Targets of Activity

hopscotch is required maternally for the establishment of the normal array of embryonic segments. In hop embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. The effect of hop on the expression of these genes is stripe-specific. This is the first evidence for stripe-specific regulation of pair-rule genes by a tyrosine kinase (Binari, 1994).

An single enhancer sequence consisting of 500bp mapping 3.3kb upstream of the transciption start site is sufficient to direct eve expression in both stripes 3 and 7. There are 5 KNI binding sites in the 3 + 7 enhancer and 11 HB sites. HB and KNI act as repressors of stripe 3 expression, while the JAK kinase HOP, acting through the Drosophila STAT protein Marelle, is involved in activation, with the KNI and HB sites closely linked to two STAT binding sites. A model is presented in which the repressors provide short term quenching of widespread STAT activation (Small, 1996). p>The determination of sexual identity in Drosophila depends upon a system that measures the X chromosome to autosome ratio (X/A). This system relies upon the unequal expression of X-linked numerator genes in 1X and 2X nuclei. The numerators activate a special Sex lethal promoter, Sxl-Pe, in 2X/2A nuclei, but not 1X/2A nuclei. By multimerizing a conserved Sxl-Pe sequence block, a gain-of-function promoter, Sxl-PeGOF, is generated that is inappropriately active in 1X/2A nuclei. GOF activity requires the X-linked unpaired (upd) gene, which encodes a ligand for the Drosophila JAK/STAT signaling pathway. upd also functions as a numerator element in regulating wild-type Sxl-Pe reporters. The JAK kinase, Hopscotch, and the STAT DNA-binding protein, Marelle, are also required for Sxl-Pe activation (Jinks, 2000).

The numerators most important for turning on Sxl are sis-a and sis-b (scute). They are expressed throughout the embryo, and mutations in both can have quite pronounced effects on Sxl-Pe activity. However, neither of these numerators is critical for the gain-of-function activity of the Sxl-PeGOF promoter. Instead, the two numerators that contribute most to Sxl-PeGOF activity are the segmentation genes runt and upd. At the syncytial blastoderm stage, run is expressed in a broad central domain, and it is in this region that Sxl activation is defective in 2X/2A run mutants. Except for a dorsal crescent in the head, the upd expression domain closely coincides with that of run. It is in this same central run-upd domain that the highest levels of Sxl-PeGOF promoter activity are observed. Moreover, in both run and upd mutant males, Sxl- PeGOF promoter activity is severely impaired. From these findings, it can be inferred that the multimerized 72 bp fragment contains cis-acting targets for run and upd action (Jinks, 2000).

Since Upd is a secreted ligand, it is unlikely that it interacts directly with sequences in the 72 bp fragment. Instead, the data suggests that Upd acts by turning on a Drosophila JAK/STAT signaling cascade consisting of the Hop protein kinase and the Mrl transcription factor. In this model, the extracellular Upd ligand would activate the Drosophila JAK protein Hop. Hop would in turn phosphorylate the D-STAT homolog Mrl, which would then enter the nucleus and activate Sxl-Pe. That the Mrl protein is critical for the activity of Sxl-PeGOF is demonstrated by the dramatic reduction in beta-galactosidase expression seen in both 1X/2A and 2X/2A embryos derived from homozygous mrl- germline clones (Jinks, 2000).

The 72 bp fragment has a sequence that closely matches the consensus D-STAT-binding site. Hence, a plausible hypothesis is that Sxl-PeGOF is activated in 1X and 2X embryos by the binding of multiple Mrl proteins to the reiterated STAT sites in the multimerized fragment. Since there are also potential target sites for Runt in the 72 bp fragment, it is possible that Runt and Mrl collaborate in promoter activation. There are precedents in mammals for synergistic interactions between STAT and other transcription factors. Although a definitive answer will require further study, it is interesting that Sxl-PeGOF is not activated in male embryos in the dorsal crescent region of the head where upd but not run is expressed (Jinks, 2000).

Since Sxl-PeGOF has regulatory properties not seen in other Sxl-Pe promoter constructs, an obvious question is whether the JAK/STAT signaling pathway is a part of the normal X/A counting system. Several lines of evidence suggest that it is. (1) Genetic studies indicate that the upd gene is an X chromosome-counting element. Deletions that remove upd show female lethal interactions with mutations in the numerator genes sis-a and sis-b, and with Sxl. (2) As would be expected for an X chromosome-counting element, deletion of upd in females heterozygous for either sis-a or sis-b compromises the activity of wild-type Sxl-Pe reporter constructs. (3) The gain-of-function hopTum allele enhances the activity of the Sxl-Pe promoter in 2X/2A embryos. Moreover, consistent with the idea that a target site for the JAK/STAT signaling pathway is contained in the multimerized 72 bp fragment, the minimal Sxl-Pe0.4kb promoter (from which the 72 bp fragment is derived) is activated by the hopTum-1 mutation. (4) The Sxl autoregulatory feedback loop is not properly turned on in 2X/2A embryos when the maternally derived mrl gene product is absent. The observed defects in SXL protein expression are regional and for the most part overlap with the domain in which the JAK/STAT signaling pathway would be activated by upd expression. (5) The failure to properly activate the Sxl autoregulatory feedback loop in the absence of maternal mrl appears to be due to a marked reduction in Sxl-Pe activity. For the full-length promoter construct, Sxl-Pe3.0kb, beta-galactosidase expression is almost completely eliminated except in the very anterior of the embryo. In this context, it should be noted that Sxl-Pe contains two consensus STAT/Mrl-binding sites, in addition to the one found in the minimal 0.4 kb promoter. Conceivably these two upstream sites could provide additional targets for Mrl binding and promoter activation in vivo (Jinks, 2000).

The gene encoding the JAK/STAT ligand, upd, is required in the zygote to activate Sxl-Pe. Hence, like other numerators, it is the dose of the upd gene product produced in 1X and 2X embryos that is critical to the X chromosome-counting mechanism. The JAK kinase, hop, and the STAT transcription factor, mrl, have a different function in the counting process. These experiments show that the mrl gene is required in the mother's germline, not in the zygote. The available evidence suggests that this is also true for the X-linked hop gene. Since the products of these two genes would be deposited in constant amounts in the egg during oogenesis, they correspond to signal transduction elements like da. While the findings indicate that the JAK/STAT pathway plays an important role in the choice of sexual identity, the effects of mutations in the pathway do not seem to be as great as those observed for mutations in other components of the X/A counting system. For example, mutations that disrupt the maternal deposition of DA essentially eliminate both Sxl-Pe activity and SXL protein expression in female embryos. By contrast, when maternal mrl is removed, Sxl-Pe is not completely turned off, and SXL protein expression can still be detected, particularly in the termini. This suggests that the JAK/STAT pathway plays a secondary rather than a primary role in X chromosome counting (Jinks, 2000).

It is now clear that transcription factors involved in many different aspects of development, from segmentation to neurogenesis, have been coopted by the sex determination system in Drosophila. These genes generally have cell-autonomous activities and, consequently, are readily adaptable to a process that requires counting the number of chromosomes in each nucleus. Hence, it is somewhat surprising that a JAK/STAT signaling pathway, which depends upon the production and reception of an extracellular ligand, has also been incorporated into the counting system. Moreover, the apparent ligand, upd, corresponds to the X chromosome-counting element. Since Upd is secreted, it could potentially influence the counting process not only in the nucleus that produced the protein to begin with but also in adjacent nuclei. Supporting this possibility, it has been found that upd mutant cells can generate a normal pattern when adjacent to wild-type cells. Except under special circumstances (e.g., in gynandermorphs where 1X and 2X cells are in close proximity), counting elements that function nonautonomously need not have detrimental consequences and might even offer some advantages. For example, the signaling cascade may respond in a nonlinear fashion to variations in the dose of the ligand. In this case, the JAK/STAT pathway may provide a mechanism for magnifying the initial 2-fold difference in the amount of ligand produced in 1X/2A versus 2X/2A nuclei. In addition, signaling between adjacent nuclei might compensate for stochastic differences in numerator expression and might further amplify the signal by a relay mechanism (Jinks, 2000).

The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signaling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signaling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. The function and regulation of four-jointed (fj) during eye patterning has been analyzed. Loss-of-function clones or ectopic expression of four-jointed results in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicate that localized four-jointed expression is required at the time during development when ommatidial polarity is being determined. In contrast, complete removal of four-jointed function results in only a mild ommatidial polarity defect. four-jointed expression has been found to be regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. It is concluded that the clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal (Zeidler, 1999b).

Both in situ hybridization for fj transcripts and the lacZ activity patterns revealed by enhancer traps in the fj locus indicate that fj is normally expressed most strongly in a broad domain around the dorsoventral midline of the eye imaginal disc). To determine whether this localized expression is functionally significant, fj was ectopically expressed during eye development. Ectopic expression of fj was driven at the poles of the eye during eye patterning using an optomotor-blind driver. This results in dorsoventral inversions of ommatidial polarity at both the dorsal and ventral poles of the eye, often with three or more rows of ommatidia inverted (Zeidler, 1999b).

The expression pattern of fj, and the phenotypes that were observed for loss-of-function and gain-of-function of fj activity, indicate a role for fj function in ommatidial polarity determination along the dorsoventral axis. Recent studies have revealed functions for the N, JAK/STAT and Wg pathways as regulators of ommatidial polarity determination, with the current model suggesting that Notch and Upd are positive regulators of a graded signal that is highest at the equator, whereas Wg is a negative regulator of such a factor (or factors). The fj gene is therefore a good candidate for being a downstream target of regulation by one or more of these pathways. Consistent with this, fj is regulated by the JAK/STAT and Wg pathways. In clones mutant for the Drosophila JAK homolog hop, which lack JAK function, a reduction in fj expression is observed. Although JAK is a cell-autonomously acting signal-transduction component, the effect on fj expression is not cell-autonomous, with greatest downregulation being observed in the center of the clone. In accordance with downregulation in hop clones, clones of cells ectopically expressing the JAK ligand Upd result in activation of fj expression. Conversely, ectopic expression of Wg (which is predicted to be a negative regulator) results in downregulation of fj expression. Activated N can nonautonomously activate fj expression. Taken together, these results indicate that fj is regulated by all three of these pathways in a manner consistent with mediating their functions in dorsoventral polarity determination (Zeidler, 1999b).

One of the noteworthy aspects of fj regulation by the Notch and JAK/STAT pathways is that it is non-autonomous, even when it is studied using cell-autonomously acting signaling components such as the intracellular domain of N, Nintra. One possible explanation for this non-autonomy would be that fj is able to activate its own expression via an autoregulatory loop. To test this hypothesis, fj was ectopically expressed in the presence of a fj enhancer trap and it was found that fj was indeed able to activate its own expression. The activation of fj expression by ectopic expression of fj is non-autonomous, again consistent with the proposed secreted nature of the fj gene product. In addition to the N, JAK/STAT and Wg pathways, the only other gene reported to non-autonomously influence ommatidial polarity is frizzled (fz). A possible mechanism for non-autonomy of fz function would be via regulation of fj expression. The expression of fj was examined in fz loss-of-function clones and in clones of cells ectopically expressing fz, but in neither case is there any change in fj expression (Zeidler, 1999b).

Protein Interactions

The Drosophila STAT homolog is marelle, also known as DSTAT. Marelle is a dual function protein, interacting with Hopscotch via a shc homology 2 domain and also serving as a transcriptional activator (Hou, 1996 and Yan, 1996a).

The genetic and molecular data regarding outstretched and its relationship to Hopscotch and Stat92E (Marelle) are all consistent with the predicted role of Os as a ligand that activates the JAK signaling cascade. To directly investigate this hypothesis, os was expressed in Drosophila cells, which were then assayed for tyrosine phosphorylation of Hop. The cell line chosen for this experiment is the Clone 8 (Cl.8) line, derived from developing wing imaginal disc. For cells to respond to Os by phosphorylating Hop, it was hypothesized that some transmembrane receptor would be required to bind Os by an extracellular domain, and be associated with Hop on the intracellular domain. Since no such receptor has yet been identified in flies, cells were chosen that were derived from a tissue known to be responsive to such a signal. It has been shown that Om(1E) protein from Drosophila ananassae, overexpressed in the wing disc, causes defects (Juni, 1996), suggesting that a receptor for this Os homolog must be present in the wing discs of D. ananassae. Thus, the D. melanogaster wing disc-derived Cl.8 cell line seemed a likely candidate to express a receptor for Os. To show Os-dependent tyrosine phosphorylation of Hop, anti-Hop immunoprecipitates from os-transfected cells were prepared and tested for reactivity with the anti-phosphotyrosine antibody 4G10. Whereas Hop protein is detectable in all samples, Hop is tyrosine phosphorylated only in immunoprecipitates prepared from os-transfected cells. Transfection of cells with os lacking a signal sequence does not result in Hop phosphorylation, consistent with the notion that Os is required extracellularly for signaling to occur. To further prove that extracellularly provided Os is necessary and sufficient to observe Hop phosphorylation, Cl.8 cells were cocultured with S2 cells transiently transfected with Os. After thorough removal of the nonadherent S2 cells, Hop immunoprecipitates were prepared from Cl.8 cell lysates and analyzed. Hop phosphorylation is only seen when Cl.8 cells are cultured in the presence of os-transfected S2 cells. Identical results are obtained when Cl.8 cells are grown in the presence of conditioned medium taken from os-transfected 293T cells. These data are consistent with the hypothesis that Os is an extracellular ligand that binds a membrane-bound receptor to activate the JAK signaling pathway (Harrison, 1998).

In the mammalian system, JAK proteins are bound to monomeric cytokine receptors through the membrane-proximal domain. Signaling is triggered when cytokine binding induces receptor dimerization. This brings the receptor-associated JAK kinases into apposition, enabling them to transphosphorylate each other. The JAK kinases, now activated, phosphorylate a distal tyrosine on the receptor. This receptor phosphotyrosyl residue is subsequently recognized by the SH2 domain of the STAT proteins, drawing them into the receptor complex, where they are activated through phosphorylation on the tyrosine residue by JAKs (Chen, 2002).

To show Domeless/Mom-dependent activation of the Hop/Stat92E pathway, the tyrosine phosphorylation of Mom, Hop, and Stat92E was examined. S2 cells were co-infected with V5-epitope-tagged Mom, Hop, and Stat92E with either Upd-V5 or vector alone. Anti-Stat92E immunoprecipitates were prepared and tested for reactivity with the anti-phosphotyrosine antibody 4G10. Whereas Upd, Mom, Hop, and Stat92E proteins are detectable in the transfected samples, increased tyrosine phosphorylation of Mom, Hop, and Stat92E is detected in immunoprecipitates prepared from Upd-V5- and Mom-V5-transfected cells. These data are consistent with the hypothesis that Mom is a receptor of Upd that activates the Hop/Stat92E signal transduction pathway (Chen, 2002).

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, a genome-wide RNA interference (RNAi) screen was carried out in cultured Drosophila cells. One hundred and twenty-one genes were identified whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, it was found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, a key negative regulator of Drosophila JAK/STAT signaling was identified, protein tyrosine phosphatase PTP61F; it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. This study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway (Baeg, 2005).

An important step in the JAK/STAT signal transduction pathway is the dephosphorylation of the signaling molecules JAKs and STATs. In mammals, several PTPs have been implicated in the dephosphorylation of JAK and/or STAT proteins both in the cytoplasm and in the nucleus. In contrast, no PTPs have been identified that regulate JAK/STAT signaling in Drosophila. PTP61F was identified as a strong negative regulator in the screen. Knockdown of PTP61F by RNAi resulted in a more than fourfold increase in STAT92E-dependent reporter activity. PTP61F encodes the Drosophila homolog of mammalian PTP-1B, which has been shown to attenuate insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues of JAKs and/or STATs in mammalian tissue culture. Therefore the hypothesis was tested that PTP61F might serve as the tyrosine phosphatase for Hop. A dramatic increase was observed in tyrosine phosphorylation of Hop upon RNAi knockdown of PTP61F, suggesting that Hop may be a substrate of PTP61F. A significant increase was detected in STAT92E phosphorylation in cells treated with dsRNA against PTP61F. This is consistent with the notion that STAT92E is a downstream target of Hop, although the possibility that both Hop and STAT92E may be targets of PTP61F cannot be ruled out (Baeg, 2005).

In both mammals and Drosophila, SOCS, a negative regulator of the JAK/STAT pathway, has been shown to be transcriptionally activated by JAK/STAT signaling, thus generating a negative feedback loop. This prompted an examination of the expression pattern of PTP61F and whether its expression is responsive to JAK/STAT signaling in vivo. It was found PTP61F is expressed in a striped pattern, reminiscent of the STAT92E expression pattern. In addition, overexpression of Upd under the control of prd-Gal4 resulted in a dramatic increase in PTP61F transcript levels in the paired domain. Furthermore, levels of the PTP61F transcript were greatly reduced in embryos lacking Hop activity, suggesting that PTP61F transcription is dependent on active JAK/STAT signaling. Taken together, these results demonstrate that PTP61F expression responds to JAK/STAT signaling in vivo (Baeg, 2005).

These data suggested that loss of PTP61F would result in an increase in JAK/STAT signaling. Thus, the genetic interaction between PTP61F and canonical components of the JAK/STAT pathway was examined, using Df(3)ED4238, a deficiency uncovering the PTP61F gene. The interaction was tested in the Drosophila eye following overexpression of Upd using GMR-Gal4 driver, which causes a dramatic overgrowth and deformation of the adult eye. The severity of this phenotype is proportional to the strength of the JAK/STAT-mediated signal, because removing one copy of STAT92E significantly suppresses the GMR-Upd eye phenotype. Consistent with PTP61F being a negative regulator of the JAK/STAT signaling pathway, flies heterozygous for Df(3)ED4238 showed an enhanced deformed eye phenotype. A PTP61F transgene rescues this enhanced deformed eye phenotype in flies heterozygous for Df(3)ED4238. In addition, the PTP61F transgene also rescues lethality in flies carrying UAS-Upd GMR-Gal4/+; Df(3)ED4238/+, presumably caused by leaky expression of UAS-Upd in conjunction with PTP61F deficiency (Baeg, 2005).

The genetic interaction between PTP61F and Hop was examined. Flies carrying a dominant hyperactive Hop allele (HopTum-l) display decreased viability and the formation of melanotic tumors. This tumor formation phenotype is sensitive to gene dosage. Previous studies have shown that reducing the levels of positive regulators, such as STAT92E, Cdk4, and CycE, increases the viability and/or decreases tumor formation. Therefore both viability and melanotic tumor formation were monitored in females heterozygous for HopTum-l and these results were compared to females heterozygous for both HopTum-l and Df(3)ED4238. Removing one copy of PTP61F in HopTum-l heterozygous females leads to a significant decrease in survival rate and a dramatic enhancement in the formation of melanotic tumors. Altogether, these results demonstrate that PTP61F is a bona fide negative regulator of the JAK/STAT pathway in Drosophila (Baeg, 2005).

hopscotch: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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