1 School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow
Street, Dundee, DD1 5EH, UK
2 Institute of Biological Sciences, University of Tsukuba,Tsukuba, Ibaraki
305-8572, Japan
3 Department of Biology, Osaka University, Machikaneyama 1-16, Toyonaka, Osaka
560-0043, Japan
4 Novartis Foundation (Japan) for the Promotion of Science, Roppongi, Minato-ku,
Tokyo 106-0032, Japan
Author for correspondence (e-mail:
j.g.williams{at}dundee.ac.uk)
Accepted 24 March 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dictyostelium, DIF, STAT, Stress response
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When Dictyostelium cells are subjected to hyperosmotic shock,
guanylyl cyclase activity increases and cGMP levels within the cell rise
(Kuwayama et al., 1996;
Oyama, 1996
;
Roelofs and Van Haastert,
2002
). Analysis of KI-8, a chemically induced mutant that is
defective in cGMP accumulation, suggested that osmotically induced elevation
of intracellular cGMP leads to phosphorylation of the myosin II heavy chain
(Kuwayama et al., 1996
). This
conclusion was supported by the fact that 8-bromo-cGMP induces myosin
phosphorylation and prevents osmotic lethality in KI-8 cells. The gene
encoding, sGC, a soluble guanylyl cyclase that is activated by osmotic stress,
has been isolated, but the relationship between sGC activity, myosin
phosphorylation and the response to osmotic stress was not reported
(Roelofs et al., 2001
;
Roelofs and Van Haastert,
2002
).
Intracellular cAMP levels also rise in response to hyperosmotic shock and
this is mediated by the DokA protein (Ott
et al., 2000; Schuster et al.,
1996
). A null mutant for DokA (a dokA- strain) displays reduced
viability when exposed to high osmolarity, and artificial elevation of the
intracellular cAMP concentration, by exposure to 8-bromo-cAMP, is
osmoprotective in dokA- cells (Ott et al.,
2000
). DokA is a hybrid histidine kinase. On exposure to osmotic
stress, DokA is activated by serine phosphorylation and it functions as a
negative regulator of the two-component system that controls intracellular
cAMP concentration (Chang et al.,
1998
; Thomason et al.,
1998
; Thomason et al.,
1999
; Wang et al.,
1996
). DokA is believed to regulate this system, the RdeA:RegA
system, by acting as a phosphatase for RdeA
(Ott et al., 2000
).
The link between the above three response systems is unclear. In a dokA-
strain cGMP accumulates after hyperosmotic stress and the cells show a normal
shrinkage reaction (Schuster et al.,
1996; Ott et al.,
2000
). This suggests that cGMP functions either upstream of DokA
or in a parallel pathway. Also, PTP3 is phosphorylated in response to osmotic
shock in dokA- cells (Gamper et al.,
1999
), suggesting that DokA does not lie in an upstream part of
the PTP3 modification pathway.
In fission yeast, mammalian cells and plant cells, a variety of cellular
stresses activate mitogen-activated protein (MAP) kinase cascades that
function by regulating the activity of specific transcription factors
(Toone and Jones, 1998;
Waskiewicz and Cooper, 1995
).
There is, however, no evidence for MAP kinase involvement in the hyperosmotic
stress response of Dictyostelium. In addition to activating MAP
kinase cascades, animal cells activate specific Janus kinase-signal transducer
and activator of transcription (JAK-STAT) signalling pathways when subjected
to osmotic or oxidative stress (Bode et
al., 1999
; Carballo et al.,
1999
; Gatsios et al.,
1998
). The mechanisms are not known in detail but their activation
by osmotic shock is thought to be triggered by the cell shrinkage associated
with hyperosmosis and is again thought to involve a MAP kinase cascade
(Gatsios et al., 1998
;
Bode et al., 1999
). STAT
proteins contain three highly conserved domains: a DNA binding site, an SH2
domain and a site of tyrosine phosphorylation (reviewed by
Bromberg and Chen, 2001
;
Chatterjee-Kishore et al.,
2000
; Horvath,
2000
). On tyrosine phosphorylation, most often by a member of the
JAK family, STAT monomers dimerise, via mutual SH2 domain:phosphotyrosine
interactions, and accumulate in the nucleus.
Dictyostelium uses STATs but no JAKs have been reported, and the
only known roles for the STATs are developmental. The Dd-STATa protein becomes
tyrosine phophorylated and accumulates in the nucleus when extracellular cAMP
binds to its cell-surface serpentine receptor
(Araki et al., 1998;
Kawata et al., 1997
). DIF
(differentiation-inducing factor) is a chlorinated hexaphenone, produced by
the developing cells, which was originally identified by its ability to induce
stalk cell differentiation in a monolayer assay system
(Kay and Jermyn, 1983
;
Morris et al., 1987
;
Town et al., 1976
). DIF
induces the differentiation of pstO cells
(Thompson and Kay, 2000
), a
band of prestalk cells lying in the rear part of the prestalk region,
immediately behind the pstA cells (Early
et al., 1993
). Dd-STATc is activated by DIF and accumulates in the
nuclei of pstO cells (Fukuzawa et al.,
2001
). The Dd-STATc null mutant has an accelerated rate of early
development and a defect in the regulation of entry into terminal development
(Fukuzawa et al., 2001
)
Dd-STATc functions as a transcriptional repressor that prevents ectopic
pstA-specific gene expression in the pstO cells
(Fukuzawa et al., 2001
).
Our previous study showed that there is a low level of tyrosine
phosphorylation of Dd-STATc in cells newly set up for development
(Fukuzawa et al., 2001).
Tyrosine phosphorylation was quickly lost as the cells entered development but
reappeared several hours later. We suggested that its presence in newly
developing cells might be a stress response induced by the manipulations
necessary to transfer the cells out of growth medium. To determine whether
Dd-STATc is stress inducible we have characterised its response to
hyperosmotic shock, because this is the best-characterised
Dictyostelium stress-response pathway. We show that Dd-STATc is
indeed stress activated and that it mediates specific stress-induced gene
transcription.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemical staining, western and northern transfer
Immunohistochemical detection was performed as described previously
(Araki et al., 1998). For
Dd-STATc, the 7H3 monoclonal antibody
(Fukuzawa et al., 2001
) was
used and for Dd-STATa monoclonal antibody D4 was used
(Araki et al., 1998
). For
western analysis, 2x107 cells were lysed in 100 µl of SDS
sample buffer and proteins were separated by SDS-PAGE and then transferred
onto a Hybond C 'extra' membrane (Amersham Bio-science, Amersham, UK). The
membrane was blocked with 5% skimmed milk in TBS containing 0.5% Tween-20 for
30 minutes and then reacted overnight with primary antibody, CP22 - a
monoclonal antibody that recognises tyr922 of Dd-STATc only when it is in its
phosphorylated form (Fukuzawa et al.,
2001
). The membranes were incubated with secondary antibodies
followed by a reagent that allows chemiluminescent detection (Pierce and
Warriner, Chester, UK). For checking the amount of Dd-STATc, membranes were
stripped and reprobed with 7H3 antibody. Northern analysis was performed as
described by Fukuzawa and Williams (Fukuzawa and Williams, 1997), except that
total RNA at 15 µg per lane was used.
Microarray analysis
The PCR products from 334 cDNA clones (see supplementary data at
jcs.biologists.org/supplemental),
chosen from a set of Dictyostelium-expressed sequence tags
(Morio et al., 1998)
(http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html),
were used as probes in the microarray. The cDNA clones were amplified in a
first PCR reaction using primers flanking the vector sequences. The resultant
PCR products were used as templates for a second PCR reaction, using nested
primers, to generate probes. The first and second PCR reactions were run for
35 and 40 cycles, respectively, with a 2 minute extension time using
'TITANIUM' Taq DNA polymerase (Clontech, BD Bio-science, Oxford, UK). These
DNA probes were printed on CMT-GAPS2 coated slides (Corning Ltd, UK) using a
MicroGrid-II microarray robot (BioRobotics, Cambridge, UK). Fluorescently
labelled cDNA targets were prepared using the methods described at
http://cmgm.stanford.edu/pbrown/protocols/4_yeast_RNA.html.
They were produced from 50-
60 µg of total RNA with the use of oligo-dT
primers, Superscript II DNA polymerase (Life Technologies, Invitrogen Ltd,
Paisley, UK) and Cy3- or Cy5-conjugated dCTP (Amersham Pharmacia, Amersham,
UK). Unincorporated dyes were removed using QIAquick spin columns (QIAGEN Ltd,
W. Sussex, UK) and the targets were purified with microcon-30 filters
(Millipore Ltd, Watford, UK) before resuspending in 20 µl of hybridization
solution (5xSSC, 0.1% SDS, 50% formamide). Slide processing and
hybridisation were performed according to the instructions at
http://www.corning.com/lifesciences.
The denatured targets were deposited on the microarrays after the addition of
37.5 µg of yeast tRNA (GICBCO BRL, Invitrogen Ltd, Paisley, UK) and 50
µg of oligo-dA (Amersham Pharmacia) and covered with coverslips.
Hybridisation was carried out overnight in sealed chambers (Corning) at
42°C. The slides were then washed three times, each for 5 minutes, with
0.1xSSC, 0.1% SDS warmed in a water bath at 50°C and rinsed in
0.1xSSC twice before drying. The microarrays were scanned in an
Arrayworx scanner (Applied Precision, Issaquah, USA). The images were analysed
visually and probes showing a clear enrichment for one or other dye were
subjected to secondary analysis by northern transfer.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
After hyperosmotic shock, Dd-STATc translocated to the nucleus with initial kinetics of accumulation that are as rapid as with DIF; the peak of nuclear accumulation was reached approximately 2 minutes after DIF addition (Fig. 1B). Again, as would be expected from the respective activation kinetics (Fig. 1A), Dd-STATc did not detectably exit from the nucleus when cells were treated with sorbitol but it did rapidly exit the nucleus in cells treated with DIF (Fig. 1B).
The biological relevance of the osmotic-stress-inducible activation of
Dd-STATc was assessed by measuring the relative sorbitol sensitivities of
Dd-STATc null cells and control cells
(Fukuzawa et al., 2001). The
strains were either left untreated or exposed to 200 mM sorbitol for 2 hours
(Schuster et al., 1996
).
Samples were then plated onto bacterial lawns to determine the number of
viable cells. The parental strain displayed an apparently higher level of
resistance than the null strain: parental=84±17%, versus
null=59±21% (mean±s.d. from eight experiments, with each
viablity determination assayed in triplicate). However, there is a very large
amount of intrinsic biological variability in this assay and, as can be seen,
the difference was not statistically significant. At the level of
discrimination of this assay, which we consider to be a difference in
viability equal to or greater than twofold, the results would seem to indicate
that the Dd-STATc null mutant is not abnormally sensitive to hyperosmotic
stress.
Specificity of the response: Dd-STATa is not activated by osmotic
stress but other cellular stresses activate Dd-STATc
To determine whether the osmotic stress response of Dd-STATc reflects the
existence of a generalised STAT activation pathway, we determined whether
osmotic stress also activates Dd-STATa. Cells were treated with various
inducers and the nuclear translocation of Dd-STATa was analysed, using
Dd-STATc as a positive control (Fig.
2). As expected, nuclear translocation of Dd-STATa was induced by
extracellular cAMP but not by DIF. Nuclear translocation of Dd-STATa was also
not induced when cells were treated with sorbitol
(Fig. 2). The osmotic stress
response is therefore specific to the DIF-inducible STAT, Dd-STATc.
|
We next determined whether stress conditions other than hyperosmotic shock lead to activation of Dd-STATc. We analysed the tyrosine phosphorylation of Dd-STATc protein isolated from cells exposed to a 33°C heat shock or to the uncoupling reagent di-nitro phenol (DNP). Dd-STATc became specifically tyrosine phosphorylated after both treatments but with different efficiencies, relative to a DIF-treated control (Fig. 3A,B). In the case of heat shock there was a slight (2 to 3 minutes) delay relative to DIF (cf. Fig. 1A), but this was probably caused by the lag period, during which the cells equilibrated at the higher temperature. The extent of the activation at peak level was somewhat greater in the heat-shock-induced cells than in DIF-induced cells (Fig. 3A). DNP was also a slower activator than hyperosmotic stress (Fig. 3B). However, this reflected an intrinsically weaker response because the level of activation never reached that observed with heat shock or osmotic shock. Nonetheless, the results show that Dd-STATc is activated by a generalised stress-response pathway.
|
Nuclear translocation of Dd-STATc in response to sorbitol requires
its specific tyrosine phosphorylation
Although we initially suggested that tyrosine phosphorylation of Dd-STATc
is not necessary for its regulated nuclear accumulation
(Fukuzawa et al., 2001),
subsequent work showed that a mutant form of Dd-STATc, in which the site of
tyrosine modification is altered to a phenylalanine residue (a Y to F mutant),
does not accumulate in the nucleus after DIF treatment
(Fukuzawa et al., 2001
). To
determine whether this is also true for stress induction, a single copy of the
Dd-STATc gene, and of its Y to F mutant form, were transformed into a Dd-STATc
null strain. In both cases GFP was fused at the N-terminus of the Dd-STATc
protein to yield GFP:STATc and GFP:STATc-YF. When the two strains were exposed
to sorbitol, GFP:STATc cells showed translocation of the GFP fusion protein to
the nucleus, while the fusion protein remained cytosolic in GFP:STATc-YF cells
(Fig. 4). Thus, the key event
in stress-induced nuclear translocation of Dd-STATc is, just as in the case of
DIF induction, the activating tyrosine phosphorylation.
|
8-Bromo cGMP induces rapid tyrosine phosphorylation and nuclear
translocation of Dd-STATc
Because of the evidence that intracellular cAMP and cGMP both act as second
messengers in the cellular responses to hyperosmotic shock, we determined the
effects of their respective membrane-permeant analogues. Exposure of cells to
concentrations of 8-bromo cGMP of >1 mM for just 3 minutes
(Fig. 5A) induced tyrosine
phosphorylation of Dd-STATc and, at the highest concentration studied (20 mM),
activation and nuclear accumulation occurred almost as rapidly as with DIF or
sorbitol (cf. Fig. 5B,C with
Fig. 1A,B). There was no
apparent nuclear efflux of Dd-STATc when cells were treated 8-bromo cGMP, that
is, the efflux kinetics mirror that of sorbitol rather than that of DIF.
Again, activation was selective for Dd-STATc, because Dd-STATa was
unresponsive to 8-bromo cGMP (Fig.
2).
|
In contrast to the robust induction of Dd-STATc observed after 3 minutes of induction with 8-bromo cGMP, 8-bromo cAMP barely induced activation of Dd-STATc at this time (Fig. 5A). However, when 20 mM 8-bromo-cAMP was added, in a kinetics experiment, tyrosine phosphorylation and nuclear translocation were detected at the later time points, although the peak levels were lower and were reached more slowly than with 8-bromo cGMP (Fig. 5B,C). Thus, the induction kinetics for Dd-STATc are more consistent with a second messenger role for cGMP than for cAMP.
Analysis of mutants in the known cAMP-mediated and cGMP-mediated
intracellular response pathways
We next studied mutants in the cAMP- and cGMP-mediated stress-response
pathways using western transfer (Fig.
6). After probing the filters with CP22, the monoclonal antibody
that recognises tyr922 of Dd-STATc only when it is in its phosphorylated form,
we re-probed the filter with 7H3, a general Dd-STATc antibody, to check
equivalence of loading. Although equal amounts of total protein were loaded
onto the gel, the amounts of total Dd-STATc protein varied between different
mutants. However, there was very little variation between time-point samples
taken from the same strain. Below, we therefore score the results in a
plus/minus fashion and disregard any quantitative differences in the absolute
induced level of Dd-STATc.
|
We first analysed a strain that lacks DokA, the histidine kinase that forms
part of the cAMP-mediated stress-response pathway. The dokA- strain showed
Dd-STATc tyrosine phosphorylation in response to sorbitol treatment
(Fig. 6). DokA is believed to
function by regulating protein kinase A (PKA) activity, so we also analysed a
strain that is defective in PKA-mediated responses to changes in intracellular
cAMP concentration. A15:Rm cells are transformed with a dominant-negative form
of the R subunit of PKA that blocks C subunit function
(Harwood et al., 1992). The
response of Dd-STATc to sorbitol stress was found to be normal in A15:Rm cells
(Fig. 6). As a further check
for any involvement of PKA, we analysed a null mutant for the catalytic
subunit of PKA (Mann et al.,
1992
), and this also displayed sorbitol-inducible activation of
Dd-STATc (Fig. 6).
We next analysed mutants in the cGMP pathway. The sgc gene encodes
a guanylyl cyclase activity that is inducible by hyperosmotic stress
(Bosgraaf et al., 2002).
Activation of Dd-STATc is responsive to hyperosmotic shock in the sGC null
mutant (Fig. 6). Gca
encodes a guanylyl cyclase that is not stress inducible but, in order to rule
out absolutely some form of functional redundancy with sgc, we
analysed the double null, sGC-/GCA-, strain
(Bosgraaf et al., 2002
). The
strain was stress inducible for Dd-STATc activation
(Fig. 6). Finally, to determine
whether there is functional redundancy between the cGMP and cAMP pathways, we
generated and analysed a dokA-/sGC- strain. This strain was also stress
inducible for Dd-STATc activation (Fig.
6).
Microarray analysis identifies two genes that are osmotic stress
induced and Dd-STATc dependent
Although there has been a significant body of work characterising the
hyperosmotic stress response in Dictyostelium there has, to our
knowledge, been no report of associated gene expression changes. Indeed,
proteomic analysis suggested that there may be none
(Zischka et al., 1999). Given
the results presented above, we were nonetheless encouraged to search for
genes that might be regulated when Dd-STATc is activated by hyperosmotic
stress. We screened for such genes using a microarray of 334 expressed
sequence tags (ESTs) derived from the Dictyostelium cDNA sequencing
project (Morio et al., 1998
).
The cDNA library used for this project was prepared from slug stage cells and
the ESTs were selected because they have all previously been described in
published papers. Information obtained from any positive signals seemed more
likely, therefore, to be more functionally interpretable.
The microarrays were hybridised with a mixed probe prepared using RNA
isolated from cells that were either untreated or exposed to sorbitol for 15
minutes. We identified two ESTs that reproducibly showed a higher signal with
the probe from osmotically stressed cells and that could be confirmed by
northern transfer. These derive from the rtoA
(Brazill et al., 2000) and the
gapA genes (Adachi et al.,
1997
). Both genes are rapidly induced by osmotic stress but
neither gene was inducible by DIF (Fig.
7).
|
We next analysed the induction of the two stress-regulated genes in the
Dd-STATc null strain. Expression of both genes was noninducible by
hyperosmotic stress in the Dd-STATC null strain
(Fig. 7). Although osmotic
stress inducibility is lost in Dd-STATc disruptants, the gapA and
rtoA genes show their normal patterns of semiconstitutive gene
expression in the null strain (data not shown)
(Adachi et al., 1997;
Wood et al., 1996
).
Differential effects of 8-bromo-cGMP and 8-bromo-cAMP on gapA and
rtoA gene expression
Because Dd-STATc is rapidly activated by 8-bromo-cGMP, and because Dd-STATc
is required for stress-induced transcription of gapA and
rtoA, we determined whether 8-bromo-cGMP treatment would activate
gapA and rtoA gene expression
(Fig. 8). The addition of
8-bromo-cGMP produced a detectable increase in the concentration of both
rtoA and gapA mRNA after 15 minutes of induction. By 30 minutes of
induction the expression of rtoA was strongly induced, whereas that
of gapA was less strongly induced. 8-Bromo-cAMP was a much less
potent inducer of the expression of both genes than 8-bromo-cGMP
(Fig. 8). As expected, neither
cyclic nucleotide was effective in inducing gapA or rtoA
gene expression in Dd-STATc null cells but the sgc null mutant
remained fully stress inducible for expression of both genes (data not
shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cGMP is the most likely candidate for second messenger in the stress
pathway
We were able to mimic the effect of sorbitol using 8-bromo cGMP, but
8-bromo-cAMP is a much less effective inducer. Cyclic AMP and cGMP binding
proteins often show a degree of cross-reactivity (reviewed by
Francis and Corbin, 1999).
Hence, we suggest that cGMP is the primary second messenger but that it
interacts with an intracellular cGMP binding protein that also binds cAMP at
low relative affinity. We took advantage of the fact that there are mutants in
the cAMP-mediated stress-response pathways, to further investigate the weak
8-bromo cAMP response. The dokA- strain lacks the histidine kinase that is an
element in the intracellular cAMP mediated pathway and it is hypersensitive to
osmotic shock. We also studied two differently generated strains that are
defective in PKA-mediated signalling. Both forms of mutant show a normal
sorbitol-induced nuclear translocation response for Dd-STATc.
We were not able to perform a similarly definitive genetic analysis for the cGMP-mediated osmotic stress pathway because there are no molecularly defined mutants that block the stress pathway. However, we have analysed a null strain for sGC, the only known osmotically inducible guanylyl cyclase, and found that Dd-STATc is stress activated. We have also analysed a double null mutant with the other known guanylyl cyclase GCA, and we have ruled out possible redundancy between the cGMP and cAMP pathways by analysing a DokA/sGC double mutant.
Because of the above results, we favour the notion of a cGMP-regulated signalling pathway that utilises a guanylyl cyclase that is yet undiscovered; the genome project is not yet complete and biochemical analysis may also have failed to reveal the proposed enzyme. The conclusion that cGMP is the second messenger is supported by our analyses of specific gene activation because, as we go on to discuss, the two stress-inducible genes we identify are much better induced by 8-bromo-cGMP than by 8-bromo-cAMP.
The rtoA and gapA genes are stress inducible
We identified genes that are regulated by hyperosmotic stress using
microarrays bearing the PCR products derived from 334 characterised
Dictyostelium cDNAs. We identified two ESTs, gapA and rtoA,
that are strongly inducible by hyperosmotic stress and by 8-bromo cGMP. Thus,
as in other organisms, hyperosmotic stress induces gene expression changes in
Dictyostelium. The previous failure of two-dimensional gel analysis
to reveal such changes (Zischka et al.,
1999) presumably reflects the greater sensitivity, and reliability
of sample comparison, afforded by the microarray technique.
The principal aim of our study was to determine whether there are
stress-induced gene transcripts that are regulated by Dd-STATc. It was not
intended as a comprehensive microarray analysis, an endeavour that can only be
properly undertaken when the genome sequence is complete. However, the
identity of even this very limited sample of regulated transcripts is of
interest. rtoA encodes a protein that affects cell type choice and it
is thought to bring about this effect by acting on the cell cycle
(Wood et al., 1996). RtoA acts
to promote vesicle fusion and in rtoA null strains cytosolic pH
regulation during the cell cycle is perturbed
(Brazill et al., 2000
). GAPA is
a RasGAP-related protein that is needed for normal cytokinesis
(Adachi et al., 1997
;
Lee et al., 1997
;
Sakurai et al., 2001
). GAPA is
homologous in sequence to mammalian IQGAPs and these are effectors for Rac and
CDC42 - Rho family members that regulate the actin cytoskeleton
(Epp and Chant, 1997
;
Kuroda et al., 1996
;
Osman and Cerione, 1998
).
The known cellular functions of GAPA and RtoA are perhaps consistent with a role in re-structuring the cell after hyperosmotic shock but they are not likely to act in isolation; we assayed only a small number of ESTs and there seems certain to be additional Dd-STATc-regulated genes. GAPA, RtoA and these putative co-induced proteins presumably act in concert to help confer hyperosmotic stress resistance. Also, because Dd-STATc null cells are not hyper-sensitive to the lethal effects of sorbitol treatment, we believe that there may be functional redundancy with other osmoprotective, stress-activated pathways.
The stress-response mechanism can be uncoupled from developmental and
DIF-inducible gene expression
In the absence of stress, expression of the gapA and rtoA
genes in the Dd-STATc null strain is normal. Hence, gene transcription during
growth and development presumably uses different promoter elements from those
used for stress-induced gene expression
(Fig. 9). Furthermore, even
though Dd-STATc transitorily accumulates in the nucleus in response to DIF,
the gapA and rtoA genes are completely non-DIF-inducible
(Fig. 9). Possibly,
gapA and rtoA activation require the relatively prolonged
nuclear persistence of Dd-STATc that is observed after osmotic shock but not
after DIF treatment. Alternatively, hyperosmotic stress may bring about
additional changes, beyond those bought about by DIF, that result in specific
gene activation. There may be, for example, transcriptional co-activators for
Dd-STATc that are recruited to the promoters after osmotic shock but that are
not activated by DIF signalling. Distinguishing these possibilities will
require detailed promoter analyses of the gapA and rtoA
genes.
|
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adachi, H., Takahashi, Y., Hasebe, T., Shirouzu, M., Yokoyama,
S. and Sutoh, K. (1997). Dictyostelium IQGAP-related
protein specifically involved in the completion of cytokinesis. J.
Cell Biol. 137,891
-898.
Aizawa, H., Katadae, M., Maruya, M., Sameshima, M.,
Murakami-Murofushi, K. and Yahara, I. (1999). Hyperosmotic
stress-induced reorganization of actin bundles in Dictyostelium
cells. Genes Cells 4,311
-324.
Araki, T., Gamper, M., Early, A., Fukuzawa, M., Abe, T., Kawata,
T., Kim, E., Firtel, R. A. and Williams, J. G. (1998).
Developmentally and spatially regulated activation of a Dictyostelium
STAT protein by a serpentine receptor. EMBO J.
17,4018
-4028.
Bode, J. G., Gatsios, P., Ludwig, S., Rapp, U. R., Haussinger,
D., Heinrich, P. C. and Graeve, L. (1999). The
mitogen-activated protein (MAP) kinase p38 and its upstream activator MAP
kinase kinase 6 are involved in the activation of signal transducer and
activator of transcription by hyperosmolarity. J. Biol.
Chem. 274,30222
-30227.
Bosgraaf, L., Russcher, H., Smith, J. L., Wessels, D., Soll, D. R. and van Haastert, P. J. (2002). A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium EMBO J. 2,4560 -4570.[CrossRef]
Brazill, D. T., Caprette, D. R., Myler, H. A., Hatton, R. D.,
Ammann, R. R., Lindsey, D. F., Brock, D. A. and Gomer, R. H.
(2000). A protein containing a serine-rich domain with vesicle
fusing properties mediates cell cycle-dependent cytosolic pH regulation.
J. Biol. Chem. 275,19231
-19240.
Bromberg, J. and Chen, X. (2001). STAT proteins: signal tranducers and activators of transcription. Methods Enzymol. 333,138 -151.[Medline]
Carballo, M., Conde, M., El Bekay, R., Martin-Nieto, J.,
Camacho, M. J., Monteseirin, J., Conde, J., Bedoya, F. J. and Sobrino, F.
(1999). Oxidative stress triggers STAT3 tyrosine phosphorylation
and nuclear translocation in human lymphocytes. J. Biol.
Chem. 274,17580
-17586.
Chang, W. T., Thomason, P. A., Gross, J. D. and Newell, P.
C. (1998). Evidence that the RdeA protein is a component of a
multistep phosphorelay modulating rate of development in
Dictyostelium. EMBO J.
17,2809
-2816.
Chatterjee-Kishore, M., van den Akker, F. and Stark, G. R. (2000). Association of STATs with relatives and friends. Trends Cell Biol. 10,106 -111.[CrossRef][Medline]
Early, A. E., Gaskell, M. J., Traynor, D. and Williams, J.
G. (1993). Two distinct populations of prestalk cells within
the tip of the migratory Dictyostelium slug with differing fates at
culmination. Development
118,353
-362.
Epp, J. A. and Chant, J. (1997). An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast. Curr. Biol. 7,921 -929.[Medline]
Francis, S. H. and Corbin, J. D. (1999). Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit. Rev. Clin. Lab. Sci. 36,275 -328.[Medline]
Fukuzawa, M., Hopper, N. and Williams, J. G.
(1997). CudA: a Dictyostelium gene with
pleiotropic effects on cellular differentiation and slug behaviour.
Development 124,2719
-2728.
Fukuzawa, M., Araki, T., Adrian, I. and Williams, J. G. (2001). Tyrosine phosphorylation-independent nuclear translocation of a Dictyostelium STAT in response to DIF signaling. Mol. Cell 7,779 -788.[CrossRef][Medline]
Gamper, M., Howard, P. K., Hunter, T. and Firtel, R. A. (1996). Multiple roles of the novel protein tyrosine phosphatase PTP3 during Dictyostelium growth and development. Mol. Cell. Biol. 16,2431 -2444.[Abstract]
Gamper, M., Kim, E., Howard, P. K., Ma, H., Hunter, T. and
Firtel, R. A. (1999). Regulation of Dictyostelium
protein-tyrosine phosphatase-3 (PTP3) through osmotic shock and stress
stimulation and identification of pp130 as a PTP3 substrate. J.
Biol. Chem. 274,12129
-12138.
Gatsios, P., Terstegen, L., Schliess, F., Sussinger, D. H.,
Kerr, I. M., Heinrich, P. C. and Graeve, L. (1998).
Activation of the Janus kinase/signal transducer and activator of
transcription pathway by osmotic shock. J. Biol. Chem.
273,22962
-22968.
Harwood, A. J., Hopper, N. A., Simon, M. N., Bouzid, S., Veron, M. and Williams, J. G. (1992). Multiple roles for cAMP-dependent protein kinase during Dictyostelium development. Dev. Biol. 149,90 -99.[Medline]
Horvath, C. M. (2000). STAT proteins and transcriptional responses to extracellular signals. Trends Biochem. Sci. 25,496 -502.[CrossRef][Medline]
Howard, P. K., Sefton, B. M. and Firtel, R. A. (1993). Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes. Science 259,241 -244.[Medline]
Jungbluth, A., Eckerskorn, C., Gerisch, G., Lottspeich, F., Stocker, S. and Schweiger, A. (1995). Stress-induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine-53 adjacent to the DNase I binding loop. FEBS Lett. 375,87 -90.[CrossRef][Medline]
Kawata, T., Shevchenko, A., Fukuzawa, M., Jermyn, K. A., Totty, N. F., Zhukovskaya, N. V., Sterling, A. E., Mann, M. and Williams, J. G. (1997). SH2 signaling in a lower eukaryote: A STAT protein that regulates stalk cell differentiation in Dictyostelium. Cell 89,909 -916.[Medline]
Kay, R. R. and Jermyn, K. A. (1983). A possible morphogen controlling differentiation in Dictyostelium. Nature 303,242 -244.[Medline]
Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N.,
Iwamatsu, A. and Kaibuchi, K. (1996). Identification of IQGAP
as a putative target for the small GTPases, Cdc42 and Rac1. J.
Biol. Chem. 271,23363
-23367.
Kuspa, A. and Loomis, W. F. (1992). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc. Natl. Acad. Sci. USA 89,8803 -8807.[Abstract]
Kuwayama, H., Ecke, M., Gerisch, G. and van Haastert, P. J. M. (1996). Protection against osmotic stress by cGMP-mediated myosin phosphorylation. Science 271,207 -209.[Abstract]
Lee, S., Escalante, R. and Firtel, R. A.
(1997). A Ras GAP is essential for cytokinesis and spatial
patterning in Dictyostelium. Development
124,983
-996.
Mann, S. K. O., Yonemoto, W. M., Taylor, S. S. and Firtel, R. A. (1992). DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 89,10701 -10705.[Abstract]
Morio, T., Urushihara, H., Saito, T., Ugawa, Y., Mizuno, H., Yoshida, M., Yoshino, R., Mitra, B. N., Pi, M., Sato, T. et al. (1998). The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development. DNA Res. 5, 335-340.[Medline]
Morris, H. R., Taylor, G. W., Masento, M. S., Jermyn, K. A. and Kay, R. R. (1987). Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum. Nature 328,811 -814.[CrossRef][Medline]
Nayler, O., Insall, R. and Kay, R. R. (1992). Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum. Eur. J. Biochem. 208,531 -536.[Abstract]
Osman, M. A. and Cerione, R. A. (1998). Iqg1p,
a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the
actin cytoskeleton. J. Cell Biol.
142,443
-455.
Ott, A., Oehme, F., Keller, H. and Schuster, S. C.
(2000). Osmotic stress response in Dictyostelium is
mediated by cAMP. EMBO J.
19,5782
-5792.
Oyama, M. (1996). cGMP accumulation induced by
hypertonic stress in Dictyostelium discoideum. J. Biol.
Chem. 271,5574
-5579.
Rivero, F., Koppel, B., Peracino, B., Bozzaro, S., Siegert, F.,
Weijer, C. J., Schleicher, M., Albrecht, R. and Noegel, A. A.
(1996). The role of the cortical cytoskeleton: F-actin
crosslinking proteins protect against osmotic stress, ensure cell size, cell
shape and motility, and contribute to phagocytosis and development.
J. Cell Sci. 109,2679
-2691.
Roelofs, J. and van Haastert, P. J. (2002).
Characterization of two unusual guanylyl cyclases from Dictyostelium.
J. Biol. Chem. 277,9167
-9174.
Roelofs, J., Meima, M., Schaap, P. and van Haastert, P. J.
M. (2001). The Dictystelium homologue of mammalian
soluble adenylyl cyclase encodes a guanylyl cyclase. EMBO
J. 20,4341
-4348.
Sakurai, M., Adachi, H. and Sutoh, K. (2001). Mutational analyses of Dictyostelium IQGAP-related protein GAPA: possible interaction with small GTPases in cytokinesis. Biosci. Biotechnol. Biochem. 65,1912 -1916.[CrossRef][Medline]
Schuster, S. S., Noegel, A. A., Oehme, F., Gerisch, G. and Simon, M. I. (1996). The hybrid histidine kinase DokA is part of the osmotic response system of Dictyostelium. EMBO J. 15,3880 -3889.[Abstract]
Thomason, P. A., Traynor, D., Cavet, G., Chang, W. T., Harwood,
A. J. and Kay, R. R. (1998). An intersection of the cAMP/PKA
and two-component signal transduction systems in Dictyostelium.
EMBO J. 17,2838
-2845.
Thomason, P. A., Traynor, D., Stock, J. B. and Kay, R. R.
(1999). The RdeA-RegA system, a eukaryotic phospho-relay
controlling cAMP breakdown. J. Biol. Chem.
274,27379
-27384.
Thompson, C. R. L. and Kay, R. R. (2000). The role of DIF-1 signaling in Dictyostelium development. Mol. Cell 6,1509 -1514.[Medline]
Toone, W. M. and Jones, N. (1998).
Stress-activated signalling pathways in yeast. Genes
Cells 3,485
-498.
Town, C. D., Gross, J. D. and Kay, R. R. (1976). Cell differentiation without morphogenesis in Dictyostelium discoideum. Nature 262,717 -719.[Medline]
Wang, N., Shaulsky, G., Escalante, R. and Loomis, W. F. (1996). A two-component histidine kinase gene that functions in Dictyostelium development. EMBO J. 15,3890 -3898.[Abstract]
Waskiewicz, A. J. and Cooper, J. A. (1995). Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals. Curr. Opin. Cell Biol. 7, 798-805.[CrossRef][Medline]
Watts, D. J. and Ashworth, J. M. (1970). Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem. J. 119,171 -174.[Medline]
Wood, S. A., Ammann, R. R., Brock, D. A., Li, L., Spann, T. and
Gomer, R. H. (1996). RtoA links initial cell type choice to
the cell cycle in Dictyostelium. Development
122,3677
-3685.
Zischka, H., Oehme, F., Pintsch, T., Ott, A., Keller, H.,
Kellermann, J. and Schuster, S. C. (1999). Rearrangement of
cortex proteins constitutes an osmoprotective mechanism in
Dictyostelium. EMBO J.
18,4241
-4249.