(2002)
in
Molecular and Medical Parasitology
(Marr, J.
, Nilsen, T.
, and Komuniecki, R., eds)
, pp. 241-276, Academic Press, New York
|
25.
|
Keister, D. B.
(1983)
Trans. R. Soc. Trop. Med. Hyg.
77,
487-488[Medline]
[Order article via Infotrieve]
|
26.
|
Kaul, D.,
Rani, R.,
and Shegal, R.
(2001)
Mol. Cell. Biochem.
225,
167-169[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Smith, M. W.,
Aley, S. B.,
Sogin, M.,
Gillin, F. D.,
and Evans, G. A.
(1998)
Mol. Biochem. Parasitol.
95,
267-280[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Abel, E. S.,
Davids, B. J.,
Robles, L. D.,
Loflin, C. E.,
Gillin, F. D.,
and Chakrabarti, R.
(2001)
J. Biol. Chem.
276,
10320-10329[Abstract/Free Full Text]
|
29.
|
Combet, C.,
Jambon, M.,
Deleage, G.,
and Geourjon, C.
(2002)
Bioinformatics
18,
213-214[Abstract/Free Full Text]
|
30.
|
Sambrook, J.,
Fritsch, E, F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, pp. 7.37-9.57, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
|
31.
|
Chakrabarti, R.,
McCracken, J. B., Jr.,
Chakrabarti, D.,
and Souba, W. W.
(1995)
Gene (Amst.)
153,
163-199[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Dorin, D.,
Alano, P.,
Boccaccio, I.,
Ciceron, L.,
Doerig, C.,
Sulpice, R.,
Parzy, D.,
and Doerig, C.
(1999)
J. Biol. Chem.
274,
29912-29920[Abstract/Free Full Text]
|
33.
|
Antonsson, B.,
Marshall, C. J.,
Montessuit, S.,
and Arkinstall, S.
(1999)
Anal. Biochem.
267,
294-299[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Haycock, J. W.,
Ahn, N. G.,
Cobb, M. H.,
and Krebs, E. G.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
2365-2369[Abstract]
|
35.
|
Zhang, W.,
and Liu, H. T.
(2002)
Cell Res.
12,
9-18[Medline]
[Order article via Infotrieve]
|
36.
|
Luongo, D.,
Mazzarella, G.,
Della, R. F.,
Maurano, F.,
and Rossi, M.
(2002)
Mol. Cell. Biochem.
231,
43-50[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Eriksson, M.,
and Leppa, S.
(2002)
J. Biol. Chem.
277,
15992-16001[Abstract/Free Full Text]
|
38.
|
Wang, X. M.,
Zhai, Y.,
and Ferrell, J. E., Jr.
(1997)
J. Cell Biol.
137,
433-443[Abstract/Free Full Text]
|
39.
|
Zecevic, M.,
Catling, A. D.,
Eblen, S. T.,
Renzi, L.,
Hittle, J. C.,
Yen, T. J.,
Gorbsky, G. J.,
and Weber, M. J.
(1998)
J. Cell Biol.
142,
1547-1558[Abstract/Free Full Text]
|
40.
|
Harrison, R. E.,
and Turley, E. A.
(2001)
Neoplasia
3,
385-394[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Liu, F.,
Austin, D. A.,
Mellon, P. L.,
Olefsky, J. M.,
and Webster, N. J.
(2002)
Mol. Endocrinol.
16,
419-434[Abstract/Free Full Text]
|
42.
|
Erlandersen, S. L.,
and Feely, D. E.
(1984)
in
Giardia and Giardiasis: Biology, Pathogenesis, and Epidemiology
(Erlandersen, S. L.
, and Meyer, E., eds)
, pp. 33-63, Plenum Publishing Corp., New York
|
43.
|
Recio, J. A.,
Paez, J. G.,
Maskeri, B.,
Loveland, M.,
Velasco, J. A.,
and Notario, V.
(2000)
Cancer Res.
60,
1720-1728[Abstract/Free Full Text]
|
44.
|
Kim, H. J.,
Miyazaki, M.,
Man, W. C.,
and Ntambi, J. M
(2002)
Ann. N. Y. Acad. Sci.
967,
34-42[Abstract/Free Full Text]
|
45.
|
Kotzka, J.,
Muller-Wieland, D.,
Roth, G.,
Kremer, L.,
Munck, M.,
Schurmann, S.,
Knebel, B.,
and Krone, W.
(2000)
J. Lipid Res.
41,
99-108[Abstract/Free Full Text]
|
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