? Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2
3EJ, UK
* These authors contributed equally to this work
Author for correspondence (e-mail:
jec24{at}cam.ac.uk)
Accepted 4 April 2003
![]() |
SUMMARY |
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Key words: JAK/STAT, Signalling competence, Receptor dimerisation, Protein interaction
![]() |
INTRODUCTION |
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The Drosophila JAK/STAT pathway is functionally identical to the
vertebrate pathway (Heinrich et al.,
1998) but has less components to each level
(Castelli-Gair Hombría and Brown,
2002
; Luo and Dearolf,
2001
). There is only one confirmed element to each level of the
signalling pathway. The ligand is encoded by the unpaired
(upd) gene (Harrison et al.,
1998
), the receptor by domeless (dome)
(Brown et al., 2001
), the JAK
kinase by hopscotch (hop)
(Binari and Perrimon, 1994
),
and the transcription factor by the signal transduction and activator of
transcription (STAT) stat92E gene
(Hou et al., 1996
;
Yan et al., 1996
). UPD
directly binds DOME (Chen et al.,
2002
) and this is thought to bring into proximity the receptor
associated JAK tyrosine kinase HOP. The JAK proteins are thought to
phosphorylate the receptor, creating a docking site for the inactive
cytoplasmic STAT proteins. The STATs become phosphorylated
(Chen et al., 2002
), dimerise
and translocate to the nucleus where they induce gene transcription. The
identical mutant phenotypes of these genes suggest that most of the basic
elements of this pathway have been isolated
(Castelli-Gair Hombría and Brown,
2002
). This view is reinforced by the failure to find other JAK or
STAT proteins in the Drosophila genome databases
(Dearolf, 1999
).
In vertebrates, the active form of the cytokine receptors of the JAK/STAT
pathway have been shown to function as homo- or hetero-dimeric complexes
(Stahl et al., 1994). As DOME
is the only confirmed receptor in Drosophila and is required for all
JAK/STAT functions analysed (Brown et al.,
2001
; Ghiglione et al.,
2002
; Johansen et al.,
2003
), we suspected that it might be forming homo-dimers. To test
whether homo-dimerisation of DOME occurs in Drosophila, and to find
how it is controlled during development, we decided to use a technique that
would allow us to observe dimerisation in situ. The technique that we have
applied is a ß-galactosidase (ß-gal) complementation assay developed
for the study of protein interactions in vertebrate cell cultures
(Blakely et al., 2000
;
Mohler and Blau, 1996
). We
show that this technique, which we rename ßlue-ßlau to distinguish
from other ß-gal complementation assays, allows detection of protein
interactions in whole embryos. Using this technique we present evidence that
DOME homo-dimerises in the ectoderm in tissues where the JAK/STAT pathway is
active. In agreement with vertebrate results, DOME dimerisation is not ligand
induced. Our results indicate that there is a correlation between the tissues
where DOME dimerises and those that can respond to ligand activation,
suggesting that receptor dimerisation confers competence for ligand
activation. These results are very important as they reveal a novel level of
control for cytokine receptor activation.
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MATERIALS AND METHODS |
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Df(1)os1A was used as a null allele of upd. Searches of
the genome databases indicate the presence in Drosophila of three
other genes with related sequence to upd clustered in the 17 region
of the X chromosome: cg5988, cg5963 and cg15062
(Castelli-Gair Hombría and Brown,
2002). Using in situ and PCR we have determined that
Df(1)os1A deletes all four genes (data not shown). On the basis of
the identical phenotype of this deficiency to loss of function for JAK/STAT
signalling, Df(1)os1A can be considered as a null mutant for all
JAK/STAT ligands.
Constructs
UAS-dome or UAS-dome
were
made by using the 5' Nco1 site in the domeless cDNA
(Brown et al., 2001
) fused to
the Nco1 site in the ß-galactosidase
or
mutant genes (Mohler and Blau,
1996
). This fusion was subcloned into the fly transformation
vector pUAST and transgenic stocks made. The fusion proteins are missing the
last 60 amino acids of the carboxyl terminal end of DOME. We tested the
functionality of the hybrid proteins in dome217;
Klu-Gal4/UAS-dome
or dome217;
Klu-Gal4/UAS-dome
flies. On some occasions, as well as
rescuing the mutant phenotype, high levels of ectopic UAS-dome
(Brown et al., 2001
) as well as
the UAS-dome
and UAS-dome
lines
gave dominant negative phenotypes.
Stainings
Complementation was visualised by incubating embryos with the chromogenic
substrate 5-bromo-4-chloro-3-indolyl-ß-D-galactosidase (X-gal). X-gal
stainings were done by fixing dechorionated embryos in a mixture of heptane/1%
glutaraldehyde in PBS for 2 minutes. The glutaraldehyde solution was then
removed and the embryos were devitelinised by adding 70% ethanol and shaking
vigorously in a glass vial. Devitelinised embryos were transferred to a fresh
vial and washed in 70% ethanol, rehydrated in PBS and transferred to X-gal
buffer (Ashburner, 1989). After
adding X-gal substrate from a 20 mg/ml solution in dimethylformamide to a
final concentration of 1.2 mg/ml, the reaction was developed by incubating at
37°C for 1-3 hours depending on the driver line. Cuticle preparations, RNA
in situ hybridisation and antibody stainings were performed as previously
described (Brown and Castelli-Gair
Hombría, 2000
). Only embryos upto stage 15 (0-14 hours)
were analysed; these are the stages for which JAK/STAT requirement during
development is well understood.
Analysis of protein stability
hs-Gal4; UAS-dome UAS-dome
embryos were heatshocked for 15 minutes, 30 minutes or 1 hour to test whether
some tissues accumulate more hybrid protein than others. The embryos were
allowed to develop for different periods and stained with
anti-ß-galactosidase (anti-ß-gal) to detect the expression of the
hybrid proteins. The protein disappeared from all tissues simultaneously with
the exception of the amnioserosa where the protein could be detected at stages
when the rest of the embryo had no detectable ß-galactosidase. Embryos
were analysed upto stage 14. Later stages were not studied because of the
existence of cryptic promoters in the hs-Gal4 line.
![]() |
RESULTS |
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We fused the or the
mutants to the DOME
carboxyl end and expressed the hybrid receptors using the GAL4 system
(Brand and Perrimon, 1993
). The
hybrid receptors are functional as they can rescue dome mutant
phenotypes (Fig. 1A-C). To
determine the sub-cellular localisation of the fusion proteins, we expressed
them in the large, polarised, ectodermal cells of the hindgut and the salivary
gland using the h-Gal4 line. The hybrid receptors localise mainly to
the apical membrane, although some protein can be detected in the cytoplasm
(Fig. 1D-E).
|
The above experiments show that the ßlue-ßlau technique can be used to detect protein interaction in whole animals with sub-cellular resolution. Our results also indicate that in the embryonic ectoderm, DOME is differentially transported to the apical membrane where it homo-dimerises.
DOME homo-dimerisation is developmentally controlled
To investigate the regulation of DOME homo-dimerisation during embryonic
development we compared the expression of the hybrid proteins as detected by
anti-ß-gal antibodies (Fig.
2, left column) with the regions where they complement as tested
by X-gal staining (Fig. 2,
right column). As upd is expressed in the ectoderm
(Harrison et al., 1998), we
first used the ubiquitous ectodermal drivers da-Gal4 and
69B-Gal4 (Brand and Perrimon,
1993
; Wodarz et al.,
1995
) to express the tagged DOME proteins. At early stages, both
Gal4 lines have similar spatio-temporal dynamics and they revealed identical
developmentally regulated patterns of DOME homo-dimerisation. da-Gal4
drives expression of the fusion proteins in most ectodermal cells
(Fig. 2A,C,E). However, not all
of these cells developed the blue coloration after X-gal staining
(Fig. 2B,D,F).
69B-Gal4, which drives expression slightly later
(Fig. 2G,I), revealed very
similar patterns of dimerisation to da-Gal4
(Fig. 2, compare D with H and F with
J).
|
Recently, Gal4VP16 lines have been generated that are stronger inducers of expression. We have used arm< <Gal4VP16 (see Materials and Methods) and compared it to arm-Gal4. Although dimerisation in arm-Gal4 embryos is observed in the same patterns as 69B- and da-Gal4, the VP16 line initially has the same patterns of dimerisation, but at later stages results in ubiquitous dimerisation.
The above results show that the pattern of dimerisation is not dependent on
the Gal4 line used to express the hybrid proteins, and confirm that the
ß-gal part of the hybrid protein is not driving dimerisation
(Blakely et al., 2000).
However, care should be taken when using very strong VP16 inducer lines, as
they can generate artefactual staining.
Homo-dimerisation of DOME as revealed with the ßlue-ßlau
technique occurs in the salivary glands, the tracheal pits, the posterior
spiracles, the foregut, the hindgut, the oenocytes, the Malpighian tubules,
the midline, the anal pads and the amnioserosa
(Fig. 2 and not shown). The
JAK/STAT pathway is required for the development of the trachea, the posterior
spiracles (Brown et al., 2001),
the Malpighian tubules, the foregut and the hindgut
(Johansen et al., 2003
),
showing that the regions where DOME homo-dimerisation is detected coincide
with the regions where the receptors are active. To date, a requirement for
the JAK/STAT pathway has not been reported in the salivary glands,
amnioserosa, oenocytes, midline or anal pads. This may reflect that
dimerisation of the receptor in certain tissues does not automatically lead to
the activation of the pathway, or perhaps that a function for the pathway is
still to be discovered in these tissues.
Given that excessive levels of hybrid protein expression can result in artefactual dimerisation, we studied whether the hybrid proteins have the same stability in all cells. We used a hs-Gal4 line to drive homogeneous levels of expression and tested for protein perdurance. After heat-shock induction, we allowed the embryos to develop and monitored the presence of the proteins in different tissues with an anti-ß-gal antibody (Materials and Methods). The protein disappeared with a similar dynamic from all tissues studied except from the amnioserosa where it lasted for a longer period (data not shown). Because the amnioserosa is one of the tissues where we observe X-gal staining, it is conceivable that protein stability in the amnioserosa leads to protein accumulation and artefactual dimerisation. To test whether increased expression of normal Gal4 proteins results in the appearance of novel areas of X-gal staining, we have studied embryos in which the hybrid proteins are expressed with both da-Gal4 and 69B-Gal4 lines simultaneously. Despite the increased level of protein expression driven by these two strong Gal4 lines, no new areas of expression were detected (not shown). Thus, a wide range of strong Gal4 lines can be used safely with the ßlue-ßlau technique without obtaining artefactual protein dimerisation.
JAK/STAT pathway activation is also required for segmentation during early development. Expression of the fusion proteins using maternal-Gal4 lines failed to show any complementation (data not shown). This failure may be because of aberrant folding or insufficient expression of the hybrid proteins driven by the maternal-Gal4 line or reflect a difference in DOME complex formation at the blastoderm stage. Whatever the reasons for not being able to detect dimerisation at early stages during embryogenesis, the above results show that the ßlue-ßlau technique can be used to detect protein-protein interactions in whole embryos from, at least, the extended germ band stage. Our data show that DOME homo-dimerises. The dimerisation is not ubiquitous, but is developmentally controlled in space and time. Among the tissues in which we detect dimerisation are the ones that have been shown to require JAK/STAT function during embryonic development, proving that the dimerisation we detect is functionally significant.
DOME homo-dimerisation is ligand independent
To determine whether ectopic ligand can induce ectopic homo-dimerisation of
the receptor, we used the same drivers to simultaneously express UPD and the
receptor fusions in the ectoderm. No changes in the pattern of X-gal staining
were observed (Fig. 3A,B
compared to Fig. 2B,D), showing
that ectopic UPD cannot induce DOME dimerisation even though UPD has been
shown to bind DOME in cell culture and activate the JAK/STAT pathway
(Chen et al., 2002).
|
To further test whether upd, or any unknown ligands not deleted by
Df(1)os1A, could be responsible for the observed receptor
dimerisation, we have expressed a dominant negative DOME receptor with the
cytoplasmic and transmembrane domains deleted (domeDN). As inferred
from its vertebrate equivalent, this construct encodes a soluble protein
(Narazaki et al., 1993). DOME
receptors lacking the cytoplasmic domain behave as a dominant negative
(Brown et al., 2001
;
Ghiglione et al., 2002
;
Silver and Montell, 2001
),
probably because they sequester the ligand(s), making them unavailable for the
endogenous receptor. Expression of domeDN does not affect receptor
dimerisation in any of the tissues studied
(Fig. 3E-F), confirming that
DOME homo-dimerisation is ligand independent.
Correlation between receptor dimerisation and competence for JAK/STAT
activation
To analyse whether the state of receptor dimerisation affects the
sensitivity of the cells to the ligand, we compared the effects of ectopic UPD
expression on cells that have the receptor in a dimerised form as judged by
the ßlue-ßlau technique, with its effects in cells where it is not
dimerised. DOME dimerises in the amnioserosa
(Fig. 2F,J), an extra embryonic
membrane required for germ band retraction and dorsal closure during
development (Lamka and Lipshitz,
1999). However, in wild-type embryos, UPD is not expressed in the
amnioserosa (Harrison et al.,
1998
) and mutants in the JAK/STAT pathway have no defects in
dorsal closure (Binari and Perrimon,
1994
; Brown et al.,
2001
; Harrison et al.,
1998
; Hou et al.,
1996
; Yan et al.,
1996
), suggesting that the pathway is not required for amnioserosa
development. Despite this, ectopic ligand expression using da-Gal4
results in embryos with a dorsal hole and incomplete retraction of the germ
band (Fig. 4A arrowhead). These
phenotypes are similar to those in mutants for hindsight, a gene
required for amnioserosa development
(Lamka and Lipshitz, 1999
).
Both the amnioserosa and the cells of the abutting leading edge are required
for dorsal closure. To distinguish which cells are responsible for the
observed phenotype, we expressed UPD with the amnioserosa-specific line
c381.611-Gal4 and the leading edge-specific line LE-Gal4.
Only expression of UPD with the amnioserosa line resulted in dorsal hole
phenotypes (not shown). These results show that the cells of the amnioserosa
are competent to receive the UPD signal even though they do not activate the
JAK/STAT pathway during normal development. Also, the capacity of the
amnioserosa cells to respond to UPD expression confirms that the dimerisation
observed in this tissue using the ßlue-ßlau technique is
physiological.
|
Looking for other putative targets of JAK/STAT signalling, we found that
ventral veinless (vvl)
(Anderson et al., 1995;
de Celis et al., 1995
) is
expressed in a small region of the hindgut in a similar pattern to that of
upd (Fig. 4G). In
upd or stat92E mutant embryos vvl is not activated
in the hindgut (Fig. 4H and not
shown), suggesting that vvl is a target of the JAK/STAT pathway.
Given that all the cells of the hindgut show DOME dimerisation
(Fig. 2D,H), we tested whether
they could all respond to the ligand. Ectopic upd expression results
in ectopic vvl activation throughout the hindgut
(Fig. 4I). Taken together, the
above results suggest that UPD expression is only capable of activating
JAK/STAT function in cells in which the receptors are in a pre-dimerised state
as detected by the ßlue-ßlau technique.
![]() |
DISCUSSION |
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Two different hypotheses have been proposed for the induction of
dimerisation in vertebrates. Our results are not consistent with the view that
JAK/STAT receptors become activated by ligand-induced dimerisation
(Fig. 5A), but suggest that the
receptors dimerise prior to receptor activation
(Fig. 5B). The patterned
expression of X-gal shows that pre-dimerisation is a developmentally regulated
process that occurs mainly, but not exclusively, in cells that activate the
pathway. Previous reports with the IL-2 , ß and
subunits
(Damjanovich et al., 1997
),
the growth hormone (Gent et al.,
2002
) and the erythropoietin
(Livnah et al., 1999
)
receptors have presented evidence for preformed receptor complexes using three
different techniques: FRET, immunoprecipitation and crystallography.
Crystallographic evidence indicates that the erythropoietin receptor can
dimerise in the absence of ligand (Livnah
et al., 1999
). In this case, only the extracellular domains of the
receptor are apposed. Ligand binding changes the extracellular conformation
and, as a result, the intracellular domains come together allowing JAK
phosphorylation (Fig. 5B).
Although these results suggest that ligand binding induces an extracellular
conformational change in the pre-associated receptor complex allowing receptor
activation, they do not provide information about when and where
pre-association occurs or whether it has a physiological role. Our experiments
allow us to visualise receptor pre-association in situ and suggest that
pre-association is essential for functionality.
|
Pre-association could be mediated by unknown ligands, membrane-spanning
proteins or by intracellular proteins. Given that a deficiency deleting
upd and three other homologous genes has an identical phenotype in
the ectoderm to the inactivation of dome, stat92E or hop, it
is unlikely that there are any other ectodermal ligands. This strongly
indicates that other proteins play a role in the formation of the receptor
pre-associated complexes. Recent results show that cytoplasmic scaffold
proteins are fundamental for specificity of the signaling responses
(Harris et al., 2001). We
propose that cytoplasmic `clamp' proteins expressed in a developmentally
regulated pattern are responsible for cytokine receptor pre-association, and
thus for deciding the competence of a cell to respond to a particular
ligand.
The ßlue-ßlau technique efficiently detects protein
interactions in whole organisms
In recent years several techniques have been developed to detect protein
interactions in vivo (Ayoub et al.,
2002; Hu et al.,
2002
; Mochizuki et al.,
2001
; Pelletier et al.,
1998
; Rossi et al.,
1997
) but have only been used in cell culture. Three main issues
should be taken into account when considering the usefulness of these
techniques. First, sensitivity; second, the physiological significance of the
results obtained; and third, whether the technique can detect protein
interactions in all cellular compartments.
It could be argued that the areas in which we observe dimerisation are
those where higher levels of hybrid protein are expressed. The experiments we
present discard this possibility because not all strongly expressing areas
lead to dimerisation. For example, in Fig.
2A the head expresses high levels of hybrid protein but there is
no dimerisation in this area in Fig.
2B; and the same can be said for the head and segmental grooves in
Fig. 2G and
Fig. 2H. Moreover, lines of
very different origin give consistent results regardless of them being strong
(da-Gal4) or weak ectodermal inducers (h-Gal4) (compare
tracheal pits of Fig. 2B and
Fig. 2L). The lack of
correlation between high levels of hybrid protein expression and dimerisation
is further confirmed by the absence of dimerisation when using strong
mesodermal drivers (Fig. 2M and
Fig. 2N). Finally, increasing
the levels of expression by simultaneously expressing with 69B-Gal4
and da-Gal4 does not result in novel areas of dimerisation (not
shown). This supports that, as has been described in cell culture experiments
(Blakely et al., 2000;
Rossi et al., 1997
), the
ßlue-ßlau technique reveals the intrinsic ability of the proteins
tested to dimerise. However, it should be pointed out that when using a very
strong Gal4VP16 activator line we have observed unspecific dimerisation. This
result shows that precaution should be taken to use different inducing lines,
as unspecific dimerisation can be observed with unusually high levels of
hybrid protein expression.
The use of X-gal for the detection of protein interactions makes this technique highly sensitive as even very faint levels of blue staining can be detected against the unstained background. This also provides the advantage of not requiring any special type of microscopy. Although our results show that ßlue-ßlau can detect in whole embryos interaction between transmembrane proteins, future experiments should test whether the technique can be used for testing protein interactions in other cellular compartments.
In summary, we have shown that ßlue-ßlau is an inexpensive
technique to view protein interactions in whole organisms. By applying the
technique to DOME we have provided direct visual evidence that the JAK/STAT
receptor homo-dimerises, and that the dimerisation is not ligand induced. It
will be interesting to see whether developmental control of receptor
pre-association is a peculiarity of JAK/STAT receptors or if it is a more
general strategy to control receptor activation in other signalling pathways
(Moriki et al., 2001;
Yu et al., 2002
).
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ACKNOWLEDGMENTS |
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