From the
The cytokine signaling pathways that activate the Janus family
of tyrosine kinases (Jaks) and the ``signal transducers and
activators of transcription'' (Stats) have been well characterized
in mammalian systems. Work shown here provides evidence that an
analogous signaling pathway exists in Drosophila melanogaster.
Because many of the ligand-receptor pairs in Drosophila have
not been fully characterized, it was necessary to bypass the receptor
stimulation event that normally triggers intracellular Jak/Stat
activation. This was done by treating Drosophila Schneider 2
cells with vanadate/peroxide, which has been shown to closely mimic
some signaling events triggered by interferon
The Jak/Stat
There is growing
evidence that a Jak/Stat pathway may exist in Drosophila.
First, hopscotch, a Drosophila Jak kinase homologue
has been cloned and characterized(6) . hopscotch is a
maternal transcript, expressed in embryonic stripes. Mutations in this
gene locus cause abnormalities in the expression patterns of some of
the pair-rule and segment-polarity genes. This implicates the Jak
kinase being involved in signal transduction pathways controlling
segmentation of the developing fly.
Second, a number of growth
factor receptor homologs have been cloned from Drosophila. torso encodes a receptor tyrosine kinase that has a cytoplasmic domain
similar to that of the mammalian platelet-derived growth factor
receptor and is involved in embryonic pattern
formation(7, 8) . der, the epidermal growth
factor receptor homolog, has been cloned and genetically
characterized(9) . Mutations in the der locus cause
pleiotropic effects, implicating it in the development of a wide range
of tissues(10) . Treatment of mammalian cells with either
platelet-derived growth factor or epidermal growth factor has been
shown to activate Jak/Stat pathways, suggesting that Torso or Der may activate a Jak/Stat pathway in Drosophila as well.
Using the ligand-independent activation by vanadate/peroxide
treatment, we have attempted to identify a Stat-like activity in Drosophila Schneider 2 cells. The specific GRR binding
activity, which was seen after cell treatment, was dependent upon
tyrosine phosphorylation. This binding complex contained
phosphoproteins of 100 and 150 kDa. Detection of Stat1
The DNA elements
that were bound by the VBC share a consensus ATTTCCCNGAAA core region
that contains the GAS element described in the mammalian system (Fig. 2D)(2) . The ISRE and AP-1 elements, which
did not compete, lack this consensus sequence.
Work presented here is important to the
understanding of the mammalian Jak/Stat signaling pathways. Drosophila can provide a genetic model of Jak/Stat activation
and give insight into the significance of conserved protein sequences.
This work is also important for Drosophila development
because, like the Drosophila Jak protein, the Stat-like
protein may play a role in signal transduction during stripe formation.
, including the
activation of Jak1, Jak2, and the Stat1
protein. Evidence
presented here demonstrates that vanadate/peroxide can induce a
response region binding complex in Drosophila Schneider 2
cells. This complex contains two phosphoproteins of 100 and 150 kDa,
respectively, and shares many features with the
vanadate/peroxide-stimulated binding complex in the mammalian system.
Southern blot analysis of genomic DNA using the src homology
domain 2 (SH2) of Stat1
confirms the presence of a related gene in
the Drosophila genome.
(
)pathway is activated in
mammalian cell systems by treatment of cells with a number of different
cytokines and growth factors(1, 2) . The Stat family of
transcription activators has several common structural features,
including conserved SH2 domains. The current model of Stat activation
is that a tyrosine in the carboxyl terminus of the Stat protein is
phosphorylated and acts as an SH2 binding site upon cytokine
stimulation(3, 4) . The Stat can then form dimers,
either with itself or with another member of the Stat family.
Dimerization of the Stats is necessary for DNA binding and, ultimately,
induction of transcription. Activation of a transcription complex
containing phosphorylated Stats can also be induced by treating cells
with a combination of sodium orthovanadate and hydrogen peroxide in the
absence of cytokines or growth factors(5) .
-related
sequences in the Drosophila genome also suggested that a Stat
homolog exists in Drosophila.
Cells and Reagents
Schneider 2 cells (ATCC CRL
1963) were maintained in Schneider's Drosophila medium
(Life Technologies, Inc.), 10% fetal bovine serum (Quality Biologics).
Sodium orthovanadate and hydrogen peroxide were purchased from Sigma.
SDS-polyacrylamide gel electrophoresis was performed on the Novex
system. 4G10 antibody was purchased from Upstate Biotechnology Inc. All
other reagents were purchased from commercial sources unless otherwise
noted.
Schneider 2 Cell Treatment
A solution of 50 mM sodium orthovanadate, 500 mM hydrogen peroxide made in
Schneider medium was incubated at 24 °C for 5 min. This solution
was added to exponentially growing Schneider 2 cells to a final
concentration of 100 µM sodium orthovanadate, 1 mM hydrogen peroxide, and cells were incubated for the indicated
times at 24 °C. Cells were washed two times in phosphate-buffered
saline. Whole cell lysates were prepared by solubilizing cells for 10
min, on ice, with intermittent vortexing, in a buffer containing 20
mM HEPES, pH 7.0, 10 mM KCl, 1 mM
MgCl, 20% glycerol, 0.1% Nonidet P-40, and 1% Triton X-100.
Particulate matter was separated from soluble material by
centrifugation at 18,000
g for 5 min. Where indicated,
cells were pretreated with 500 nM staurosporin or 30 µg/ml
genistein for 30 min. Vanadate/peroxide solution (described above) was
added to cells, and the incubation continued for an additional 60 min.
Electrophoretic Mobility Shift Assays
10 µg of
soluble protein was diluted in binding buffer to a final concentration
of 10 mM Tris, pH 7.4, 5 mM MgCl, 100
mM KCl, 1 mM dithiothreitol, 50% glycerol, 0.03%
Nonidet P-40, and 0.1 mg/ml poly(dI
dC). Double-stranded probe was
labeled with [
-
P]ATP using polynucleotide
kinase. 1 ng of probe was added to the extract. A 10- or 50-fold excess
of unlabeled competitor oligonucleotide was added as indicated. For
sequences of oligonucleotides used, see Fig. 2D. Binding
reactions proceeded for 5 min at room temperature. Where indicated,
binding reactions were preincubated for 30 min with 5 mM
phenyl phosphate, 5 mM sodium phosphate, or Yersinia protein tyrosine phosphatase in the presence or absence of 1
mM sodium orthovanadate or 3 mM sodium tungstate.
Binding was analyzed on a 6% non-denaturing gel containing 2.5%
glycerol in 0.25
TBE (22.5 mM Tris, 22.2 mM boric acid, 0.5 mM EDTA). Gels were dried and subjected
to autoradiography.
Figure 2:
Vanadate/peroxide-inducible DNA binding
complex binds specifically to GRR and GAS elements. A,
vanadate/peroxide-induced GRR binding complexes formed in the presence
of a 10-fold (lane2) or 50-fold (lane3) excess of unlabeled GRR and a 10-fold (lane4) or 50-fold (lane5) excess of
unlabeled GAS. B, vanadate/peroxide-induced GRR binding
complexes formed in the presence of a 50-fold excess of unlabeled SIE (lane2), a 50-fold excess of unlabeled ISRE (lane3), or a 50-fold excess of unlabeled AP-1 (lane4). C, vanadate/peroxide-induced GAS
binding complexes formed in the presence of a 10-fold (lane2) or 50-fold (lane3) excess of
unlabeled GAS and a 10-fold (lane4) or 50-fold (lane5) excess of unlabeled GRR. D,
comparison of the enhancer sequences used for electrophoretic mobility
shift assays and competitions. A consensus sequence was determined by
comparing sequences that competed for the GRR binding
complex.
Protein Purification and SDS-PAGE Analysis
Soluble
cell lysate from activated or control Schneider 2 cells was allowed to
bind to heparin-agarose (Sigma) for 1 h at 4 °C. The agarose was
washed with cell lysis buffer, and the vanadate/peroxide-induced
binding complex (VBC) was eluted with a salt gradient from 0 to 300
mM NaCl in cell lysis buffer. Biotinylated GRR oligonucleotide
was bound to streptavidin-agarose according to standard protocols.
Partially purified VBC was allowed to incubate with GRR-agarose in the
presence of 200 µg/ml salmon sperm DNA (Digene Diagnostics, Inc.)
for 1 h at 4 °C. Affinity beads were washed 3 times with cell lysis
buffer. Protein was solubilized using SDS-PAGE sample buffer. After
denaturation, proteins were separated on a 4-20% acrylamide gel
and transferred to polyvinylidene difluoride membranes (Immobilon,
Millipore) by electroblotting. Blots were subjected to standard Western
blotting procedures using biotinylated 4G10 anti-phosphotyrosine,
streptavidin-conjugated horseradish peroxidase, and ECL (Amersham
Corp.) for detection.
Southern Analysis
10 µg of normal human
genomic DNA, 10 µg of Drosophila genomic DNA, or 2.5
µg of Drosophila genomic DNA was digested using EcoRI. Southern blot analysis, at a stringency reduced by 14
°C, was performed as described(11) . The probe was generated
by the polymerase chain reaction using oligonucleotide primers
(5`-TACTGTGTTCATCATACTGTC-3` and 5`-TGGAATGATGGATGCATCATGGGCTT-3`),
spanning nucleotides 1913-2446 of human Stat1, and labeled
by nick translation.
Vanadate/Peroxide Treatment of Drosophila Schneider 2
Cells Induces Binding of a Specific Complex to the GRR of the Fc
Receptor Promoter
We have previously shown that
vanadate/peroxide will mimic the action of interferon on
monocytes(5) . We have also detected vanadate/peroxide
stimulation of GRR-binding proteins in a number of different cell
types, including HeLa, U266, THP1, U937, Daudi, and fibroblast
lines.
(
)Because Drosophila interferons
have yet to be identified, we activated potential Stat-like proteins
with vanadate/peroxide, thereby bypassing a ligand/receptor
interaction. Treatment of Schneider 2 cells with vanadate/peroxide
resulted in the formation of a complex that specifically bound to the
GRR in a time-dependent manner (Fig. 1). Cells required 20 min of
exposure to vanadate/peroxide for assembly of the complex as measured
by electrophoretic mobility shift assays. DNA binding activity
increased for up to 120 min of incubation (Fig. 1, lanes
1-8). Longer incubation times did not increase the intensity
of the shift (data not shown). The intensity of the Drosophila shift was similar to the intensity of shifts detected in mammalian
cells; however, the Drosophila shift complex migrated slightly
faster on the native gel than did the mammalian shift complex (data not
shown). The binding activity was induced in the presence of
cycloheximide suggesting that new protein synthesis was not necessary
for activity (data not shown). These findings suggest that the
appearance of DNA binding activity is an early step in the activation
of this pathway.
Figure 1:
Vanadate/peroxide-inducible GRR
binding complex in Drosophila Schneider 2 cells. Whole cell
lysates were made from untreated Schneider 2 cells (lane1), cells treated with vanadate/peroxide for increasing
amounts of time (lanes 2-8), or cells treated for 1 h
with either hydrogen peroxide alone (lane9) or
vanadate alone (lane10). Whole cell lysates from
these cells were analyzed by electrophoretic mobility shift assay (as
described under ``Materials and
Methods'').
To determine whether the induced complex
bound specifically to the GRR, we performed competition experiments
using specific unlabeled oligonucleotides. A 10-fold excess of either
unlabeled GRR or a closely related -activated sequence (GAS) from
the IRF1 promoter competed for labeled GRR binding (Fig. 2A, lanes2 and 4),
while only a slight decrease in binding was observed when a 50-fold
excess of a high affinity SIE (13) was added (Fig. 2B, lane2). Addition of either
unlabeled ISRE or AP-1 did not effect the GRR binding complex (Fig. 2B, lanes3 and 4). A
DNA binding complex was also detected when a labeled IRF1/GAS element
was used in the electrophoretic mobility shift assay. Addition of
either unlabeled GRR or GAS to this binding reaction competed for the
labeled GAS binding complex (Fig. 2C, lanes
2-4). These data suggest that the VBC has approximately the
same affinity for the GAS and GRR sites as judged by competition
analysis but has a lesser affinity for the SIE sequence.
Electrophoretic mobility shift assays supported these findings where
the GAS and GRR elements showed strong binding and the SIE showed
slight but detectable binding (data not shown).
Tyrosine Phosphorylation Is Necessary for the Formation
of the Binding Complex
It has been clearly established that the
Stat-like proteins are phosphorylated on tyrosine residues in response
to cytokine stimulation of cells and that this phosphorylation is
necessary for an active complex to be
assembled(1, 14, 15, 16, 17, 18, 19, 20) .
A functional SH2 domain of Stat1 and phosphorylation of tyrosine
701 are necessary for dimer formation in response to interferon
(4) . Phosphopeptides corresponding to sequences
surrounding tyrosine 701 can block dimer formation. It has been
speculated that this disruption is due to the SH2 domain of Stat1
binding to the phosphopeptide instead of binding to phosphotyrosine
701. Phenyl phosphate can also disrupt Stat complexes, presumably by
competing for SH2 domain binding(20) . Phenyl phosphate added to
vanadate/peroxide-stimulated extracts of Schneider 2 cells inhibited
the DNA binding activity of the VBC (Fig. 3A, lane2), while equimolar amounts of sodium phosphate or a
10-fold higher concentration of sodium chloride had no effect on the
DNA binding activity (Fig. 3A, lanes3 and 4). The inhibition of DNA binding activity by phenyl
phosphate can be reversed by removing the salt by dialysis (data not
shown). These results are consistent with the Drosophila vanadate/peroxide-inducible complex containing a protein with an
SH2 domain.
Figure 3:
Tyrosine phosphorylation is necessary for
maintenance of the shift complex. A, whole cell lysates from
vanadate/peroxide-stimulated Schneider 2 cells were untreated (lane1), treated with 5 mM phenyl phosphate (lane2), treated with 5 mM sodium phosphate (lane3), or treated with 50 mM sodium chloride (lane4). B, whole cell lysates from
vanadate/peroxide-stimulated Schneider 2 cells were untreated (lane1), treated with YOP51 (lane2), or
treated with YOP51 in the presence of 1 mM sodium
orthovanadate (lane3) or 3 mM sodium
tungstate (lane4). C, Schneider 2 cells
were pretreated with no addition (lane1), 500 nM staurosporin for 30 min (lane2), or 30
µg/ml genistein for 30 min (lane3). Cells were
then treated with vanadate/peroxide for 60 min. Whole cell lysates from
all experiments were analyzed by electrophoretic mobility shift
assay.
Other assays have been used in the mammalian system to
confirm that Stat-like proteins are tyrosine-phosphorylated and that
this phosphorylation is important for maintaining a DNA binding
activity(1, 20, 21) . These assays have included
the treatment of activated extracts with the tyrosine-specific
phosphatase, YOP51, and pretreatment of cells with the tyrosine kinase
inhibitor, staurosporin(21) . Treatment of a Schneider 2 cell
lysate from vanadate/peroxide-treated cells with the tyrosine-specific
phosphatase, YOP51, disrupted the DNA binding activity (Fig. 3B, lane2). This inhibition was
reversed by the addition of either 1 mM sodium vanadate or 3
mM sodium tungstate, which are inhibitors of tyrosine-specific
phosphatases. The VBC was also inhibited by pretreatment of the
Schneider 2 cells with the tyrosine kinase inhibitors, genistein or
staurosporin (Fig. 3C, lanes2 and 3). These data suggest that a tyrosine kinase is involved in
the formation of the Drosophila DNA binding complex and that
tyrosine phosphorylation is necessary for DNA binding activity.
Vanadate/Peroxide Induces the Tyrosine Phosphorylation of
Two Major GRR-binding Proteins, pp100 and pp150
VBC was
partially purified as described under ``Materials and
Methods'' and adsorbed to a GRR oligonucleotide affinity resin.
The loss of the VBC activity from the extract was monitored by
electrophoretic mobility shift analysis. The DNA binding complex, which
specifically adsorbed to the GRR column, was analyzed by Western
blotting with 4G10, a monoclonal antibody that recognizes
phosphotyrosine. Two inducibly phosphorylated proteins of approximate
molecular mass of 100 and 150 kDa were detected (Fig. 4). Either
of these sizes would be appropriate for a Drosophila Stat
since the mammalian Stat proteins range in molecular mass from 84 kDa
(Stat 4) to 113 kDa (Stat 2)(22, 23) . Two minor bands
of 120 and 130 kDa were not consistently detected and could be due to
degradation of pp150.
Figure 4:
Vanadate/peroxide-stimulated tyrosine
phosphorylation of two GRR-binding proteins. Partially purified
vanadate/peroxide-stimulated binding complex was bound to a GRR
affinity column in the presence of salmon sperm DNA to compete for
nonspecific DNA-binding proteins. Proteins adsorbed to the column were
eluted in SDS sample buffer and separated by SDS-PAGE. The proteins
were transferred to Immobilon and probed with 4G10. The two dominant
proteins that were inducibly phosphorylated are
indicated.
Southern Analysis Demonstrates That a Drosophila Gene
Exists Which Hybridizes to the Human Stat1
Based upon functional evidence presented here, we
sought to determine whether the Drosophila genome harbors DNA
sequences related to human Stat1 SH2 Domain
Sequence
. Ten µg of normal human and Drosophila genomic DNA were digested with EcoRI, and
the products were separated on agarose gels. To correct for differences
in genomic complexity of both species, 2.5 µg of Drosophila genomic DNA were also analyzed. Southern blot analysis was
performed using the SH2 domain coding sequence of human Stat1
as a
probe. As shown in Fig. 5, three major bands of 3, 4.4, and 10
kilobases, respectively, were observed in Drosophila DNA in
addition to several minor bands. The strongly hybridizing fragments
were also detectable in 2.5 µg of Drosophila genomic DNA
ensuring specificity of hybridization. These findings suggest that the Drosophila genome contains at least one and perhaps three
genes related to the mammalian Stat1
SH2 domain.
Figure 5:
Stat1-related sequences identified in Drosophila genomic DNA by reduced stringency hybridization. 10
µg of human genomic DNA (lane1), 10 µg of Drosophila genomic DNA (lane2), or 2.5
µg of Drosophila genomic DNA (lane3)
was digested with EcoRI and separated on a 0.8% agarose gel.
The SH2 domain of human Stat1 was labeled by nick translation and
used as a probe. The mobility of molecular weight standards (
HindIII) is indicated on the left.
In summary, we
have identified a unique DNA binding activity in Drosophila
melanogaster. This activity resembles the Stat-like activity that
has been extensively characterized in the mammalian system. In both the
mammalian and the Drosophila systems, vanadate/hydrogen
peroxide treatment of cultured cells induces a specific GRR binding
complex whose formation is dependent upon tyrosine phosphorylation.
Detection of Stat1-related sequences in the Drosophila genome raises the possibility that, as in the mammalian system,
Stat1
-like proteins are responsible for this activity. Work is in
progress to obtain the protein sequence and to clone the cDNA encoding
such novel Stat proteins.
response region; GAS,
-activated site; SIE, serum-inducible element; ISRE,
interferon-stimulated response element; DER, Drosophila epidermal growth factor receptor; PAGE, polyacrylamide gel
electrophoresis; VBC, vanadate/peroxide-induced binding complex.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.