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

Gene name - hopscotch

Synonyms - janus kinase (JAK), Tumorous (Tum)

Cytological map position - 10B 6-8

Function - signal transduction-non receptor tyrosine kinase

Keyword(s) - JAK/STAT pathway

Symbol - hop

FlyBase ID:FBgn0004864

Genetic map position - 1-34.6

Classification - janus kinase family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene
Recent literature
Hoi, C.S., Xiong, W. and Rebay, I. (2016). Retinal axon guidance requires integration of Eya and the JAK/STAT pathway into phosphotyrosine-based signaling circuitries in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 27194748
Summary:
The transcriptional coactivator and phosphatase eyes absent (Eya) is dynamically compartmentalized between the nucleus and cytoplasm. Although the nuclear transcriptional circuits within which Eya operates have been extensively characterized, understanding of its cytoplasmic functions and interactions remains limited. Previous work has showed that phosphorylation of Drosophila Eya by the Abelson tyrosine kinase can recruit Eya to the cytoplasm, and that eya-abelson interactions are required for photoreceptor axons to project to correct layers in the brain. Based on these observations, this study postulated that photoreceptor axon targeting might provide a suitable context for identifying the cytoplasmic signaling cascades with which Eya interacts. Using a dose-sensitive eya misexpression background, an RNAi-based genetic screen was performed to identify suppressors. Included among the top 10 hits are non-receptor tyrosine kinases and multiple members of the Jak/Stat signaling network (hop, Stat92E, Socs36E, and Socs44A), a pathway not previously implicated in axon targeting. Individual loss-of-function phenotypes combined with analysis of axonal projections in Stat92E null clones confirm the importance of photoreceptor autonomous Jak/Stat signaling. Experiments in cultured cells detect cytoplasmic complexes between Eya and Hop, Socs36E and Socs44A; the latter interaction requires both the Src Homology 2 motif in Socs44A and tyrosine phosphorylated Eya, suggesting direct binding and validating the premise of the screen. Taken together, these data provide new insight into the cytoplasmic phosphotyrosine signaling networks that operate during photoreceptor axon guidance and suggest specific points of interaction with Eya.

Terriente-Félix, A., Pérez, L., Bray, S.J., Nebreda, A.R. and Milán, M. (2017). Drosophila model of myeloproliferative neoplasm reveals a feed-forward loop in the JAK pathway mediated by p38 MAPK signalling. Dis Model Mech [Epub ahead of print]. PubMed ID: 28237966
Summary:
Myeloproliferative neoplasms (MPNs) of the Philadelphia-negative class comprise polycythemia vera, essential thrombocythemia and primary myelofibrosis (PMF). They are associated with aberrant amounts of myeloid lineage cells in the blood, and in the case of overt PMF, with the development of myelofibrosis in the bone marrow and the failure to produce normal blood cells. These diseases are usually caused by gain-of-function mutations in the kinase JAK2. This study used Drosophila to investigate the consequences of activation of the JAK2 ortholog in hematopoiesis. The maturing hemocytes in the lymph gland, the major hematopoietic organ in the fly, was identified as the cell population susceptible to induce hypertrophy upon targeted overexpression of JAK. JAK was shown to activate a feed-forward loop including the cytokine-like ligand Upd3 and its receptor Domeless, which are required to induce lymph gland hypertrophy. Moreover, p38 MAPK signalling plays a key role in this process by inducing the expression of the ligand Upd3. Interestingly, forced activation of the p38 MAPK pathway in maturing hemocytes suffices to generate hypertrophic organs and the appearance of melanotic tumours. These results illustrate a novel pro-tumorigenic cross-talk between the p38 MAPK pathway and JAK signalling in a Drosophila model of MPNs. Based on the shared molecular mechanisms underlying MPNs in flies and humans, the interplay between Drosophila JAK and p38 signalling pathways unravelled in this work might have translational relevance for human MPNs.

Anderson, A. M., Bailetti, A. A., Rodkin, E., De, A. and Bach, E. A. (2017). A genetic screen reveals an unexpected role for Yorkie signaling in JAK/STAT-dependent hematopoietic malignancies in Drosophila melanogaster. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28620086
Summary:
A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of JAK/STAT signaling that may represent additional clinical interventions. Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK (Hopscotch (Hop)) and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, this study identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in expanded, wts and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling.
BIOLOGICAL OVERVIEW

A brief detour through one group of mammalian receptors will serve to introduce and clarify the Drosophila gene hopscotch. Mammalian cytokines, lymphokines and growth factors interact with the cytokine receptor superfamily. Despite a lack of catalytic kinase domains, these receptors combine ligand binding with the induction of tyrosine phosphorylation. This means that members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases physically associate with ligand-bound receptors. It is this association that results in tyrosine phosphorylation and activation.

Activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called signal transducers and activators of transcription (STATs). This JAK-STAT pathway is shared by all members of the cytokine receptor superfamily. hopscotch has been identified as the Drosophila JAK homolog. marelle, or DSTAT has been identified as the fly's STAT homolog (Ihle, 1994).

The JAK-STAT pathway regulates the expression of pair rule gene even skipped early in embryogenesis. Cooperativity in a number of positive regulator mechanisms might be required to provide an appropriate level of expression of eve in certain stripes. If the function of the JAK-STAT pathway were simply to upregulate the expression of eve in specific stripes, then the level of activation provided by the HOP-STAT system will depend on the number of STAT-binding sites present in the stripe-specific enhancer regions of eve. This might hold for activation of other pair rule genes as well. If the mechanism of activation of the JAK-STAT pathway is conserved between mammals and Drosophila, then HOP should be activated by its interaction with a membrane-bound receptor lacking a kinase domain. Because Hunchback and Knirps set the anterior and posterior borders of eve stripe 3, the JAK-STAT pathwy is not needed to spatially activate eve. It is not known which, if any, receptor is required for activation of the JAK-STAT pathway early in embryogenesis, but it is clear that the pathway is established maternally (Hou, 1997).

hopscotch has been identified as one of more than 50 Drosophila oncogenes, that is, genes that cause tumors. Tumorous-lethal (Tum-l), a hopscotch mutation, causes formation of melanotic tumors and proliferative defects in larval blood cells. Tum-l is an X-linked dominant mutation that causes melanotic tumor formation and temperature sensitive lethality. The larval tissues that produce blood cells are the lymph glands, a group of small organs arranged in lobed pairs on either side of the heart (the dorsal vessel). Undifferentiated stem cells produce two classes of mature blood cells. The first class is composed of podocytes and lamellocytes, macrophage-like cells involved in encapsulation and phagocytosis of foreign objects. The second class comprise crystal cells, involved in melanization. Tum-l causes hypertrophy (enlargement) of larval lymph glands and premature differentiation of lamellocytes.

HOP can cause neoplasia (literally "new growth;" figuratively, tumors) through one of two distinct mechanisms. The first is mutational. The original hop mutation is a single nucleotide amino acid substitution in the hop gene. The second mechanism is overexpression. Overproduction of wild type HOP by fusion of the gene to a heat shock promoter and expression during the second or third instar period also results in tumor formation. Overexpression of D-raf results in a similar phenotype to overexpression of hop. Is there a link between the two? Vertebrate epidermal growth factor signaling induces the activation of JAK1. It is tempting to speculate that DER/Torpedo/EGF-R signaling, known to transduce through the ras/raf pathway, may activate HOP as well (Harrison, 1995).

The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis

During Drosophila oogenesis, border cells perform a stereotypic migration. Slbo, a C/EBP transcription factor, is required for this migration. Drosophila Stat92E has been identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. By clonal analysis for Stat92E and hop mutants it has been found that the JAK/STAT pathway is required in border cells for their migration. The activating ligand for the pathway, Unpaired, is expressed in polar cells. Polar cells are specialized cells that can induce border cell fate in anterior follicle cells. On its own, ectopic expression of Unpaired can induce ectopic expression of border cell markers, including Slbo. However, Stat92E mutant cells still express normal levels of Slbo protein, thus Stat92E must regulate other targets critical for border cell migration (Beccari, 2002).

Production of ectopic polar cells by exposing early egg chambers to increased Hedgehog expression appears sufficient to induce ectopic migrating border cells at stage 9. A slbo-lacZ enhancer trap is induced in extra migrating clusters at stage 9. Similar ectopic border cell clusters have been observed in egg chambers with clones of follicle cells mutant for costal2, a negative regulator of the Hedgehog signal transduction pathway. Thus the presence of polar cells, and absence of posteriorizing signal from the oocyte, may be sufficient for the induction of border cells at the appropriate developmental stage. What signals from polar cells may be responsible for induction of border cell fate in adjacent follicle cells? There is good evidence that Upd is a key signal from polar cells: Upd is specifically expressed in polar cells and acts non cell autonomously; ectopic expression of Upd induces two border cell markers; and the JAK/STAT pathway is required in border cells. Previous studies of the JAK/ STAT pathway in Drosophila have indicated that Upd expression induces Stat92E activation through the JAK kinase Hop and that the effects of Upd can be explained in this manner. Ectopic expression of Upd induces ectopic expression of Slbo. Since the JAK/STAT pathway is required in border cells and thus must be active there, Upd regulated Stat92E may normally contribute to Slbo up-regulation in border cells (Beccari, 2002).

JAK signaling is somatically required for follicle cell differentiation in Drosophila

Janus kinase (JAK) plays several signaling roles in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. Consistent with unpaired expression, reduced JAK pathway activity results in the fusion of developing egg chambers. A primary defect of these chambers is the expansion of the polar cell population and concomitant loss of interfollicular stalk cells. These phenotypes are enhanced by reduction of unpaired activity, suggesting that Unpaired is a necessary ligand for the JAK pathway in oogenesis. Mosaic analysis of both JAK pathway transducers, hopscotch and Stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Moreover, JAK activity is also necessary for the initial commitment of epithelial follicle cells. Many of these roles are in common with, but distinct from, the known functions of Notch signaling in oogenesis. Consistent with these data is a model in which Notch signaling determines a pool of cells to be competent to adopt stalk or polar fate, while JAK signaling assigns specific identity within that competent pool (McGregor, 2002).

The somatic cells of the ovary consist of multiple subpopulations, each with its own function(s) in the developing egg. While the germline cyst is dividing and developing within the germarium, a monolayer of somatic cells surrounds the cyst as it moves posteriorly through the germarium. As the cyst becomes enveloped by the somatic cells, the egg chamber pinches off from the germarium, entering the vitellarium. At that time, approximately 5-8 somatic cells differentiate into stalk. These flattened, disc-shaped cells are stacked together to form the spacer between successive cysts. Stalk cells connect the anterior end of a more mature egg chamber to the posterior end of the next younger chamber. Also at that time, molecular markers can distinguish the stalk cells from the polar cells, which arise from the same precursors. The polar cells are arranged as two pairs of follicle cells, one pair at either end of each chamber near the stalk cells. While the stalk cells and polar cells cease proliferation at the end of the germarium, the remaining follicle cells, which are referred to as epithelial follicle cells, divide approximately five times to expand the pool of follicle cells. Those epithelial cells later differentiate into various subpopulations with specific functions in the vitellarium. Those subpopulations are pre-patterned with mirror image symmetry along the anterior-posterior axis of the egg. Imposed on that pre-pattern, signaling from the oocyte by the TGFalpha molecule Gurken stimulates the induction of posterior polarity on the somatic cells at the posterior end. The result is an egg with coordinated polarities of the somatic and germline cells. This coordination is essential for the proper localization of maternal determinants that pattern the resulting embryo (McGregor, 2002).

Strikingly, unpaired is expressed very specifically within the ovary. After egg chambers pinch off from the germarium, upd is restricted to the two pairs of polar cells found at the anterior and posterior tips of the egg. In the germarium, upd is expressed in a cluster of somatic cells at the posterior of region 3. Presumably these are the cells that give rise to the stalk and polar cells. Expression in the polar and border cells persists until egg maturation. In situ hybridization to Stat92E RNA reveals that Drosophila STAT is expressed in both the germarium and the vitellarium. Expression in the germarium occurs in all follicle cells in region 2a and 2b; it then begins to be restricted to terminal follicle cells in region 3. In the vitellarium, Stat92E is expressed weakly at the termini of the egg chamber, but in a broader domain than only the two polar cells. After stage 9, Stat92E is strongly expressed in the nurse cells, consistent with the maternal role of Stat92E in the segmentation of the early embryo. Moreover, weak ubiquitous expression of hop is detectable in the follicular epithelium. These data are consistent with a potential role for JAK signaling in oogenesis (McGregor, 2002).

The loss of JAK pathway function in the somatic cells of the Drosophila ovary results in the fusion of adjacent cysts and/or the mislocalization of the oocyte within a cyst. Based on molecular markers for cell identity, mutations in hop or Stat92E cause the loss of stalk cells and an increase in the number of polar cells. This shift in cell fates correlates with the fusion of adjacent cysts. An allelic series of hop mutant combinations shows a range of phenotypic severity, from occasional fusion of two adjacent chambers to complete fusion of all cysts with no morphological distinction between germarium and vitellarium. The severity of the visible phenotypes is reflective of the severity of the follicle cell fate transformations. Effects on fate range from frequent appearance of one extra polar cell in the weakest mutation to consistent appearance of a dozen or more extra polar cells in more severe alleles. Phenotypes seen in mutant clones of hop and Stat92E ovaries are similar to those seen in the heteroallelic combinations of hop mutations. By using the directed mosaic technique, clone production is limited specifically to the somatic cells, thereby demonstrating that the activity of the JAK pathway is required in the follicle cells. Mosaic analysis also demonstrates that the adoption of proper epithelial cell fates requires JAK activity (McGregor, 2002).

All follicle cell subpopulations in an egg are derived from approximately three stem cells in the germarium of each ovariole. While still in the germarium, a common pool of distinct stalk and polar cell precursors is set aside from the epithelial follicle cells. Those precursors then differentiate into either stalk or polar cells. The remaining epithelial cells are pre-patterned with mirror image symmetry along the anteroposterior axis, with three distinct subpopulations at each end. The symmetry is broken at stage 6 when Gurken in the oocyte stimulates EGF receptor in the posterior terminal cells to determine posterior polarity of the egg. The three anterior terminal cell populations then become border cells, stretched (nurse cell-associated) cells, and centripetal cells. The posterior terminal cells are essential for the reorganization of the cytoskeleton in the oocyte. Those cells send an unknown signal to the germline that stimulates the reversal of microtubular polarity in the egg which is necessary for the migration of the oocyte nucleus to the anterior and for the correct localization of polarity determinants in the egg (McGregor, 2002).

Loss of JAK pathway signaling clearly influences the terminal fate of the stalk/polar cell precursors. In heteroallelic mutant combinations of hop, the number of polar cells increases while the number of stalk cells decreases. However, the sum of stalk cells plus polar cells remains approximately the same as in wild type, indicating that loss of JAK signaling is not influencing proliferation of the precursor pool, nor is it causing recruitment of epithelial follicle cells to a polar fate. This suggests a model in which the normal function of the JAK pathway is to promote the adoption of stalk cell fate in a subset of the stalk/polar cell precursor pool. JAK pathway activation may either instruct the adoption of stalk cell fates or prevent the adoption of polar cell fate. Current data do not distinguish between these alternatives (McGregor, 2002).

A second role for JAK signaling in the follicle cells was highlighted by analysis of mosaics. In chambers of the vitellarium, the immature cell marker Fas III is rapidly downregulated in all but the polar cells. However, the epithelial follicle cells do not begin to express markers of terminal differentiation until stage 7. Indeed, these cells continue to proliferate through stage 6. Nonetheless, the loss of Fas III in the epithelial cells beginning around stage 2 suggests that the identity of these cells has already begun to change. Presumably they become preliminarily committed to an epithelial follicle cell fate. In hop or Stat92E mutant clones, younger chambers retain high levels of Fas III in all the mutant cells. In more mature egg chambers (stage 7 or later) there is a consistent lack of Fas III expansion in mutant cells. The transient nature of the increase in Fas III expression suggests that the mutant cells remain in an immature state until later stages. In this model, JAK pathway activity would be necessary for the preliminary commitment step in epithelial cell differentiation that occurs after the egg chamber pinches off from the germarium. At approximately stage 7, the normal stage for terminal differentiation, the Fas III-positive JAK mutant cells lose Fas III expression, presumably because they are cued to differentiate by another signal. The consequence of loss of JAK signaling on terminal epithelial cell fates remains to be investigated (McGregor, 2002).

Several signaling pathways have been implicated in the patterning of the follicular epithelium. The best characterized are the Notch, EGFR and Hedgehog pathways. In the earliest of these activities, strong expression of hh in the terminal filament and cap cells at the anterior tip of the germarium stimulates the proliferation of the somatic stem cells. Loss of Hh signaling results in reduced follicle cell number and consequent failure to properly encapsulate the germline cyst. Recent work has demonstrated that the normal role of Hh in the ovaries is as a somatic stem cell factor and that it is necessary for the proliferation of somatic stem cells (McGregor, 2002).

After Hh activity promotes the production of a pool of follicular precursors, the stalk/polar cell precursor pool is set aside from the epithelial cell pool. The stalk/polar cell precursor pool is distinct from the epithelial pool because it ceases to proliferate as the cyst reaches the posterior end of the germarium. The method by which the stalk/polar cell precursors are determined is not known, but it has been suggested that Notch signaling, enhanced by localized Fringe activity, may be involved in the process. Similar to JAK mutants, the loss of Notch activity causes chamber fusions that are apparently the result of a failure to produce stalk cells. But unlike JAK mutants, N pathway mutants also fail to produce polar cells. Therefore, N signaling is required for the differentiation of both polar and stalk cell fates (McGregor, 2002).

So what distinguishes stalk and polar cells from one another? JAK signaling induces the adoption of stalk cell fates in a subset of the stalk/polar cell precursors. Loss of JAK pathway activity expands polar cells at the expense of stalk cells, while ectopic activation of the pathway causes a reduction of polar cells. Therefore, it is proposed that JAK pathway activity determines the terminal fate of stalk and polar cells. However, JAK activity is limited in assigning stalk cell fates to only competent cells, that is, the stalk/polar cell precursor pool. Thus, another activity, perhaps N signaling, is necessary to induce competence for stalk and polar fates. Alternatively, N signaling may be primarily responsible for the assignment of polar cell fates. One could imagine a mechanism of lateral inhibition, already linked to N signaling in various tissues, in which all the cells of the precursor pool have N activity, but that the signal becomes limited to and maintained only in the polar cells. It may be the activity of the N pathway that then drives stable expression of upd and allows the induction of stalk cell fates in neighboring cells (McGregor, 2002).

While polar and stalk cell fates are adopted as chambers exit the germarium, differentiation of the epithelial follicle cell fates is not obvious until later. At approximately stage 7, epithelial follicle cells express markers for each of the terminal identities with a clear anterior-posterior orientation. But in the absence of Grk/EGFR signaling at the posterior, a symmetrical mirror image pattern of three terminal populations of epithelial fates at each end is revealed. In wild-type ovaries, up to approximately stage 6, the oocyte signals to the overlying posterior follicle cells through Gurken. The terminal follicle cells that receive the Grk signal are induced to become posterior follicle cells. The resulting posterior follicle cells then signal to the oocyte to stimulate a cytoskeletal rearrangement. The resulting microtubular polarity drives the migration of the oocyte nucleus from the posterior to the anterior and establishes the AP axis that allows the sequestration of anterior and posterior maternal products to their respective poles. The signal from the soma for polarization of the oocyte microtubules is not yet known (McGregor, 2002).

When the developing cyst exits the germarium, there is a distinct change in the epithelial cell precursors. The level of Fas III, a marker for immature follicle cells, is rapidly reduced in all epithelial cell precursors. However, these cells do not begin to express markers for new cell identities until around stage 7. Therefore, it seems that the epithelial cells become committed to a fate early in the vitellarium, but do not terminally differentiate until later. This is consistent with the fact that the epithelial follicle cells continue to divide until stage 6. Furthermore, Grk/EGFR signaling does not impose posterior identity on epithelial cells until stage 6. So the loss of Fas III in epithelial cell precursors in the early vitellarium marks an intermediate step in specific epithelial identities. JAK signaling is involved in this step, because clones of JAK pathway mutations cause the persistence of Fas III in epithelial cell precursors in the early vitellarium. The normal loss of Fas III expression in epithelial precursors of the early vitellarium may indicate the establishment of a pre-pattern of epithelial identities determined by JAK signaling. It is attractive to speculate such a role because the secreted JAK pathway ligand Upd is expressed symmetrically at the termini of the chamber. It is easy to envision a scheme in which the strength of the Upd signal received by the epithelial cell precursors determines the ultimate epithelial identity. However, these epithelial cells would remain in a proliferative, undifferentiated program until stage 7. The event that allows terminal differentiation is unclear, but could also be a N signal, as suggested above for competence of stalk and polar cells. This is consistent with the report of a pulse of Delta protein, a N ligand, that occurs at stages 5-7. Additional work will determine whether JAK signaling is instructive for specific epithelial fates (McGregor, 2002).


GENE STRUCTURE

cDNA clone length - There are two transcripts; 5.4 kb and 5.1 kb. The basis for the difference has not yet been documented (Binari, 1994).

Bases in 5' UTR -620

Exons - ten

Bases in 3' UTR - 1064


PROTEIN STRUCTURE

Amino Acids - 1177

Structural Domains

HOP has homology at its carboxyl terminus to the catalytic domain of tyrosine kinases. There is a short nuclear localization signal (amino acids 315-320) (Binari, 1994.)


hopscotch: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 February 2010

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