1 School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street,
Dundee DD1 5EH, UK
2 University College London, Ludwig Institute for Cancer Research, The Cruciform
Building, Gower Street, London WC1E 6BT, UK
Author for correspondence (e-mail:
j.g.williams{at}dundee.ac.uk)
Accepted 16 October 2003
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SUMMARY |
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Key words: Dictyostelium, STAT (signal transducer and activator of transcription) protein, SH2 domain:phosphotyrosine interaction, Growth control, Discoidin 1
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Introduction |
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The JAK-STAT signal transduction pathway is an example of `fast track'
signalling, from the plasma membrane to the nucleus, that relies upon SH2
domain-phosphotyrosine interactions (reviewed by
Bromberg and Darnell, 2000;
Chatterjee-Kishore et al.,
2000
; Horvath,
2000
). When a cytokine binds to its receptor it induces
multimerisation of the receptor chains and this activates a member of the JAK
(Janus kinase) family, that tyrosine phosphorylates the receptor at specific
positions. These tyrosine phosphorylated residues act as docking sites for the
SH2 domains of STAT proteins and the STAT proteins are themselves tyrosine
phosphorylated by the JAKs. The tyrosine phosphorylated STATs then undertake
reciprocal SH2 domain-phosphotyrosine interactions and subsequently accumulate
in the nucleus. Dimerisation has generally been thought to be dependent upon
tyrosine phosphorylation (Shuai et al.,
1994
). However, in a recent study, unphosphorylated STAT1 and
STAT3 molecules in unstimulated cells were shown to exist as homodimers
(Braunstein et al., 2003
). This
suggests that tyrosine phosphorylation re-configures a pre-formed STAT dimer,
such that it becomes biologically functional.
In Dictyostelium two STAT proteins and three SH2 domain-containing
kinases have been described (Fukuzawa et
al., 2001; Kawata et al.,
1997
; Moniakis et al.,
2001
). The Dd-STATa and Dd-STATc proteins contain, in their
C-terminal proximal regions, an SH2 domain, a DNA-binding domain and a site of
tyrosine phosphorylation. All three regions are conserved with respect to the
metazoan STATs but the N-terminal-proximal regions of the two Dictyostelium
STATs are highly diverged.
The Dd-STATa protein is activated by extracellular cAMP signalling and, at
the slug stage of development, it becomes nuclear localised in the subset of
pstA cells that constitute the slug tip
(Araki et al., 1998). Dd-STATa
functions there as an inducer of tip cell differentiation and a repressor of
stalk cell differentiation (Fukuzawa and
Williams, 2000
; Mohanty et
al., 1999
). The Dd-STATc protein is activated by the stalk cell
inducer DIF and, at the slug stage, Dd-STATc becomes nuclear localised in the
pstO cells: a band of cells that lies immediately behind the pstA cells
(Fukuzawa et al., 2001
).
Dd-STATc is a repressor that prevents pstA-specific gene expression in the
pstO region (Fukuzawa et al.,
2001
).
During the hybridisation screen that yielded Dd-STATc
(Fukuzawa et al., 2001) we
isolated a third STAT: Dd-STATb. Here, we analyse Dd-STATb and show that,
despite its highly unusual SH2 domain, it is a regulator of cell growth and of
specific gene expression. We also analyse its biochemical properties and
present evidence to suggest that it uses an unorthodox activation pathway.
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Materials and methods |
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Molecular modelling
A model of the Dd-STATb SH2 domain was built on the basis of the
crystallographic structure of STAT1 (Chen
et al., 1998). Initial alignment was made using the ClustalW
method, this alignment was manually changed to take into account structural
information from STAT1 and the Src SH2 domains. The final alignment was then
used to construct the model by using the suite of programs within
QuantaTM. The target (Dd-STATb) protein and template (STAT1) were aligned
by hand within QuantaTM. Where the target sequence matched the template
molecule, the residue coordinates from the template were transformed directly
to the target. Where no equivalent atoms were found in the template molecule
for the target protein, reference was made to a side chain rotamer library.
This defines the most common conformation found for each side chain type
(Summers and Karplus, 1989
).
Gaps in the target sequence were subjected to local energy minimisation to
bring the core ends together and to alleviate local conformational strain.
Although insertions in the target sequence were modelled by searching a
fragment database of high-resolution structures (<1.5 Å) to find an
appropriate template. The final structure was subjected to 500 steps of
steepest gradient minimisation by the CHARMM program to make minor shifts in
the coordinate positions, thereby alleviating steric clashes between atoms and
obtaining a reasonable peptide geometry.
Creation of mutations in Dd-STATb
Dd-STATb cDNA fragments were cloned into the Dictyostelium vector
pDXA and manipulated in E. coli. The Y to F mutation was created by
PCR amplification using a mismatched primer, while the L to R mutation was
created by site directed mutagenesis using the `GeneEditor' kit (Promega,
Ltd.). The mutated fragments, and the unmutated equivalent, were then cloned
under the transcriptional control of the Actin 15 promoter and transformed
into Dictyostelium using G418 selection.
Western transfer, immunoprecipitation and immunohistochemical staining
Western analysis was performed essentially as in Fukuzawa et al.
(Fukuzawa et al., 2001) using
2x107 cells. The membrane was blocked with 5% milk powder
then reacted overnight with the primary antibody C:STATb, a monoclonal
antibody raised against the C-terminal 15 amino acids of Dd-STATb. It was used
as a 1 in 20 dilution of the culture medium from C:STATb hybridoma cells. The
majority of immunohistochemical analyses also used the C:STATb antibody but a
few experiments employed a purified polyclonal antibody, pC:STATb. This was
raised against the C-terminal 15 residue peptide that was used to raise
C:STATb and affinity purified using the same peptide
(Araki et al., 1998
).
Immunoprecipitation was performed using cell lysates prepared from growing
cells again, as described by Araki et al.
(Araki et al., 1998
).
For immunochemical analysis, cells or developmental structures were fixed with 50% methanol/KK2 for 5 minutes, then with 100% methanol for 5 minutes. They were then stained with culture medium from the C:STATb hybridoma cells and detected using a goat anti-mouse secondary antibody labelled with Alexa Fluor 488 (Molecular Probes, Oregon, USA). The pC:STATb antibody was detected using a goat anti-rabbit secondary antibody labelled with Alexa Fluor 594 (Molecular Probes, Oregon, USA).
Generation of Dd-STATb null strains
The construct used to disrupt the Dd-STATb contains a genomic fragment with
1.4 kb of DNA upstream of the blasticidin resistance cassette and 0.55 kb
downstream of the blasticidin resistance cassette. The blasticidin resistance
cassette interrupts the Dd-STATb gene at its unique BamHI site, at
nucleotide 2536 within the coding region. The disruptant DNA fragment was
excised with SalI and HindIII and electroporated into
Dictyostelium. This procedure gave a very high proportion (90%)
of homologous integrants.
Analysis of gene expression
Microarray analysis was performed using the PCR products from 1700 cDNA
clones, chosen from a set of Dictyostelium-expressed sequence tags
(Morio et al., 1998)
(http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html).
The cDNA clones were amplified 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. These DNA probes were then
hybridised and analysed as described by Araki et al.
(Araki et al., 2003
) The images
were analysed using GeneSpring (Silicin Genetics) and probes showing a twofold
or greater enrichment (see text) were subjected to secondary analysis by
northern transfer. This was performed as described elsewhere
(Fukuzawa et al., 1997
), using
total RNA at 10 µg per lane but with `ExpressHyb' solution (Clontech, Palo
Alto, USA) used as the hybridisation buffer.
Sedimentation analysis of Dd-STATb
Extracts from cells growing at 2x106/ml were layered on
10%-40% glycerol gradient and centrifuged for 40 hours at 285,000
g in a Beckman SW41 rotor
(Shuai et al., 1994).
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Results |
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To further analyse the Dd-STATb SH2 domain peculiarities, a model based on
the crystallographic structure of STAT1
(Chen et al., 1998) was built.
The model illustrates the effects of the arginine to leucine change in more
detail. Fig. 2A shows the
C
of Dd-STATb and the C
of STAT1 superimposed, with the arginine
and leucine residues highlighted. The large insertion (insert-1) is also
obvious. The model identifies another shorter, region of insertion (insert 2),
which was not as apparent from sequence alignment alone. The superposition of
the Src-SH2 domain and its bound phosphopeptide with Dd-STATb shows that the
ßB5-leucine residue probably has little or no interaction with the
tyrosine phosphate (Fig. 2B).
However, arginine12 (which is either a lysine or arginine in the other SH2
domain sequences, Fig. 1B) is
still able to form interactions with phosphotyrosine. The model
(Fig. 2C) shows that the large
insertion (insert 1) is predicted to have little or no effect on dimerisation
or DNA binding. It may also be relevant that the regions of the two insertions
are highly variable between STAT1 and STAT3
(Fig. 2D).
|
|
In order to determine whether the Dd-STATb null mutation is phenotypically silent because of mutually redundancy with Dd-STATa or Dd-STATc, we determined the phenotypes of double mutants of Dd-STATb with the other two STATs. A Dd-STATa-/Dd-STATb-double mutant shows the same developmental behaviour as a Dd-STATa-strain and a Dd-STATc-/Dd-STATb-strain is indistinguishable from a Dd-STATc-strain (data not shown). Thus, Dd-STATb does not appear to be functionally redundant with the other two known Dictyostelium STATs. One important caveat must, however, be applied in the case of Dd-STATa. Dd-STATa null cells arrest development early in culmination. Hence, redundancy between Dd-STATb and Dd-STATa in later development is intrinsically non-assayable.
Absence of the Dd-STATb protein places cells at a growth disadvantage
Despite our inability to detect any defect in the growth or development of
Dd-STATb-cells, we reasoned that Dd-STATb must have a function that gives
wild-type strains a selective advantage. Otherwise, its retention over
evolutionary time would be very difficult to explain. We therefore performed
growth competition experiments, in which mixtures of Dd-STATb- and Dd-STATb+
cells were repeatedly transferred to fresh medium after growth to saturation.
The fractional representation of Dd-STATb+ cells was determined at the end of
each cycle of growth by immunostaining.
Initial experiments showed that the assay is extremely sensitive to intrinsic variations in the growth rate of the control, `parental' strain. Hence, the experimental design we eventually adopted was to analyse entire pools of blasticidin resistant colonies, generated using the Dd-STATb disruption construct. Each pool derived from a separate transformation and contained the progeny of approximately 100-200 `founder' clones. The Dd-STATb disruption construct is very efficient and, at the start of each experiment, 80% to 90% of the cells contained a disrupted Dd-STATb gene. The remaining 10%-20% were cells where Dd-STATb was expressed normally and where the blasticidin resistance gene had presumably integrated, non-homologously, at random sites in the genome (`random integrants'). We reasoned that, if large numbers of cells were analysed (to average out the occasional effects that random integration of the vector might have on cell growth in particular clones), the random integrants would provide the best available `isogenic' controls.
The results presented in Fig
4A are from a typical serial passage experiment and
Fig. 4B is a summary of the
results of four additional experiments. The proportion of Dd-STATb+ cells rose
from 15% to just over 80% during the course of four cycles of growth to
saturation. Thus, Dd-STATb null cells are at a selective growth disadvantage
as compared with control cells. It must, however, be stressed that this is a
very subtle growth defect that is only revealed when Dd-STATb-cells are placed
in competition with Dd-STATb+ cells, by growth through repeated cycles;
parallel comparisons of the growth rates of separate Dd-STATb+ and
Dd-STATb-cell populations, over just one growth cycle, are simply not
sensitive enough to detect the difference.
|
Micro-array analysis reveals gene expression changes in Dd-STATb null cells
The growth stage function of Dd-STATb, implied by the above competition
studies, was analysed further using a microarray bearing PCR products from
1700 ESTs (Morio et al.,
1998). The microarray was hybridised with equal amounts of
parental and Dd-STATb-cell cDNAs, prepared using RNA from cells growing
axenically. Thirty-eight ESTs showed hybridisation signals that differed at
least twofold, between the Dd-STATb null strain and the random integrant, and
that duplicated when the direction of dye labeling was reversed
(Table 1).
|
The northern transfer was performed, using RNA prepared from cells at different stages during growth to saturation. As expected, Dd-STATb-cells show a lower than normal level of expression of HGPRT and a higher than normal level of expression of smlA, discoidin 1 and DdCAD-1 (Fig. 5A). Because the level of discoidin 1 overexpression is relatively small, and only becomes manifest at low cell densities, we also compared discoidin 1 expression levels in five separate disruptant clones and five separate random integrant clones. All the Dd-STATb-strains display a several fold higher level of discoidin 1 expression than the random integrants (Fig. 5B).
|
|
When cells are exposed to a hyper-osmotic shock, Dd-STATc is activated and
sediments on a glycerol gradient with the apparent molecular weight of a dimer
(Fukuzawa et al., 2001;
Araki et al., 2003
). This
provides a convenient size marker; because a Dd-STATc dimer has a predicted
molecular weight of 214 kDa, whereas a Dd-STATb dimer has a predicted
molecular weight of 260 kDa. Hence, gradient analysis of Dd-STATb was
performed, using samples isolated from cells stimulated with sorbitol.
The glycerol gradient fractions were subjected to sequential western blot analysis, using antibodies directed against Dd-STATc and Dd-STATb. The sorbitol induction in this experiment was very efficient and most of the Dd-STATc protein sedimented as a dimer (Fig. 7). The Dd-STATb protein also sedimented in this region of the gradient; the DdSTATc peak is in fraction 11, whereas the DdSTATb protein sediments slightly more quickly and its peak lies between fractions 11 and 12. Thus, allowing for the relatively low resolution afforded by the gradient separation technique, the sedimentation rate of Dd-STATb is consistent with its being part of a dimer; although it could of course be monomeric Dd-STATb complexed with another protein or proteins.
|
|
The unmutated (wild type), LR and YF forms of the Dd-STATb protein were expressed under the control of a semi-constitutive promoter in Dd-STATb null cells. All three constructs produce proteins of the size expected for Dd-STATb (data not shown). We tried repeatedly to determine whether the different constructs correct the growth defect of Dd-STATb null cells, by performing co-cultivation with random integrant cells. Unfortunately, this proved impossible because of widely differing growth rates in cells overexpressing the control (i.e. the unmutated) Dd-STATb protein. The transformants were selected using G418. Hence, the integrated constructs are present at a high and variable copy number. We believe that the growth competition assay system is very sensitive to this variation in Dd-STATb copy number, perhaps because of a dominant-negative effect of the overexpressed Dd-STATb protein on cell growth rate.
Because we could not obtain reproducible growth results, using clones transformed with the wild-type construct, we could not study the biological behaviour of the two mutants further and this same problem also precluded the use of microarray analysis to study the Dd-STATb mutants. We therefore analysed the biochemical and cytological properties of the two mutant proteins. Both proteins sediment on glycerol gradients in the approximate position expected for a homodimer (Fig. 9) and both are nuclear enriched (Fig. 10). Hence, in so far as we are able to assay it, the two mutations do not seem to interfere with Dd-STATb function.
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Discussion |
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There are no apparent genetic interactions between Dd-STATb and Dd-STATa or between Dd-STATb and Dd-STATc; the double mutants display phenotypes that are indistinguishable from the Dd-STATa and Dd-STATc-null phenotypes. In addition, co-immunoprecipitation and genetic studies provide no evidence for heterodimerisation of Dd-STATb with either Dd-STATa or Dd-STATc. Our inability to detect a developmental defect in the Dd-STATb null strain, and the absence of additive effects in the Dd-STATb double mutants with Dd-STATa and c, led us to employ a growth competition assay to search for a role for Dd-STATb. This revealed a phenotype; Dd-STATb null (Dd-STATb-) cells are at a growth disadvantage when subjected to multiple cycles of co-cultivation with Dd-STATb+ cells.
The weak growth phenotype led us to perform micro-array analysis using RNA
from growing Dd-STATb+ and Dd-STATb-cells. Twenty-nine genes, from the total
of 1700 non-redundant ESTs analysed, were overexpressed in Dd-STATb null cells
while nine genes were underexpressed. There is a clear preponderance of
overexpressed genes and, in this context, all three Dictyostelium
STATs share one interesting characteristic; they lack the C-terminal
transactivation domains that are a general feature of metazoan STATs. This may
explain why Dd-STATa and Dd-STATC also serve as transcriptional repressors
(Mohanty et al., 1999;
Fukuzawa et al., 2001
).
The microarray results were confirmed for HGPRT, a gene that is
underexpressed in the null strain, and for three of the genes that are
overexpressed: smlA, discoidin 1 and Dd-CAD-1. The SmlA protein controls the
secretion of a factor that regulates the number of cells that participate in
the formation of individual developing structures
(Brock et al., 1996). We see no
effect on territory size because of the overexpression of smlA but, in the
microarray assay, we surveyed only about 15% of the expressed genes in the
organism and any number of other changes could be occurring to ameliorate the
effects of the quantitative change in SmlA levels. Dd-CAD-1 is a
Ca2+-dependent cell adhesion molecule
(Wong et al., 1996
).
Interestingly, growth conditions have a significant effect on the expression
of Dd-CAD-1 (Yang et al.,
1997
). In addition, the three discoidin I genes are particularly
well characterised as markers of the growth-development transition (under the
hybridisation conditions used, the probe probably recognises the transcripts
of all three discoidin 1 genes, so we will assume we are analysing their
composite behaviour).
Discoidins I, Iß and I
encode developmentally regulated
lectins. The three genes are not expressed in bacterially grown cultures at
low cell densities but cells growing in axenic culture express the discoidin
I
and I
genes at a low level
(Devine et al., 1982
). Two
different protein factors, PSF and CMF, serve as cell density sensors,
regulating discoidin gene expression
(Rathi et al., 1991
;
Blusch et al., 1995
) and the
signalling pathway has been extensively characterised; PKA, RasG and G
2
all function as modulators of discoidin I gene expression
(Primpke et al., 2000
;
Secko et al., 2001
;
Blusch et al., 1995
), and the
promoter of the discoidin I
gene has been dissected into its functional
components (Vauti et al.,
1990
). It will be of interest to determine how Dd-STATb fits into
this complex regulatory network.
The above studies show that Dd-STATb is nuclear enriched, that it regulates gene expression, both during growth and development, and that it is required for optimal cell growth. The fact that Dd-STATb is a cellular regulator was not, however, at all predictable from its structure. Dd-STATb contains a DNA-binding domain and a site of tyrosine phosphorylation that are well conserved relative to metazoan STATs but the SH2 domain displays two highly unusual features that might have been expected to abrogate its function: a 15 amino acid insertion and the substitution of an otherwise invariant arginine residue.
In the absence of a three-dimensional structure for Dd-STATb, it is
difficult to judge the extent of the functional disruption caused by the
structural variation and therefore a modelling study was carried out. The
substituted arginine residue (R175 in pp60c-src) is
universally conserved among SH2 domains, it makes direct ionic interactions
with the phosphate group of the phosphotyrosine and is the residue that is
usually subjected to site-specific mutation when an SH2 domain is to be
inactivated (Bibbins et al.,
1993; Bradshaw et al.,
1999
; Shuai et al.,
1993
; Tian and Martin,
1996
). Indeed, the equivalent arginine residue fulfils the same
function, of binding phosphotyrosine, in the most divergent SH2 domain
described to date; that of the Cbl oncogene, a highly abnormal SH2 domain that
was only clearly recognised as such when its three dimensional structure was
determined (Meng et al.,
1999
).
The presence of such an unusual SH2 domain in Dd-STATb is intriguing, because SH2 domains were discovered in the metazoa and Dictyostelium is the only non-metazoan species shown to possess functional SH2 domains. The SH2 domain of Dd-STATb could, therefore, be providing an insight into ancestral SH2 domains; lost during animal evolution but retained in Dictyostelium. However, it is equally possible that the form of SH2 domain found in Dd-STATb arose after the divergence of metazoa and protozoa and that it affords Dictyostelium a signalling potential not possessed by animals.
There are metazoan precedents that may provide insights into the mode of
action of the Dd-STATb SH2 domain. The fact that STAT1 and STAT3 homodimerise
prior to their activation (Braunstein et
al., 2003) indicates that STAT proteins have an intrinsic capacity
for self association. However, the STAT homodimers so formed do not bind to
DNA (Braunstein et al., 2003
),
hence they are biologically non-functional. By contrast, the SH2 domain of
SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome,
functions by binding to a specific sequence within the cytoplasmic tail of the
SLAM (Coffey et al., 1998
;
Nichols et al., 1998
;
Sayos et al., 1998
).
Structural and biochemical analysis shows that the recognition site within
SLAM is bound by the phosphotyrosine binding pocket of SAP in a mode that does
not require tyrosine phosphorylation (Poy
et al., 1999
; Sayos et al.,
1998
). This interaction is possible because the SAP binding site,
within the SLAM receptor, contains additional residues, upstream of the site
of tyrosine phosphorylation, that are not present in orthodox SH2
domain-binding sites and that are specifically recognised by the SAP SH2
domain.
The above example shows how an SH2 domain can functionally interact with a
non-tyrosine phosphorylated ligand but SLAP is a highly unorthodox, `free' SH2
domain protein; as its name implies it is comprised of only an SH2 domain with
a very small C-terminal extension. However, there is a prior study with an R
to L mutant form of an SH2 domain within the context of a larger protein. When
R175 within the Src SH2 domain is mutated to leucine, binding to a Src
phosphopeptide is almost completely eliminated
(Bibbins et al., 1993).
Surprisingly, binding to a peptide from the PDGF receptor (PD751) is only
marginally reduced. Furthermore, binding of the R to L mutant SH2 domain to
the PDGF receptor peptide occurs via a mechanism that is again independent of
tyrosine phosphorylation.
The fact that SH2 domains can, under some circumstances, interact with non-tyrosine phosphorylated ligands is of course relevant only if Dd-STATb is not tyrosine phosphorylated in vivo. Limited support for this idea comes from our inability to detect tyrosine phosphorylation of Dd-STATb, using an antibody specific for phosphotyrosine to probe immunoprecipitated Dd-STATb protein (N.V.Z. and J.G.W., unpublished). This result should not, however, be over-interpreted, because a very low level of tyrosine phosphorylation may not have been detected but could be biologically significant. A much more telling result derives from mutational analysis. The Y to F mutant of Dd-STATb functions sediments as a dimer and is nuclear enriched. In combination, these facts suggest that Dd-STATb functions by a mechanism that is significantly different from the standard STAT paradigm.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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