Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, BC V6T 1Z3, Canada
* Author for correspondence (e-mail: gweeks{at}unixg.ubc.ca)
Accepted 27 June 2002
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Summary |
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The Rap1-depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4 M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a role for Rap1 in the generation of cGMP. Dictyostelium Rap1 formed a complex with the Ras-binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4 M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2 mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature but not in response to low osmolarity or high temperature.
Key words: Stress responses, Differentiation, Rap1 activation, Antisense, cGMP, Dictyostelium, Viability
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Introduction |
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In Drosophila, there is a single rap gene, and
loss-of-function mutations are lethal at the larval stage
(Hariharan et al., 1991). This
lethality can be rescued by expressing rap under the control of a
heat shock promoter, and cell proliferation was unimpaired but morphogenesis
and cell movement were abnormal during development in the absence of Rap1
function (Asha et al.,
1999
).
In Dictyostelium, the available evidence suggests that there is
also a single rap gene, rapA, previously designated
rap1, encoding a protein, Rap1, 75% identical to mammalian Rap1A
(Robbins et al., 1990). The
overexpression of rapA results in cells with a variety of
cytoskeletal defects, including a flattened cell morphology and failure to
contract in response to contraction stimuli
(Rebstein et al., 1993
). In
addition, cells overexpressing activated and dominant-negative forms of Rap1
exhibited alterations in phagocytosis and fluid phase endocytosis
(Seastone et al., 1999
). To
provide a more definitive assessment of Rap1 function in
Dictyostelium, we attempted to disrupt the rapA gene by
standard procedures. However, these attempts failed, suggesting that
rapA might be an essential gene in Dictyostelium. In the
present study, we have expressed a rapA antisense construct under the
control of the folate repressible discoidin promoter
(Blusch et al., 1992
) and have
examined the effects of Rap1 depletion on cell function. We have also
demonstrated that Rap1 is activated in response to hyperosmotic stress and low
temperature.
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Materials and Methods |
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Vector construction and transformation
To create the pVEII-AS5'construct, the 5' portion of
the rapA gene (nucleotides -15 to +218) was synthesized by PCR using
the oligonucleotides 5'-TGCTCTAGAGCTCGAATTCATCATGCC-3' and
5'-TGCTCTAGAGCAGTAAATTGTTCAGTACG-3' as primers and rapA
cDNA (Rebstein et al., 1993)
as the template. Both primers contained KpnI sites, and the PCR
product was inserted into the KpnI-digested pVEII vector.
The resulting constructs contained the antisense rapA DNA linked to
the folate repressible discoidin promoter. The orientation and promoter-gene
fusions were confirmed by sequencing.
The pVEII-AS5' vector was introduced into Ax-2 cells by electroporation, and single cell transformants were selected in 96-well plates in HL-5 medium supplemented with 10 µg/ml G418 and 1 mM folate. Six pVEII-AS5' transformants were obtained, and each was plaque purified on a lawn of K. oxytoca. The isolated transformants were cultured on 10 cm Petri dishes in HL-5 growth media, supplemented with 100 µg/ml G418 and 1 mM folate for three days and then maintained on HL-5 media containing 10 µg/ml G418 and 1 mM folate. The cultures were frequently divided into fresh growth media to maintain low cell density.
Cell size determination
Exponentially growing cells were centrifuged at 600 g,
washed three times with KK2 and resuspended at
1x106 cells/ml. The cell suspensions were left on ice for 15
minutes to produce isolated, spherical cells and then viewed through a
microscope. The cells were photographed and their diameters determined from
prints.
Shock conditions
Exponentially growing cells were centrifuged at 600 g,
washed three times with KK2 and resuspended in the same buffer at
3.0x107 cells/ml and shaken at 160 rpm for 1 hour at
22°C. To induce hyperosmotic shock, sorbitol was added to a final
concentration of 0.4 M. Aliquots were removed periodically and analyzed for
either cell viability, cGMP content or the level of activated Rap1.
To induce hypo-osmotic shock, cells that had been shaken at 22°C for 1 hour in KK2 were centrifuged and resuspended in ddH2O. Cells were subjected to temperature shock by placing cells at 8°C for cold shock or 30°C for heat shock. Aliquots were removed at various time points and analyzed for the level of activated Rap1.
cGMP assay
Levels of cGMP were determined essentially as described previously
(Oyama, 1996). 100 µl
aliquots of cells that had been subjected to osmotic shock were added to 100
µl of 3.5% perchloric acid and the mixture incubated on ice for 30-60
minutes with occasional vigorous shaking. The solution was neutralized by the
addition of 50 µl of 50% saturated KHCO3 and the mixture
incubated for 60 minutes on ice with occasional vigorous shaking. The
suspensions were centrifuged at 14,000 g for 10 minutes at
4°C and 100 µl of the supernatant was assayed for cGMP content using
the Amersham Pharmacia Biotech radioisotope dilution assay.
Western blot analysis
Between 5x106 and 1x107 cells were washed
twice in KK2, resuspended in 1% SDS, and the protein concentration
was determined using the Bio-Rad protein assay. A 20 µg protein aliquot of
each sample was mixed with an equal volume of SDS sample buffer (0.5%
ß-mercaptoethanol; 0.5% SDS; 50 mM Tris-Cl, pH 6.8; 12.5% glycerol, and
0.04% bromophenol blue), boiled for 5 minutes and then fractionated by
SDS-polyacrylamide gel electrophoresis
(Laemmli, 1970) using 12%
gels. After electrophoresis, the proteins were transferred to nitrocellulose
membranes for 1 hour and probed with either polyclonal Rap1 antibody
(Rebstein et al., 1997
) or a
monoclonal phosphotyrosine antibody
(Ingham et al., 1998
). The
membranes were incubated for 1 hour at room temperature in TBS-Tween (25 mM
Tris-Cl, pH 8.0, 1.0% NaCl, 1% Tween 20) containing 5% powdered milk
(Carnation) for Rap1 detection or 4% BSA for phosphotyrosine detection. The
Rap1 primary antibodies were diluted 1:2,000 in TBS-Tween containing 0.5%
powdered milk and incubated with the nitrocellulose membranes overnight at
room temperature. The phosphotyrosine antibodies were diluted 1:200 in
TBS-Tween and incubated with the membranes overnight at 4°C. The membranes
were washed three times for 5 minutes in TBS-Tween and exposed to a secondary
antibody (donkey anti-rabbit IgG conjugated to horseradish peroxidase or
donkey anti-mouse IgG conjugated to horseradish peroxidase) diluted 1:10,000
in TBS-Tween. The bound antibodies were detected by an enhanced
chemiluminescence assay (Amersham). Blots were scanned using a ScanJet-II
scanner (Hewlett-Packard, USA). and densitometry was performed using Image
Quant (V.1.2) software for MacIntosh.
Binding of bacterially expressed Rap1 to GST-RalGDS
The mammalian Rap-binding domain (RBD) of RalGDS was expressed in
Escherichia coli as a GST fusion protein as described previously
(McLeod et al., 1998).
Exponentially growing cultures of bacteria were induced with 0.1 M IPTG for 16
hours at 22°C. The cells were lysed by sonication for two minutes in 50 mM
Tris-Cl, pH 7.5; 150 mM NaCl; 1% TritonX-100; 1 mg/ml lysozyme and 0.1 mg/ml
DNase I. The resulting cell lysate (10-50 µl) was incubated with 10 µl
of glutathione-Sepharose beads (Pharmacia) at 4°C for one hour, and the
beads were then washed three times with wash buffer (20 mM Tris-Cl, pH 7.6;
150 mM NaCl, 0.1% Triton X-100; 10 µg/ml leupeptin; 1 µg/ml aprotinin
and 1 mM PMSF).
Rap1 was also expressed as a GST fusion protein in E. coli. The
original rapA cDNA was complete at the 3' end, but the 5'
end was truncated at an EcoRI site, 12 bp downstream of the ATG
translation initiation site of the gene
(Robbins et al., 1990). The
5' end was completed through a single oligonucleotide mutagenesis, which
eliminated the internal EcoRI site through a conservative change at
nucleotide +15 and introduced an EcoRI site at the 5' end of
the completed cDNA. The rapA construct was treated with
EcoRI, and the 700 bp fragment was ligated to an
EcoRI-digested pGEX-1 vector. This gst-rapA construct was
transformed into E. coli. Exponentially growing cultures of bacteria
were induced with 0.1 M IPTG for 4 hours at 37°C and the bacteria lysed as
described above. Cell lysate (0.5 ml) was mixed with 50 µl of packed
glutathione-Sepharose beads at 4°C for 1 hour and the beads then washed
three times with wash buffer. The bound GST-Rap1 was treated with 10 units/ml
thrombin in 50 mM Tris-HCl (pH 7.6); 150 mM NaCl; 2 mM CaCl2 to
hydrolyze the peptide bond between Rap1 and GST, and the beads were removed by
centrifugation. The supernatant containing the bacterically expressed Rap1 was
concentrated using a Centricon 10 (Amicon).
The Rap1 protein (1 µg) was incubated with either 1 mM GDP or 1 mM
GTP in 20 mM Tris-HCl (pH 7.6); 10 mM EDTA; 5 mM MgCl2; 1 mM DTT
(Dithiothreitol); 0.1 mM PMSF and 10% (v/v) glycerol in a total volume of 20
µl for 30 minutes at 30°C, and MgCl2 was then added to a
final concentration of 20 mM to stabilize the binding. 210 ng Rap1 protein was
then incubated with 100 ng of GST-RalGDS(RBD) bound to glutathione-Sepharose
beads in 20 µl of binding buffer containing 100 µg BSA, 100 mM NaCl, 0.5
mM GTP or GDP, 6.5 mM EDTA, 12.5 mM MgCl2. After 2 hours at
4°C, the beads were pelleted and the unbound material in the supernatant
was removed. The beads were washed five times with 0.5 ml of ice-cold 10 mM
Tris-HCl, pH 7.6; 5 mM MgCl2; 1 mM DTT; 0.1 mM PMSF; 10% glycerol
and 0.05% Triton X-100. An equal volume of SDS sample buffer was added to both
the beads and the unbound fraction. Both fractions were subjected to SDS-PAGE
and western blotting as described above.
Binding of native Rap1 to GST-RalGDS
Samples of Dictyostelium cells were pelleted and resuspended in 30
mM HEPES (pH 7.8), 10 mM KCl, 10% sucrose, 1% TritonX-100 and protease
inhibitor (Roche). The protein concentration was determined using the BioRad
protein assay. Cell lysate (5-100 µg protein) was incubated for 1 hour at
4°C with 20 µg GST-RalGDS(RBD) that had been pre-bound to
glutathione-Sepharose beads. After three washes with 30 mM HEPES (pH 7.8), 200
mM KCl, the beads were incubated with an equal volume of SDS sample buffer and
the solubilized material subjected to SDS-PAGE and western blotting.
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Results |
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|
Rap1 protein levels in the pVEII-AS5'-1 transformant were
found to gradually decline during growth without folate, reaching 20% of
their initial value by 10 days and being barely detectable beyond 12 days
(Fig. 2). These results
indicate that the reduction in Rap1 protein level correlated with reduced cell
growth. A similar decrease in viability and Rap1 protein level was observed in
the two other pVEII-AS5' transformants that were examined in
detail (data not shown). As a control, samples were also assayed for RasG by
western blot analysis, as this protein is another member of the
Dictyostelium Ras subfamily and any non-specific effect of the
rapA antisense construct would be expected to be manifested as a drop
in RasG level. Only a slight decrease in RasG was observed during the course
of the experiment.
|
During the period of declining growth in axenic media, the cells were tested for growth on bacterial lawns. The size of the plaques formed on the bacteria were considerably smaller as the time of depression increased, suggesting that growth on bacteria was also impaired. Both pinocytosis and phagocytosis were considerably reduced in the pVEII-AS5'-1 transformant (data not shown), which may account for the slow growth rate in both axenic media and on bacteria. In addition to the defects in cell growth, the majority of pVEII-AS5'-1 cells were smaller than the parental Ax-2 cells, although about 5% of the cells were considerably larger (data not shown).
Effect of the reduction in Rap1 level on the response of cells to
hyperosmolarity
In view of the effect of Rap1 depletion on cell viability and cell size, we
determined the resistance of the Rap1-depleted cells to hyperosmotic
conditions. To ensure that the cells used in these tests were viable, we used
cells that contained 40% of the normal level of Rap1. When these
partially Rap1-depleted cells were exposed to 0.4 M sorbitol for 120 minutes,
only
15% of the transformant cells survived. By contrast, under identical
conditions all wild-type Ax-2 cells remained viable
(Fig. 3A).
|
Dictyostelium cells respond to hyperosmolarity by reducing their
cell volume by 50% within 5 minutes
(Zischka et al., 1999). This
rapid reduction in cell volume correlates with myosin phosphorylation and the
activation of guanylyl cyclase (Kuwayama
et al., 1996
). The importance of the guanylyl cyclase activation
in osmoregulation is indicated by the observation that some of the
osmosensitive mutants that have been identified are deficient in cGMP
production. Furthermore, these mutants become less osmosensitive in the
presence of the cell-permeable cGMP analog, 8-Br-cGMP
(Kuwayama et al., 1996
). We
found that cGMP levels were reduced in the pVEII-AS5'-1
transformant relative to the wildtype following treatment with 0.4 M sorbitol
(Fig. 3B), suggesting a
possible requirement for Rap1 in the accumulation of cGMP. Furthermore,
addition of 8-Br-cGMP to Rap1 depleted cells enhanced their survival following
sorbitol treatment (Fig. 3A). These results suggest that Rap1 might be important in regulating the
accumulation of cGMP during the response to osmotic shock. Consistent with
this idea was the finding that cells overexpressing the constitutively
activated Rap1, Rap1(G12V), produced more cGMP in response to sorbitol
addition than did wild-type cells (Fig.
3B).
During osmotic shock, there is also a dramatic increase in actin tyrosine
phosphorylation (Zischka et al.,
1999). The data in Fig.
4 shows that the expected increase in actin tyrosine
phosphorylation occurred in wild-type cells in response to osmotic shock.
Under these conditions, actin is by far the major protein to be tyrosine
phosphorylated (Zischka et al.,
1999
). However, in pVEII-AS5'-1 cells the basal
pre-shock level of actin tyrosine phosphorylation was considerably higher than
the pre-shock level in wild-type cells, and actin phosphorylation increased
only slightly after osmotic shock, suggesting actin tyrosine phosphorylation
was deregulated in Rap1-depleted cells.
|
RalGDS binds specifically to activated Rap1
To ascertain if Rap1 is activated in response to hyperosmolarity, we
adopted the GST-RalGDS(RBD) pull-down assay that had been used to determine
Rap1 activation in mammalian cells (Franke
et al., 1997). The rationale for this approach was the fact that
Dictyostelium Rap1 and mammalian Rap1 have identical effector domains
(Robbins et al., 1990
). To
demonstrate that RalGDS(RBD) binds specifically to the activated
Dictyostelium Rap1, we compared the binding of bacterially expressed
GST-RalGDS(RBD) fusion protein to Rap1 in lysates of wild type cells with Rap1
in lysates of cells overexpressing the constitutively activated Rap1 (G12V).
As shown in Fig. 5A, there was
appreciably more RalGDS(RBD)-bound Rap1 in the lysates from the
rap1(G12V) transformant than in the lysate from the wild-type cells
relative to the total amounts of Rap1 present in these extracts. These results
indicated that RalGDS(RBD) preferentially bound to activated Rap1.
|
To determine if there was any binding of RalGDS(RBD) to GDP-bound Rap1, bacterially expressed Rap1 was equilibrated with 1 mM GTP or 1 mM GDP prior to binding to RalGDS(RBD). The proportion of Rap1 that binds to RalGDS(RBD) is high in the presence of GTP but low in the presence of GDP (Fig. 5B). Rap1 did not bind to the control GST protein. Hence, interaction with mammalian RalGDS (RBD) is clearly a good measure of the amount of activated Rap1 in the Dictyostelium cell.
Rap1 activation in response to hyperosmotic stress
Vegetative Ax-2 cells were exposed to 0.4 M sorbitol, and cell lysates were
incubated with GST-RalGDS(RBD) bound to glutathione-Sepharose beads. As shown
in Fig. 6, the amount of Rap1
bound to RalGDS(RBD) increased within 5 minutes of exposure to 0.4 M sorbitol.
The extent of Rap1 activation was reproducibly three-to-four-fold, and there
was a slight but consistent decrease in bound Rap1 during the next 5 minutes
(Figs 6 and
7). Levels of bound Rap1
increased again as the sorbitol shock continued (Figs
6 and
7). The initial kinetics of
Rap1 activation correlated reasonably well with the increase in the cGMP level
(Fig. 3B), a result consistent
with the possibility that Rap1 regulates the pathway that leads to the
activation of guanylyl cyclase. Treatment of cells with 2 mM EDTA enhances the
cGMP response to osmotic shock (Oyama,
1996), and we found that treatment of wild-type cells with 2 mM
EDTA prior to osmotic shock also produced an enhanced activation of Rap1
(Fig. 7). The addition of EDTA
alone had no effect on Rap1 activation (data not shown).
|
|
Rap1 is activated by cold stress but not by heat or hypo-osmotic
stress
To determine whether Rap1 activation in response to hyper-osmotic
conditions was a general or specific response to shock, vegetative Ax-2 cells
were subjected to three additional stress conditions: low temperature, high
temperature and hypo-osmotic conditions. When cells were switched from
22°C to 8°C, Rap1 activation increased after approximately 5 minutes,
and the level of activation remained high for 20 minutes
(Fig. 8). However, neither a
switch to 30°C or resuspension in H2O had a noticeable effect
on the level of activated Rap1 (Fig.
8), indicating that activation of Rap1 is not part of a general
stress response.
|
![]() |
Discussion |
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The properties of the pVEII-AS5'-1 Rap1-depleted cells
resemble those of a strain that lacks both -actinin and gelation
factor, two F-actin crosslinking proteins
(Rivero et al., 1996
). This
double mutant grows slowly, exhibits reduced phagocytosis and is more
sensitive to osmotic shock. In addition, most of the double mutant cells are
smaller and more rounded than wild-type cells, although approximately 5% of
the population are larger (Rivero et al.,
1996
). All of these characteristics are shared by the
Rap1-depleted cells. In view of this similarity, we determined the amounts of
-actinin and gelation factor in depleted cells. Gelation factor levels
were normal, but
-actinin levels were reduced (data not shown).
However, cells disrupted only in the gene encoding
-actinin exhibit
normal properties (Rivero et al.,
1996
). Thus, either essential regulatory pathways controlled by
Rap1 and by
-actinin and gelation factor intersect at some point or
there are multiple means of generating the phenotype seen in Rap1-depleted
cells. In any case, it is unlikely the deleterious effects resulting from Rap1
depletion are solely caused by the regulation of
-actinin.
The activation of guanylyl cyclase is important for the response to
hyperosmolarity (Kuwayama et al.,
1996; Oyama,
1996
), and the data we have provided indicate that Rap1 plays a
role in the activation of guanylyl cyclase. In particular, there was a reduced
accumulation of cGMP in cells expressing rapA antisense RNA in
response to osmotic shock, and the osmosensitivity of these cells was
partially reduced by the addition of the cGMP analog 8Br-cGMP. In addition, we
showed that Rap1 was activated in response to osmotic shock during cGMP
accumulation. These results suggest that Rap1 acts upstream in the pathway
that transmits the signal responsible for guanylyl cyclase activation.
Consistent with this interpretation, EDTA treatment, which stimulates cGMP
production in response to osmotic stress
(Oyama, 1996
), also stimulated
the activation of Rap1. The activation of guanylyl cyclase in response to
osmotic shock does not appear to involve the heterotrimeric G protein complex,
as gß-null cells have no deficiency in cGMP accumulation
(Kuwayama and van Haastert,
1998
). However, GTP
S stimulates guanylyl cyclase in vitro
(Janssens et al., 1988
;
Janssens et al., 1989
) and in
electro-permeabilized cells (Schoen et
al., 1996
), suggesting an interaction between guanylyl cyclase and
a GTP-binding protein. Rap1 could be this presumptive GTP-binding protein.
A putative intracellular histidine kinase, encoded by the dokA
gene is important in osmoregulation, demonstrated by the fact that a
dokA-null strain exhibits increased sensitivity to osmotic shock
(Schuster et al., 1996).
However, the dokA-null strain exhibits normal cGMP accumulation in
response to osmotic shock (Schuster et
al., 1996
), and there is now evidence that DokA acts in a
signaling pathway parallel to the cGMP pathway
(Ott et al., 2000
). We found
that Rap1-activation in the dokA-null mutant was similar to that in
the parental Ax-2 strain during the osmotic shock (data not shown), consistent
with the idea that Rap1 acts upstream of guanylyl cyclase. Although Rap1
depletion clearly affected actin tyrosine phosphorylation, there is no
evidence as yet to indicate that this phosphorylation is dependent on guanylyl
cyclase activation.
Rap1 activation does not occur under all stress conditions, as shown by the fact that there was no activation in response to low osmolarity or to high temperature (30°C). However, Rap1 was activated in response to low temperatures (8°C). These results indicate that Rap1 is not activated as part of a general stress response, signal transduction pathway.
An intriguing question is whether Rap1 has a function during
Dictyostelium differentiation. It had been shown previously that the
overexpression of Rap1 during development was capable of partially reversing
the developmental defects produced by activated RasD, suggesting a possible
role for the protein during development
(Louis et al., 1997). However,
other than the fact that the Rap1 protein levels remain constant during
development (S. M. Robbins, PhD thesis, University of British Columbia, 1991),
nothing more is known about a possible developmental function. Rap1-depleted
cells do exhibit delayed differentiation (data not shown), but this may simply
be caused by the reduced viability of these cells.
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Acknowledgments |
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