1 Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
2 Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 633, Rochester, NY 14642, USA
3 Dana-Farber Cancer Institute, M649B, 44 Binney Street, Boston, MA 02115, USA
4 Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
*Author for correspondence (e-mail: willis_li{at}URMC.Rochester.edu).
Accepted 17 June 2002
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SUMMARY |
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Key words: Drosophila, Receptor tyrosine kinase (RTK), Torso, STAT
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INTRODUCTION |
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To investigate how overactivation of an RTK results in aberrant gene expression, we chose to study the Torso (Tor) pathway in the early Drosophila embryo. Tor is a fly RTK most homologous to the mammalian PDGF receptor. During Drosophila development, Tor specifies cell fates in the terminal regions of the embryo (Duffy and Perrimon, 1994). Tor mRNA is synthesized during oogenesis, deposited into the unfertilized egg, and translated following fertilization. Tor proteins are uniformly distributed on the cell membrane of the early embryo, but are activated at the terminal regions by a ligand that diffuses from the egg poles (Casanova and Struhl, 1993
; Sprenger and Nusslein-Volhard, 1992
). Previous studies have documented that Tor activates the evolutionarily conserved Ras1/Draf/MEK/MAPK signaling cassette (Duffy and Perrimon, 1994
) to induce the expression of target genes such as tailless (tll) (Pignoni et al., 1990
; Pignoni et al., 1992
), which is essential for specifying cell fates in the terminal regions (Steingrimsson et al., 1991
). The current model is that the tll promoter is repressed in the early embryo. The MAPK pathway abrogates tll repression, thereby enabling tll activation by an unknown ubiquitous transcription factor(s) (Liaw et al., 1995
; Paroush et al., 1997
).
tll expression at the posterior end is precisely restricted in a domain from 0 to 15% of the egg length (EL) in wild-type embryos (Fig. 1A). The extent of this domain is a sensitive readout of the strength of Tor activation (Hou et al., 1995; Li et al., 1998
; Li et al., 1997
). Thus, a decrease in Tor signaling, such as caused by tor or Draf loss-of-function mutations, results in reduction or elimination of tll expression in the posterior domain in a manner reflecting the severity of the mutation (<15%EL; not shown). Conversely, in tor gain-of-function (torGOF) mutations associated with an increase in Tor signaling, expansion of the posterior tll expression domain towards the middle region of the embryo is observed (>15%EL; Fig. 1C). The signal generated by either wild-type Tor or TorGOF, as visualized by the tll expression readout, can be completely blocked by null mutations in Draf (also known as pole hole; phl) (Ambrosio et al., 1989
). Thus, it has been proposed that the major output of Tor signaling is the activation of MAPK.
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The JAK/STAT pathway was first elucidated by studying the mechanisms of interferon signaling. In the canonical model, STAT is activated by the cytoplasmic tyrosine kinase JAK, which itself is activated by a non-tyrosine kinase receptor in response to ligand binding (Darnell et al., 1994). It is now well established that activation of STAT is associated with many cancers and other human diseases (Sahni et al., 1999
; Su et al., 1997
), and indeed, activated STAT3 behaves as an oncogene in causing cellular transformation and tumor formation (Bromberg et al., 1999
).
JAK and STAT proteins are conserved between flies and humans (Binari and Perrimon, 1994; Hou et al., 1996
; Yan et al., 1996
). The hop and mrl genes were isolated in genetic screens for determining the maternal effects of zygotic lethal genes (reviewed by Hou and Perrimon, 1997
). Embryos lacking the maternal product of either hop or mrl exhibit identical morphological defects when their cuticles are examined at the end of embryogenesis. They are missing the fourth and fifth ventral abdominal denticle belts, A4 and A5, respectively (see Fig. 2B). In the early embryo, Hop and Mrl are essential for the correct expression of a number of segmentation genes including even-skipped (eve) and runt that are normally expressed in alternating parasegments, forming seven stripes along the anteroposterior axis (Hou and Perrimon, 1997
).
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MATERIALS AND METHODS |
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Co-immunoprecipitation
To extract embryonic proteins, embryos of 0 to 4 hours after egg-laying were collected and homogenized in Buffer A [10 mM Tris-HCl pH 8.0; 150 mM NaCl; 1 mM EDTA; 0.1% Triton X-100; Protease Inhibitor cocktail (Sigma), and 1 mM PMSF final concentration]. To treat embryos with vanadate, a protein tyrosine phosphatase inhibitor, sodium orthovanadate (Sigma) was added to Buffer A prior to homogenization at 1 mM final concentration. To immunoprecipitate Tor from embryo extracts, we incubated anti-Tor antibody (Cleghon et al., 1996) with wild-type and torGOF embryo extracts (200 µl), respectively, at 4°C overnight at 1:200 dilution. The immunoprecipitates were resolved by 8% SDS-PAGE and blotted with anti-Tor antibody at 1:5000 dilution (Cleghon et al., 1996
) to reveal the presence of Tor. The blot was then stripped of antibodies and reprobed with an anti-Mrl antibody (raised by immunizing rat with bacterially expressed Mrl) at 1:500 dilution to detect whether Mrl was bound to Tor in the embryo extracts.
Plasmids and fly transformants
A PCR based mutagenesis was performed on a 5.9 kb tll upstream regulatory fragment (Liaw et al., 1995) to introduce nucleotide changes in the two Mrl-binding sites. As a result, site 1 was changed from ATTCTGGGAAT to ATGCGGCCGCT to create a NotI site (underlined), and site 2 from ATTCTTCGAAAGAC to ATTCTTCGGTACC to create a KpnI site (underlined). A lacZ reporter transgene was generated by replacing the wild-type tll regulatory region with this mutant 5.9 kb fragment in a tll-lacZ fusion gene (Liaw et al., 1995
) and used to transform Drosophila by P element-mediated transformation.
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RESULTS |
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Mrl and Hop are not essential for wild-type Tor signaling
To determine whether mutations in the JAK/STAT pathway show genetic interactions with members of the Ras1/Draf pathway, we generated embryos doubly mutant for various combinations of alleles. We used two Draf mutations with reduced activities, DrafC110 and DrafPB26, as well as a null Ras1 mutation, Ras1C40B. Unlike Draf null GLC embryos, which exhibit no posterior tll and cuticle structures, DrafC110 GLC embryos have a wild-type cuticle and show a near wild-type tll expression (not shown) (see also Melnick et al., 1993
). DrafPB26 GLC embryos have reduced posterior tll expression domains to 6-10% EL, and defects in the posterior cuticle structures that include frequent deletions of A8 (not shown) (see also Melnick et al., 1993
). While most of the Ras1
C40B GLC embryos are identical to tor or Draf null embryos and exhibit no posterior tll expression and cuticle structures, about 20% of these embryos have residual posterior tll expression as well as posterior cuticle structures due to a Ras1-independent activation of Draf (see also Hou et al., 1995
).
Since the phenotypes associated with torGOF are suppressed by a null mrl mutation, we investigated whether Mrl or Hop activities are essential for the expression of tll in wild-type embryos. We found that in either mrl or hop mutant embryos, the posterior domain of tll expression, which is invariably reduced in mutations that affect Tor signaling, appears wild type (about 15% EL; Fig. 2A), indicating that the Hop/Mrl pathway is not essential for the wild-type patterns of tll expression.
These results, however, do not fully exclude the possibility that Hop and Mrl constitute a branch of the Tor signaling pathway that acts in parallel and redundant to the Ras1-MAPK branch, and that the inability to detect any influence of the JAK/STAT pathway on wild-type tll expression could result from a compensatory up-regulation of the Ras1/Draf/MEK/MAPK pathway. We therefore examined the role of the JAK/STAT pathway in a number of sensitized genetic backgrounds wherein the efficiency of Tor signaling had been compromised. First, we examined tll expression and cuticle phenotype in embryos that were doubly mutant for a hop null allele and weak alleles of Draf. Elimination of hop did not increase the severity of the Draf mutations in these assays (Fig. 2C-F). Second, we examined the phenotype of embryos doubly mutant for mrl and Ras1. A fraction (about 20%) of Ras1 null mutant embryos exhibits residual tll expression due to activation of Draf by a Ras1-independent mechanism (Hou et al., 1995; Li et al., 1998
; Li et al., 1997
). Removal of mrl activity did not enhance the Ras1 phenotypes (Fig. 2G,H). Thus, neither Hop nor Mrl appear to be required for tll expression patterns in wild-type embryos, therefore they are unlikely to be integral components of the Tor pathway. This conclusion, however, does not apply to TorGOF since we find that Mrl activity is required for the full activity of TorGOF.
TorGOF is capable of activating Mrl
The above results are consistent with the possibility that TorGOF causes Mrl activation to exert its biological functions. To test whether TorGOF can cause Mrl activation, we examined Mrl activity in Drosophila Schneider (S2) cells transfected with DNA encoding different Tor molecules. As reported previously (Yan et al., 1996), transfection of Hop into S2 cells increased Mrl DNA-binding activity in these cells (Fig. 3A, lane 4). This increase in DNA binding was specific to Mrl, as addition of an anti-Mrl antibody causes the bound complex to be supershifted (Fig. 3A, lane 6). Interestingly, transfection of Tor or TorGOF also resulted in activation of endogenous Mrl in S2 cells (Fig. 3A, lane 2 and 3). Based on the intensity of the gel shift bands, Tor and TorGOF activate Mrl to levels similar to those observed after Hop transfection (Fig. 3A, lane 4). In these transfection experiments, Tor and TorGOF similarly activated Mrl, presumably because when overexpressed in transfection experiments wild-type Tor can dimerize, mimicking the effect of TorGOF mutations. These results are consistent with our hypothesis that TorGOF causes Mrl activation in vivo.
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DISCUSSION |
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In this manuscript we demonstrate that TorGOF requires Mrl but not Hop for its ability to induce ectopic target gene expression and causing deleterious effects on embryos. In addition, we show that TorGOF can associate with and cause Mrl activation in embryos and transfected cells. These results are most consistent with a model in which TorGOF directly phosphorylates Mrl, which in turn binds to the tll promoter to exacerbate its expression levels. Activation of STAT by RTKs has previously been suggested following studies in cultured mammalian cells. For example, transfected EGF or PDGF receptors can directly interact with and activate STAT by phosphorylation (Fu and Zhang, 1993; Paukku et al., 2000
). Taken together with these studies, our results seem to suggest that the intracellular kinase domain of several RTK proteins may have an intrinsic ability to activate STAT proteins.
To account for the involvement of Mrl in tll regulation we propose that a hyperactivated RTK requires a downstream pathway that is not essential for wild-type RTK under normal physiological situations. In wild-type embryos, Tor is activated only in the two terminal regions and defines the spatial limits of tll expression domains by relieving the transcriptional repressors bound to the tll promoter. Mrl is not an essential factor for tll activation in the terminal regions, although it remains to be determined whether Mrl contributes to the activation of tll expression redundantly with other yet unidentified factors. In torGOF mutant embryos, TorGOF is constitutively active in all regions of the embryo and causes ectopic tll expression. In this case, Mrl activation is indispensable for the ectopic tll expression in the central regions of the embryo. The differential requirement for Mrl in central and terminal regions might be due to the lack of other activators of tll and/or the presence of additional repressors in the central region of the embryos. Consistent with this idea, we and others have previously shown that, in the absence of Tor signaling (such as in tor mutant embryos), tll can be induced by uniformly expressing activated forms of downstream signaling components (such as RasV12 or 14-3-3). The resulting induction of tll expression happens preferentially in the terminal regions (Greenwood and Struhl, 1997; Li et al., 1998
; Li et al., 1997
). Thus tll expression could be determined by the balance between repressors and activators that can bind to the tll promoter (Fig. 5).
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Altered gene expression is commonly found in cancerous growth. The initiation and maintenance of the changes in gene expression often require the activation of multiple signaling molecules. STAT activation is found in many human cancers or transformed cells (Bromberg et al., 1999; Campbell et al., 2001
; Catlett-Falcone et al., 1999
; Garcia et al., 1997
). In light of our finding in Drosophila, STAT activation might play essential roles for the activation of genes that are required for malignant growth and other pathological conditions. More importantly, we found that STAT activation is insignificant for the normal patterns of gene expression that are controlled by an RTK. It would be interesting to investigate if it is generally true that STAT activation is an important factor only in aberrant RTK signaling. If so, a broad implication of our results is that STAT rather than Ras, should be viewed as premier target for drug interference in the treatment of human diseases and cancers associated with hyperactivation of receptor tyrosine kinases.
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ACKNOWLEDGMENTS |
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