©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Stat-like DNA Binding Activity in Drosophila melanogaster(*)

Sharon M. Sweitzer (1)(§), Soledad Calvo (2), Matthias H. Kraus (2), David S. Finbloom (1), Andrew C. Larner (1)(¶)

From the (1)Division of Cytokine Biology, Food and Drug Administration, Bethesda, Maryland 20892 and (2)Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 , 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.


INTRODUCTION

The Jak/Stat()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) .

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-related sequences in the Drosophila genome also suggested that a Stat homolog exists in Drosophila.


MATERIALS AND METHODS

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(dIdC). 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.


RESULTS AND DISCUSSION

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).

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.

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 SH2 Domain Sequence

Based upon functional evidence presented here, we sought to determine whether the Drosophila genome harbors DNA sequences related to human Stat1. 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Research Council-Food and Drug Administration research associateship.

To whom correspondence should be addressed: Division of Cytokine Biology, Food and Drug Administration, HFM 505, 8800 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-0864; Fax: 301-402-1659.

The abbreviations used are: Jak, Janus kinase; Stat, signal transducer and activator of transcription; SH2, src homology domain 2; GRR, 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.

A. C. Larner and D. S. Finbloom, unpublished data.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.