From the Department of Molecular Biology and
Microbiology, University of Central Florida, Orlando, Florida 32826 and the § Department of Pathology, Division of Infectious
Diseases, University of California, School of Medicine,
San Diego, California 92103
Received for publication, July 24, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Since little is known of how the primitive
protozoan parasite, Giardia lamblia, senses and responds to
its changing environment, we characterized a giardial protein kinase A
(gPKA) catalytic subunit with unusual subcellular localization.
Sequence analysis of the 1080-base pair open reading frame shows 48%
amino acid identity with the cyclic AMP-dependent kinase
from Euglena gracilis. Northern analysis
indicated a 1.28- kilobase pair transcript at relatively constant
concentrations during growth and encystation. gPKA is
autophosphorylated, although amino acid residues
corresponding to Thr-197 and Ser-338 of human protein kinase A
(PKA) that are important for autophosphorylation are absent. Kinetic
analysis of the recombinant PKA showed that ATP and magnesium are
preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A
myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an
IC50 of 3 µM, suggesting an important role of
gPKA during differentiation from the dormant cyst form into the active
trophozoite. gPKA localizes independently of cell density to the eight
flagellar basal bodies between the two nuclei together with centrin, a
basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and
caudal pairs of flagella was evident only at low cell density and
higher endogenous cAMP concentrations or after refeeding with fresh
medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.
Infection with Giardia lamblia is a major global cause
of water-borne diarrheal disease (1). Nonetheless, neither its basic biology nor the pathophysiology of infection is well understood. To
date, no giardial toxin or conventional virulence factor has been
identified by biological (2) or genomic studies (3). Therefore, it is
critical to understand how the parasite survives in the external
environment and infects and colonizes a new host. G. lamblia
has two life cycle stages that are each remarkably well adapted to
survival in very different and inhospitable environments. The dormant,
quadrinucleate cyst persists for months in fresh cold water (4).
Infection is initiated by ingestion of cysts (5). Exposure of cysts to
gastric acid during passage through the host stomach triggers the rapid
and dramatic differentiation known as excystation. After entry into the
small intestine and stimulation by specific factors (6), the parasite
emerges and divides into two equivalent binucleate trophozoites that
attach to and colonize the human small intestine. The cyst wall must remain intact during passage through the stomach acid; however, once
the cyst enters the small intestine, the wall must open rapidly to
enable the parasite to emerge and attach to host enterocytes.
In the small intestine, giardial trophozoites are exposed to complex
and everchanging concentrations of hydrogen ions and nutrients, as well
as bile acids and digestive enzymes (7). As enterocytes migrate to the
tip of the villus, where they are sloughed off into the lumen, attached
trophozoites must be able to sense and respond rapidly to environmental
signals to remain in the small intestine. Since excystation entails
such rapid responses to environmental stimuli, we hypothesized that
protein kinase-mediated signaling might be very important. Thus,
cell-signaling pathways may be crucial to both giardial colonization in
the small intestine and to the cellular activation of excystation.
Although many protein kinase gene fragments have been identified by
large scale genomic sequencing (Ref. 8; see also the web site for the
Giardia Genome Project Database (Marine Biological
Laboratory, Woods Hole, MA)), there is little understanding of
their potential roles in giardial survival and growth in the small
intestine or in regulation of differentiation.
In many eukaryotic cells, cyclic AMP (cAMP)-dependent
signaling pathways play a critical role in regulating cell growth,
metabolism, and differentiation (10, 11). Protein kinase A
(PKA,1 EC 2.7.1.37), the
defining enzyme of the cyclic AMP-dependent signaling
pathway alters protein activity by phosphorylation at serine/threonine
residues within the motif RRX(S/T) (12). PKA is one of the
simplest protein kinases known because of its dissociative mechanism of
activation (13). PKA holoenzyme, a catalytically inactive form, is a
tetrameric complex containing two identical catalytic (C) subunits
bound to a homodimer of two regulatory (R) subunits (14). Cyclic AMP
binding to the R subunit results in dissociation of the holoenzyme with
release of active C subunit. The activated C subunit of PKA can
phosphorylate a number of intracellular proteins, including enzymes,
cytoskeletal proteins, ion channels, and transcription factors (15).
How the specific physiological effects of cAMP can be mediated by a
broad spectrum protein kinase such as PKA is now being elucidated (16,
17). In a number of diverse cell types, PKA is localized in proximity
to substrates by A kinase anchoring protein(s) that bind to both the R
subunit and to a specific cell structure, often cytoskeletal (18).
Targeting PKA to the proximity of phosphate acceptor protein molecules
associated with specific cell structures can also achieve rapid
responses to cAMP (16, 19).
Genes encoding three different C subunits and four different R subunits
of PKA have been identified in humans (20). In higher eukaryotes,
various combinations of R and C subunits display differences in tissue
distribution (21-23), which may determine their functional specificity. The unique distribution and subcellular localization of
PKA isoforms in higher eukaryotes may represent a functional interaction with nearby structures. In lower eukaryotes, three isoforms
of PKA C subunit have been identified from Drosophila melanogaster (24), and from Saccharomyces cerevisiae
(25). However, only one gene encoding PKA C subunit has been reported from Caenorhabditis elegans, Dictyostelium
discoidium, Leishmania major, and
Trypanosoma cruzi (26-29). Dictyostelium PKA
plays a key role during differentiation and morphogenesis (30).
Similarly, PKA plays critical roles in differentiation of
Leishmania and Trypanosoma (28, 29). In contrast,
Saccharomyces PKA appears to mediate cellular responses to
various extracellular stimuli, including nutrients and heat shock
(31).
Here we report the cloning and functional characterization of a
giardial homologue of a PKA C subunit. Thus far, only a single isoform
of gPKA C subunit has been identified in large scale sequencing efforts
with ~3-fold coverage of the genome (32). Our studies demonstrate
that, although the biochemical properties of the recombinant gPKA are
largely similar to those of the mammalian enzyme, gPKA has some
noteworthy structural differences. We found that cellular distribution
of PKA was correlated with cell density and endogenous cAMP levels. At
all cell densities, PKA localized to the eight flagellar basal bodies,
which correspond to the centrosomes of higher eukaryotic cell (33).
However, at low cell inocula and higher endogenous cAMP concentrations,
PKA also localized to marginal plates along the intracellular portions
of the anterior and caudal flagella. To our knowledge, this is the
first indication of a role for cell signaling in giardial
flagellar-associated structures. In addition, a PKA inhibitor greatly
decreased giardial excystation. Thus, PKA may play a key role in
regulating trophic behavior of the motile form, as well as cellular
activation during excystation.
Materials--
Unless specified, all materials were obtained
from Sigma, Fisher, and Life Technologies, Inc.
Giardia Cell Culture and Differentiation--
G.
lamblia (WB clone C6, ATCC no. 50803) were maintained in TYI-S-33
medium with 10% bovine serum and bile (34). Encystation and
excystation were induced essentially as described by Meng et
al. (35). Briefly, encystation was induced by growing trophozoites for one culture cycle in TYI-S-33 medium without bile
(pre-encystation). Bile-deficient medium was poured off along with
unattached trophozoites and replaced with encystation medium containing
0.25 mg/ml porcine bile and 10 mM lactic acid, pH 7.8, and
incubated at 37 °C for the time specified. Total encysting cultures
were harvested at different time points by chilling and centrifugation
and subsequently used for RNA and protein extraction. Cysts were
harvested at 66 h by washing and incubating in cold
double-distilled water to lyse any trophozoites or incomplete cysts
(35). Excystation was induced by a two-step method that models cyst
passage from the cold hypotonic freshwater external environment into
the warm, acidic stomach, pH 4.0, 20 min, 37 °C (stage 1). Stage 2 models cyst passage from the stomach into the small intestine with
exposure to slightly alkaline pH (8.0) and protease (1 mg/ml bovine
trypsin type II) for 1 h at 37 °C. Cysts were pelleted at
8300 × g and resuspended in growth medium for 60 min
at 37 °C. Emerged motile trophozoites were enumerated using a
hemocytometer. The percentage of excystation was calculated as the sum
of the motile trophozoites and partially emerged trophozoites divided
by the initial number of viable cysts.
Inhibition of Excystation--
Myristoylated PKA inhibitor
(amide 14-22, Calbiochem Novobiochem, San Diego, CA) was dissolved in
water and diluted into water (pre-excystation) or excystation
solutions. Inhibitor peptide was initially screened with the inhibitor
present during 1 h before incubation at 4 °C and added again
during stage 1 and 2 of excystation, since cysts were pelleted between
stages. However, inhibitor was not included in the emergence step in
growth medium because of possible effects on trophozoites. 50%
inhibitory concentration (IC50) was estimated by
interpolation from concentration-inhibition curves. Viability was
determined by trypan blue exclusion (35). To determine the stage(s) at
which each inhibitor was effective, cysts were pre-incubated with
inhibitor (at a higher than IC50 concentration, 10 µM) prior to stage 1 in water for 1 h at 4 °C, or
during stage 1 or stage 2 of excystation.
The mean inhibition was normalized to solvent controls for each
experiment. All experiments were repeated at least twice. Data shown
are mean (± S.D.) for at least two independent experiments. The
p values were calculated by paired two-tailed Student's
t test with the normalized comparison value set at 100% for
control excystation (35).
PCR Amplification--
A 250-base pair DNA fragment of PKA
catalytic subunit was obtained by PCR amplification of
Giardia genomic DNA using degenerate primers (upper primer,
5'-TAC(T)A(C)GXGAC(T)C(T)TXAAA(G)C(T)C-3'; lower primer,
5'-CCXAA(G)XG(C)A(C)(T)CCACCAA(G)TC-3', where
X is all four nucleotides). Primers were designed on the
basis of the consensus sequence in the catalytic subdomains VIB and IX of the PKA catalytic subunit. Designed degeneracy allowed every possible codon for the consensus amino acid residues to maximize the
probability of amplification. The amplified PKA fragment was purified,
sequenced, and used to screen a Giardia cDNA library.
Screening of a cDNA Library--
A G. lamblia WB
clone C6 cDNA library was made in DNA Sequencing and Identity Analysis--
A capillary
electrophoresis-based automated DNA sequencer (ABI Prism, 310 Genetic
analyzer; ABI, Foster City, CA) was used to sequence positive clones
from the cDNA library. A PCR-based reaction kit (ABI
PrismTM dRhodamine Terminator Cycle Sequencing Ready
Reaction), ABI prism 310 capillaries (61 cm × 50 µm, inner
diameter) and POP-6 for long-read sequencing were used according to the
manufacturer's protocol. Raw output was analyzed using ABI Prism
sequence analysis software. The entire 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.
Tertiary structure was determined using SWISS-MODEL and Swiss-Pdb
Viewer, an internet-based tool for automated comparative protein
modeling (37, 38).
Southern Blot and Northern Blot--
Genomic DNA from
Giardia was isolated using standard protocol as described by
Maniatis et al. (39). Total RNA was extracted using an RNA
isolation kit (Stratagene) according to the manufacturer's specification. 32P-Labeled gPKA cDNA probes were used
for hybridization as described previously (36). For Northern blots, a
strand-specific probe was synthesized and used as a probe for gPKA.
Briefly, 25-50 ng of the template DNA was labeled using 100 pmol of
antisense primer, [32P]dCTP, dNTP, and Klenow (exo Expression and Purification of Recombinant gPKA--
The gPKA
ORF was cloned into a T7 polymerase-driven expression vector
pET30Ek/LIC (Novagen, Madison, WI) according to the manufacturer's
protocol. Two primers (upper primer,
5'-GACGACGACAAGATGGAAGATATTCAAGTA-3'; and lower primer,
5'-GAGGAGAAGCCCGGTCTATTTAAATCCCAC-3') were designed to amplify the PKA
ORF (1.08 kb) and part of the vector sequence. The PCR product was
treated with T4 DNA polymerase and dATP only to use the exonuclease
activity of the polymerase to generate vector compatible overhangs.
Pre-digested vector and insert were annealed and used to transform BL21
(DE3) Escherichia coli cells. Induction of gPKA
overexpression in BL21 (DE3) was performed at 25 °C with 0.5 mM isopropyl-1-thio- Western Blot and Immunodepletion Experiment--
Trophozoites
and encysting cells were harvested, and total proteins (50 µg) were
used for the immunodetection of gPKA using the polyclonal antibody made
against recombinant protein. A chemiluminescence detection kit (Pierce)
was used to detect positive signals recognized by anti-gPKA antibody
using anti-rabbit HRP-conjugated secondary antibody.
Immunodepletion was done in a two-step process. Crude trophozoite
extracts (500 µg) in 100 mM Tris, pH 7.5 and 5 mM MgCl2 were diluted in TAN buffer (10 mM Tris acetate, pH 8.0, 1% Nonidet P-40, 100 mM NaCl) containing 1 mM sodium orthovanadate,
1 mM phenylmethylsulfonyl fluoride, 2 mM
n-ethylmalemide, 1 µg/ml leupeptin, and 2 µg/ml
aprotinin. Ten µg of rabbit IgG was added to the extract and
incubated for 2 h at 4 °C to remove nonspecific binding of proteins to gPKA IgG. Next, 150 µl of Protein A-agarose beads (50%
slurry) was added and incubated overnight at 4 ° C to remove rabbit
IgG. Protein A beads were separated next by centrifuging at 1,500 × g for 5 min at 4 °C. The supernatant was incubated with 50 µg of gPKA antibody for 6 h at 4 °C followed by 200 µl of Protein A beads for 18 h at 4 °C. Protein A beads were
separated by centrifugation, and the supernatant was used for the
kinase assay.
Assay of PKA Activity--
Trophozoites were grown to
confluence, harvested at 700 × g, and washed three
times with PBS. They were lysed in a medium containing 100 mM Tris, pH 7.5, and 5 mM MgCl2 and
a mixture of protease inhibitors (Sigma) using a French press (750 p.s.i.). The lysate was centrifuged at 30,000 × g, and
the supernatant was passed through a syringe filter (0.45 µm). The
filtrate was used for the kinase assay using kemptide
(Calbiochem-Novobiochem Corp) as a substrate. Fifty µg of crude
extract or purified recombinant gPKA (5 µg) was used in a reaction
mix containing 100 mM Tris, 5 mM
MgCl2, 0.2 mM ATP, 0.2 mM kemptide,
and 0.5 nM [ Immunofluorescence Analysis--
G. lamblia
trophozoites were inoculated in growth medium at 350-2000 cells/ml in
12-well tissue culture plates and allowed to grow and attach to 18-mm
glass coverslips for 65-70 h in an AnaeroPack jar (Mitsubishi Gas
Chemical Company, Inc., Japan) at 37 °C. Coverslips containing
attached trophozoites were directly fixed in 100% chilled MeOH
( cAMP Measurements--
To measure endogenous cAMP levels in
various stages of G. lamblia growth, a competitive cAMP
enzyme immunoassay (Biotrack Cellular Communications Assays, Amersham
Pharmacia Biotech) was employed. G. lamblia were cultured as
described for immunolocalization. Using the nonacetylated protocol 3 method of the Biotrack enzyme immunoassay, parasites were lysed in a
500-µl volume of lysis reagent and the amount of cAMP was determined
by the manufacturer's protocol. The enzyme immunoassay was repeated
five times with parasites from independent cultures, and cAMP
measurements were done in duplicate for each sample. Mean differences
in the amount of cAMP levels between different cell densities were
compared using a paired Student's t test, and p
values Sequence and Expression Analyses of gPKA--
Upon screening a
Giardia cDNA library with a 250-base pair probe derived
from conserved PKA sequences, a 1.28-kb fragment containing an ORF of
1.08 kb (360 amino acids) corresponding to a Giardia
homologue of PKA catalytic subunit was cloned (Fig. 1). An unusually long 189-base pair
3'-untranslated region was present in the cloned fragment (accession
no. AF181097).
The translated amino acid sequence of the ORF showed 48% identity with
E. gracilis, 45% identity with C. elegans, 43%
identity with human, and 41% identity with yeast cyclic
AMP-dependent PKA catalytic subunit with probability scores
between 3e-77 and 2e-69 as obtained from PSI-BLAST homology search of
the NCBI data base. Since the serine-threonine kinase family exhibits a
significant degree of conservation of the kinase domains, the gPKA ORF
also showed limited homology with a number of other serine threonine kinases, such as cGMP-dependent protein kinases. However,
probability scores (starting at 2e-57 for human KGPA) were
significantly lower, and gPKA does not have a cyclic GMP-binding domain.
The identification of the ORF as a PKA homologue was strengthened by
the presence of most of the key functional motifs, as predicted by
comparison with the crystal structure of bovine PKA C subunits (PKAc)
(42). The amino acid sequence analysis of gPKA shows the presence of
all 11 subdomains of the kinase domain of PKAc (Fig. 1). Because of its
early evolutionary position (43), giardial genes are frequently highly
divergent (8). Despite the lower degree of identity, gPKA exhibits the
important consensus motifs, which include the glycine-rich consensus
motif GXGXXGXV (Gly-24 to Val-31 using
the amino acid numbers of the giardial sequence) in subdomain I, the
APE (Ala-190 to Glu-192) motif in subdomain VIII and the catalytic loop
YRDLKXXN (Tyr-138 to Asn-145) in subdomain IV. The glycine
loop is involved in interactions with ATP
Analyses of Southern blots and large scale genomic sequence (32)
suggests that gPKA is a single-copy gene. Northern and Western analyses
showed that gPKA was expressed apparently at a constant concentration
during both growth and encystation as a 1.3-kb transcript and a 41-kDa
protein (data not shown).
gPKA Has a Predicted Additional Loop in the Activation
Domain--
Using an internet-based protein modeling program (37, 38),
we compared the three-dimensional structure of gPKA to the
The other major difference is the large loop in the activation domain
(subdomain VIII shown in red in Fig. 2 (A,
C, and D)). A unique stretch of 10 amino acids
from Asn-169 to Leu-178, is predicted to form an additional loop
creating a distinct change in the conformation of the activation domain
(Fig. 2, C and D). Specifically, Ser-171 (Thr-197
in the template) does not form H-bonds with Lys-153 (Lys-189) and
Thr-169 (Thr-195), which are present in the crystal structure of the
1YDS template. These ion pairs may stabilize the subdomain VIII in an
active conformation, thereby permitting proper orientation of the
substrate peptide. The presence of several H-bonds in the extra loop
indicates a highly stable configuration, which may interfere with the
binding of the PKI inhibitor. The Mg2+ binding domain DFG
(Asp-184 to Gly-186), although replaced by DLG (Asp-148 to Gly-150) in
gPKA shows a similar conformation with H-bond formation between Leu-149
and Ala-152 (Fig. 2C). Another minor difference is in the
subdomain IX representing the large Recombinant gPKA Is Catalytically Active--
We determined the
catalytic activity of the purified recombinant gPKA using histone (Fig.
3) and kemptide (see below) as
substrates. Kemptide is a synthetic heptapeptide (LRRASLG) and is
highly specific for PKA. Recombinant gPKA phosphorylated both
substrates (specific activity 6.2 ± 0.2 nmol of
phosphate/min/µmol of enzyme) and was also capable of
autophosphorylation (Fig. 3, A and B). Purified gPKA autophosphorylated in a concentration dependent manner with a
stoichiometry of 1-1.8 mol of phosphate/mol of gPKA. Phosphorylated PKA increased with incubation time and was not observed in the control
lane without enzyme. The recombinant PKA band is also absent from the
control no extract lane in the protein gel (Fig. 3A).
Apparent Km values for Mg2+ATP and
kemptide are in the micromolar range: kemptide (7.5 ± 8.5 µM), Mg2+ (127 ± 10 µM),
and ATP (11.18 ± 4.5 µM as phosphate donor). The apparent Km for ATP and the synthetic peptide
kemptide are in the range close to that of cytosolic type II PKA from
erythrocyte and from bovine heart (44, 45). The Km
for Mg+ for gPKA is in the micromolar range instead of the
millimolar range that has been reported for vertebrate PKAc (42).
Additionally, Mg2+ is preferred over Mn2+ as
the divalent cation and ATP over GTP as the phosphate donor for optimum
activity (data not shown). Kinase activity of recombinant gPKA with
histone or kemptide was not inhibited by the 6-22 amide (PKI), which is
potent inhibitor of mammalian PKAs (data not shown). However, both the
shorter myristoylated peptide inhibitor, 14-22 amide (which inhibited
giardial excystation, see below) and a PKA-specific synthetic
hexapeptide inhibitor (RGYALG) were able to inhibit phosphorylation of
kemptide by gPKA with IC50 values of 10 µM
(Fig. 3D) and 3 mM, respectively. The
IC50 of the hexapeptide inhibitor was lower than that
obtained with the PKA from rabbit skeletal muscle (46).
Catalytically Active PKA Is Present in Giardia
Trophozoites--
We measured the phosphorylating ability of the
native PKA in the crude extracts of trophozoites using kemptide as the
specific substrate (Fig. 4). PKA activity
was monitored in the presence or absence of kemptide (Fig.
4B) to eliminate phosphorylation of endogenous substrates by
PKA and other kinases. A 5-fold increase in activity was obtained with
kemptide, indicating active gPKA in the trophozoite extracts (specific
activity 1.2 ± 0.26 nmol/min/mg of protein). Exogenous cAMP at a
concentration (4 µM) slightly higher than that used for
the mammalian PKA was able to stimulate gPKA activity in the crude
extract. A 2.5-fold increase in kemptide phosphorylation was noted
following addition of cAMP in the crude extract (p < 0.02) (Fig. 4C). To confirm that the activity was due to
native PKA, we immunodepleted gPKA from crude trophozoite extracts by
immunoprecipitation using a rabbit polyclonal antibody against
recombinant gPKA. The specificity of the gPKA antibody was shown by its
reaction with the 47-kDa recombinant gPKA and with a single 41-kDa
polypeptide in the crude extract in Western blot at a dilution of
1:7000 (Fig. 4A). The idea that the cAMP-stimulated posphorylation of kemptide in crude extracts was due to gPKA was also
supported by immunodepletion studies. As expected, addition of kemptide
did not increase kinase activity of the depleted crude extract (Fig.
4B). Depletion of the native PKA was confirmed by Western
blot analysis, which shows no PKA protein band in the depleted lane
(Fig. 4A). The higher band in this lane is the residual PKA
IgG that reacted with anti-rabbit secondary antibody.
Immunolocalization of gPKA and cAMP Measurements in
Trophozoites--
The cellular localization of PKA enzyme in G. lamblia trophozoites was determined using primary antiserum
generated to purified recombinant Giardia PKA (or anti-gPKA)
and confocal microscopy. At low cell densities (achieved with inocula
of 350-700 cells/ml), distinct signals were seen in dense rods or
marginal plates along the intracellular portions (axonemes) associated
with the pairs of anterior and caudal flagella, as well as in the eight
basal bodies located between the nuclei (Fig.
5A). Localization of gPKA to
the flagellar basal bodies was confirmed by colocalization with
monoclonal anti-centrin antibody (41) (Fig. 5B). In addition to the basal bodies, anti-centrin localized along the two axonemes associated with the posterior-lateral flagella that were not recognized by gPKA (Fig. 5B). Neither antibody reacted in the region of
the ventro-lateral pair of flagella. Localization of gPKA at high cell
density (inoculum > 1400 cells/ml) also showed staining of basal
bodies. However, there was no localization to axonemes and diffuse
staining was observed in the cytosol (Fig. 5C). Refeeding cells from the higher inocula cultures with fresh medium for 10 min,
led to the localized patterns (Fig. 5A); however, exposure to exogenous dibutyryl cAMP did not (data not shown). Results from the
cAMP analyses showed that those cells having a diffuse staining pattern
(Fig. 5C) had significantly (p < 0.03 for
comparisons of 700 with 1400 and 2000 cells/ml) lower endogenous levels
of cAMP compared with those parasites with localized staining (Fig. 5A) to flagellar marginal plates components (Fig.
5D). This suggests that localization of gPKA to the flagella
is responsive to both cell density and growth factors.
Involvement of gPKA in the Excystation Process--
Since
excystation is a very rapid differentiation in response to extreme
changes in the external milieu, intracellular signaling may be more
important than changes in gene expression. Cyclic AMP is important in
cellular differentiation in other organisms, and we asked if PKA
activity might be needed for excystation. We found that a
cell-permeable myristoylated pseudosubstrate inhibitor specific for PKA
(amide 14-22) inhibited excystation with an IC50 of 3 µM when it was present throughout excystation (Fig.
6A). The effect of the PKA
inhibitor on the individual stages of excystation was also studied.
Pre-incubating cysts with the PKA inhibitor prior to excystation
stimuli was effective in preventing excystation. The inhibitor was also
effective at the low pH stage 1, which mimics passage through the
stomach, but less effective at stage 2, which mimics cyst entry into
the host small intestine (Fig. 6B). Since this stage entails
digestion of cysts with trypsin, it is likely that the peptide
inhibitor was at least partly inactivated. These studies suggest a need
for PKA activity during the early stages of excystation.
Since PKA regulates critical cellular processes in both higher and
lower eukaryotes, we characterized a homologue of a PKA catalytic
subunit of Giardia. Sequence and structural predictions showed that most of the kinase subdomains that are conserved in higher
eukaryotes, are present in gPKA, and we have also identified a
regulatory subunit homologue (data not shown), suggesting a conventional PKA holoenzyme. Despite several structural differences, functional characterization demonstrates that both recombinant and
native PKA are catalytically active and phosphorylate a PKA-specific substrates, such as kemptide as well as histone. Structural alignments, substrate specificity, and stimulation of the kinase activity of native
PKA by cAMP all suggest that the cloned ORF is indeed a
Giardia homologue of the C subunit of PKA.
In the case of a ubiquitous enzyme, such as PKA, it is important to
understand how different physiological conditions determine specificity. A prominent level of control is to localize signaling proteins to specific subcellular organelles or compartments. Thus, it
is very interesting that, in Giardia, PKA can localize to
both basal body/centrosome and specific flagellar structures. In
certain cells, protein kinase A-anchoring proteins or AKAPs can bind to a PKA regulatory subunit, which then localizes the catalytic subunit to
the proper structure. For example, AKAP450 targets PKA to centrosomes in HeLa cells (47). In bovine and human sperm, mAKAP82 localized PKA,
via its RII subunit, to the fibrous sheath (48), between the axoneme
and the surrounding mitochondrion. In contrast, PKA of a single-celled
flagellated alga, Chlamydomonas, is reported to be localized
to the radial spokes of the flagellar structure, where it may be
involved in regulating dynein ATPase (49). The location of PKA in
giardial flagellar structure appears closer to that of mammalian sperm
than to that of Chlamydomonas.
Immunolocalization of gPKA in trophozoites showed a selective
accumulation of PKA in the eight flagellar basal bodies at low and high
cell densities. Localization of PKA to the basal bodies was confirmed
by colocalization with centrin, a basal body-specific Ca2+-binding protein. Basal bodies are rodlike structures
associated with the origins of the flagellar axonemes and are involved
in cell motility. The basal bodies of flagellated cells correspond to
the centrosomes of higher eukaryotic cells (50). Centrin, which is
found in centrosomes in higher eukaryotes and basal bodies of all
flagellated and ciliated cell types including Giardia (51), is also involved in mitotic spindle pole segregation in
Saccharomyces (52). Interestingly, sequence analyses have
shown that a giardial centrin gene has a motif that predicts
phosphorylation by PKA. Indirect immunofluorescence studies revealed
that antibody specific for phosphorylated centrin reacted with the
basal bodies of dividing cells, but not interphase
cells.2 Thus, centrin in the
basal bodies may be a substrate for phosphorylation by PKA during a
specific stage of the cell cycle. In contrast, gPKA was associated with
the paired anterior and caudal flagella only at low cell densities,
which correlated with higher endogenous cAMP levels. This is the first
evidence that trophozoites can respond to signals from their
environment with changes in cAMP levels and altered localization of a
protein kinase. Since the flagellar localization was also found after
refeeding high density cultures, it is likely a response to growth factors.
Giardia has four pairs of eukaryotic flagella (5) that are responsible
for both motility and attachment. Each originates from a basal body
between the two nuclei and every pair of flagella traverses part of the
cell body and emerges at a specific location. Paraflagellar densities
or marginal plates have been observed by electron microscopy, along
much of the intracellular portion of all four pairs of flagella, but
little is known about the composition or function of the marginal
plates (53). Only the extracellular portions of the flagella are
covered with plasmalemma.
The ability of trophozoites to alter their flagellar motility in
response to external conditions may be critical to their ability to
colonize the small intestine where the availability of growth factors
is constantly changing. The anterior flagella cross anteriorly from the
basal bodies and pass through the cell's ventrolateral flange before
exiting the cell body. The ventrolateral flange is the outer margin of
the cell body that interdigitates between microvilli of the small
intestinal enterocytes (9), along with the ventral adhesive disc in
trophozoite attachment. The ventrolateral flagella, stained for
centrin, but not for PKA. Both the anterior and ventrolateral flagella
are important in forward motion of the trophozoite and continue to beat
while the parasite is attached to intestinal enterocytes or to inert
substrates. In contrast, the caudal flagella, whose marginal plates
also stained for PKA, do not appear to beat during swimming or
attachment and have a rigid appearance, very distinct from the helical
form of the other flagella (9). The caudal flagella are thought to be
associated with dorsal flexion of the "tail" of the cell body that
is involved in trophozoite detachment. Since Giardia attach to enterocytes, which migrate to the tip of the villus and are then
sloughed off, trophozoites must be able to detach, swim, and re-attach
to younger host cells to remain in the small intestine. Our finding of
association of gPKA with anterior and caudal flagella, whereas centrin
associates with the posterior-lateral flagella, may help understand
differential flagellar function. Localization of gPKA and centrin to
structures responsible for both motility and cell division may link
those functions in an ancient eukaryote. These data support a role for
PKA and cAMP in regulating giardial attachment and locomotion.
Blocking excystation with the cell permeable PKA inhibitor (amide
14-22) suggests a key role for gPKA in this critical differentiation. The role of PKA appears most critical early in excystation. Cysts are
ingested from cold freshwater and are first exposed to the highly
acidic gastric fluid (pH 2.0 to pH 4.0), which is needed for the
induction of excystation (35) and is modeled by our stage 1 in
vitro. PKA inactivation prior to or during stage 1 appears to
interfere with the induction processes (Fig. 6B). Giardial excystation entails a dramatic resumption of flagellar motility and
cell division. In vivo, excysting cells must regain their motility and attach before they are carried downstream by the flow of
intestinal fluid. Therefore, the localization of PKA in flagellar and
basal body structures may reflect a role in motility and cytokinesis in
excystation as well as in vegetative growth.
Structural analyses of gPKA suggested both a high degree of
conservation and possibly significant divergences. Key conserved motifs
include the glycine-rich motif, reactive loop, and Thr-197 residue for
autophosphorylation. The most unusual feature of gPKA is a highly
stable loop in the activation domain (subdomain VIII) of the enzyme as
indicated in the predicted three-dimensional structure (Fig.
2C). This loop may interfere with the interaction of gPKA
with PKI (6-22 amide) but not with the shorter 14-22 inhibitor or the
heptapeptide substrate, kemptide. We hypothesize that gPKA can interact
with the shorter inhibitory peptide, but not the longer, possibly
because the extra loop in the activation domain of gPKA that could
interfere with binding of the larger inhibitor. The catalytic activity
of native gPKA should be due to catalytic subunits that have
dissociated from the holoenzyme in response to increased cAMP.
Exogenous cyclic AMP at a slightly higher concentration than for the
mammalian enzyme-activated gPKA activity.
At present, the precise physiological stimuli that affect cAMP levels
and PKA activity and localization in Giardia are not known.
In Saccharomyces, the stimuli are very simple; only glucose or cytosolic acidification is known to increase cAMP concentrations. Yeast must respond to transiently favorable growth conditions that
allow stationary phase cells to switch to a growth program. Perhaps
Giardia's needs are more complex, as its environment
changes drastically during excystation. Moreover, within the small
intestine, conditions change rapidly according to whether trophozoites
are attached to young or senescent enterocytes, or are in the lumen and
according to the nutritional status of the host. Our data suggest key
roles for PKA in regulating giardial responses to its environment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAPII (Stratagene, La Jolla,
CA) using a kit (ZAP-cDNAR synthesis kit, Stratagene)
according to the manufacturer's protocol. The library was screened for
the full-length clone of PKA using 32P-labeled PKA probes
as described previously (36). Five positive plaques were obtained by
screening ~6 × 105 plaque-forming units. Positive
plaques containing inserts were plaque-purified and inserts cloned into
pBluescript plasmids by in vivo excision using ExAssist
helper phage (Stratagene). Inserts were confirmed by restriction
digestion and cross-hybridization.
)
(Stratagene). Unincorporated nucleotides were removed using a NucTrap
push column (Stratagene).
-D-galactopyranoside.
Expressed gPKA as a 47-kDa His tag and S tag fusion protein was
purified through His-Bind columns (Novagen) and used as antigen for
generation of polyclonal antibodies through a commercial vendor
(CoCalico, Reamftown, PA). The His tag and S tag sequences added 6 kDa
of molecular mass to the fusion protein.
-32P]ATP. The reaction mix was
incubated at 30 °C for 10 min and spotted (40 µl) on
phosphocellulose filter discs. Filters were washed four times for 5 min
each with 1% phosphoric acid and bound radioactivity counted in a
liquid scintillation counter (LKB). Kinase assays using recombinant
gPKA were conducted in the presence or absence of the myristoylated PKA
inhibitor 14-22 amide and a PKA-specific hexamer inhibitor (RGYALG)
(Bachem) at different concentrations. Assays using crude extract were
performed in the presence or absence of 4 µM cAMP
(Calbiochem). The catalytic parameters were measured under standard
assay condition, and the data were graphically fit to the
Lineweaver-Burk form of the Michaelis-Menten equation (40).
20 °C) for 10 min at
20 °C and further permeabilized with
PBS containing 0.5% Triton X-100 for 10 min. Permeablized trophozoites
were blocked for 1 h in blocking buffer (5% goat serum, 1%
glycerol, 0.1% BSA, 0.1% fish gelatin, and 0.04% sodium azide in
PBS), and incubated with rabbit anti-gPKA polyclonal antibodies (1/2500
in blocking buffer) and/or mouse monoclonal anti-centrin antibodies
(clone 20H5, 1/500 in blocking buffer) (41) for 1 h. At the end of
incubation, cells were washed for 5 min (four times) with PBS and
incubated with the secondary antibodies (anti-rabbit ALEXA 488 and/or
anti-mouse ALEXA 568 (Molecular Probes, Eugene, OR) each diluted in
blocking buffer 1/800) for 1 h. Next, cells were washed for 5 min
(four times) with PBS, post-fixed for 7 min with 4% paraformaldehyde
(Electron Microscopy Sciences, Ft. Washington, PA) in PBS, rinsed with
PBS, and mounted on glass slides in Prolong (Molecular Probes).
Localization of gPKA and centrin was observed on a Zeiss LSM 510 laser
scanning confocal microscope equipped with argon-krypton (455/488) and helium-neon (543/633) lasers and appropriate filter sets.
0.05 were considered significantly different.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (70K):
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Fig. 1.
Multiple sequence alignment of gPKA.
Gl, G. lamblia; Ce,
C. elegans; Eg, E. gracilis;
Hs, H. sapiens; Sc, S. cerevisiae. The solid bars above the
sequences indicate the G loop, catalytic loop, and APE sites,
respectively. The black arrow indicates the
conserved Lys-64 for ATP binding. The asterisk indicates
conserved Glu-65, which interacts with Lys-46. The triangle
shows Ser-181 instead of Thr-197 required for autophosphorylation in
other species. The diamond shows Leu instead of Phe in
sequence DFG, and the filled circle shows
Arg-264.
-phosphate oxygens and the
pseudosubstrate peptides such as the PKA inhibitor peptide (PKI). The
APE motif helps to stabilize the large C-terminal lobe of PKAc. Highly
conserved amino acid residues such as Lys-46 in subdomain II, which is
responsible for ATP-binding; Glu-65 in subdomain III, which interacts
with Lys-46; and Arg-264 in subdomain IX are also present. Among the consensus residues for interaction with the R subunit of PKA or PKI,
only His-61 in subdomain III, Arg-107 (not conserved in PKAc
), and
Arg-108 in subdomain V are present but Trp-196, Thr-197, Leu-198, and
Lys-213 (in vertebrates) in subdomain VIII are not. The consensus residue Thr-197 (in vertebrates) that is responsible for
autophosphorylation in other species is replaced by a serine residue,
which can also be phosphorylated in gPKA (Ser-181). In subdomain VII,
the Phe residue in DFG sequence is also substituted by another
hydrophobic residue Leu in gPKA. This triplet helps to orient
phosphate for transfer to the substrate by chelating the
Mg2+ ion, which bridges the
and
phosphates of the
ATP. The invariant Arg-280 (Arg-264 in gPKA) responsible for
stabilizing the large lobe of PKAc is also present.
-catalytic subunit of bovine PKA complexed with the inhibitor staurosporine (PDB accession no. 1STC) and H8 protein kinase inhibitor
(PDB accession no. 1YDS) (Fig.
2A). However, this is a
predicted three-dimensional structure based on the actual crystal
structure of the template complexed with inhibitors, which may
contribute some degree of inaccuracy. Nonetheless, the superimposed structure of gPKA indicates two major differences. A small loop (red) is in subdomain XI at the C-terminal end of the kinase
domain (Fig. 2, A and B). The interaction of the
invariant arginine corresponding to Arg-280 (Fig. 2B,
yellow arrow), which stabilizes the large lobe by
forming an ion pair with Glu-208 is predicted to be present in gPKA
(Arg-254 and Glu-182) but the hydrogen (H) bond between Arg-280 and
Asn-283 appears to be missing in gPKA. Asn-283, which maintains the
loop structure by forming a H-bond with Leu-277 in the template, is
replaced by Ser-258 forming a H-bond with Lys-251 in gPKA, thus making
the loop more relaxed than that in the template.
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Fig. 2.
Analysis of predicted three-dimensional
structure of gPKA. A, comparison of the
predicted three-dimensional structure of gPKA (blue) and
crystal structure of the -catalytic subunit of bovine PKA complexed
with staurosporine (white). Structures of gPKA and the
template are represented as blue and white
ribbons, respectively. Any change in the color of the
blue ribbon represents dissimilarities with the
template. Increasing color intensity represents increasing degree of
mismatches leading to red representing absolute differences
between gPKA and the template. Arrows indicate the
additional loop structure in gPKA. B, comparison of the
structure of the loop in subdomain IX. Blue, gPKA;
white,
-catalytic subunit of bovine PKA complexed with H8
inhibitor peptide (PDB accession no. 1YDS). Yellow
arrows indicate the invariant Arg-280 (Arg-254 in gPKA).
Red arrow indicates the H-bond that is missing
from gPKA. C, comparison of the activation domains of gPKA
(blue) and bovine PKAc-
(white). The
orange arrow indicates the H-bond in the
Mg2+-binding domain when Phe-185 is replaced by Leu-149 in
gPKA. D, structures of the activation domains of gPKA
(blue) and bovine PKAc-
complexed with H8 inhibitor
peptide (white). Orange arrows
indicate H-bonds that are missing between Lys-153 and Thr-165 and
between Lys-153 and Ser-171 (Lys-189 and Thr-195, and Lys-189 and
Thr-197 in bovine PKAc) making the loop more relaxed. Purple
arrows show the bond that is missing between Thr-165 and
Ser-171 (Thr-195 and Thr-197 in bovine PKAc). Red
loop indicates several H-bonds that are absent in bovine
PKAc. E, comparison of the subdomain IX of gPKA
(blue) and bovine PKAc-
(white).
Orange arrows indicate the invariant Asp-194
(Asp-220 in bovine PKAc). Purple arrows indicate
the H-bond that is missing between Asp-194 and Tyr-128 (Asp-220 and
Tyr-164 in bovine PKAc) needed to stabilize the catalytic loop.
-helix in the large lobe (Fig.
2E). The invariant Asp-220 (Asp-194 in gPKA), which acts to
stabilize the YRDKXXN catalytic loop by forming H-bonds with
backbone amides of Arg-165 and Tyr-164 is predicted to form H-bond only
with Arg-129 but not with Tyr-128 in gPKA. This may cause instability
in loop formation.
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Fig. 3.
Phosphorylation of histone by gPKA at
different incubation time (0-30 min). A, Coomassie
Blue-stained gel showing equal amounts of histone and gPKA in all lanes
except in lane N. N contains histone
but no extracts. B, autoradiogram showing phosphorylated
histone and gPKA. C, Lineweaver-Burk plot of the
phosphorylation of kemptide by gPKA at 30 °C. Each point
represents the mean of three separate experiments. Apparent
Km for kemptide, ATP, and Mg2+, and
specific activity of gPKA are presented in the box.
D, inhibition of phosphorylation of kemptide by
myristoylated PKA inhibitor 14-22 and by the synthetic hexapeptide
inhibitor (RGYALG). Data represented as mean ± S.D. (* indicates
p < 0.03 (n = 3) for the myristoylated
inhibitor and p < 0.005 (n = 2) for
the hexapeptide; ** indicates p < 0.04).
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Fig. 4.
Detection of active gPKA in trophozoite
extracts. A, Western blot. Detection of gPKA in
trophozoite extracts before and after immunodepletion of gPKA by PKA
antibody. The PKA IgG band in the depleted extract lane is the
remaining gPKA antibody recognized by the anti-rabbit secondary
antibody. B, kinase activity in crude extracts and
immunodepleted extracts using kemptide as the substrate. Data
represented as mean ± S.D. (n = 3). C,
effect of exogenous cAMP on the kinase activity in the crude extract.
Data represented as mean ± S.D. (p < 0.02, n = 4).
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Fig. 5.
Immunolocalization of gPKA and analysis of
cAMP levels in G. lamblia trophozoites.
A, at low cell density, anti-gPKA recognizes the
intracellular portions of caudal flagella (CF), anterior
flagella (AF), and the basal bodies (BB) located
between the nuclei. B, centrin localization to the
posteriolateral flagella. Colocalization of centrin and gPKA was
observed to the basal bodies shown in yellow and indicated
by an arrow. C, gPKA was associated with basal
bodies (arrow) and the cytosol, but not the flagella, at
high cell densities. D, trophozoites that had localized
(L) staining to caudal flagella and anterior flagella had
significantly higher (*) levels of endogenous cAMP, compared with
trophozoites that appeared to have diffuse (d) staining in
the cytosol. Data are represented as mean femtomoles of
cAMP/105 cells ± S.D. (n = 5).
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Fig. 6.
Effect of myristoylated PKA inhibitor (amide
14-22) on excystation. A, effect of various
concentrations of the inhibitor on excystation. The inhibitor was
present at all stages of excystation. Data are represented as mean ± S.D. *, p < 0.03 (comparison of untreated with 1 and 3 µM concentrations of the inhibitor). B,
effect of the inhibitor peptide (14-22) on various stages of
excystation. The inhibitor was present at 10 µM only
during the stage indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank F. Ahmadi for able technical help, Dr. J. Salisbury for the anticentrin antibody, and Dr. W. Lingle for help with the confocal analyses.
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FOOTNOTES |
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* This work was supported by Grants GM53835, AI42488, and DK35108 from the National Institutes of Health and funds from the 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.
AF181097.
¶ To whom correspondence should be addressed: Dept. of Molecular Biology and Microbiology, Biomolecular Research Annex, University of Central Florida, 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, December 4, 2000, DOI 10.1074/jbc.M006589200
2 W. L. Lingle and J. L. Salisbury, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PKA, protein kinase A; gPKA, giardial protein kinase A; PKAc, protein kinase A C subunit; PDB, Protein Data Bank; R, regulatory; C, catalytic; PCR, polymerase chain reaction; PKI, protein kinase A inhibitor peptide; PBS, phosphate-buffered saline; ORF, open reading frame; kb, kilobase pair(s).
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