InteractiveFly: GeneBrief

Suppressor of cytokine signaling at 36E: Biological Overview | References

BIOLOGICAL OVERVIEW

Socs36E, which encodes a negative feedback inhibitor of the JAK/STAT pathway, is the first identified regulator of niche competition in the Drosophila testis. The competitive behavior of Socs36E mutant cyst stem cells (CySCs) has been attributed to increased JAK/STAT signaling. This study shows that competitive behavior of Socs36E mutant CySCs is due in large part to unbridled Mitogen-Activated Protein Kinase (MAPK) signaling. In Socs36E mutant clones, MAPK activity is elevated. Furthermore, it was found that clonal upregulation of MAPK in CySCs leads to their outcompetition of wild type CySCs and of germ line stem cells, recapitulating the Socs36E mutant phenotype. Indeed, when MAPK activity is removed from Socs36E mutant clones, they lose their competitiveness but maintain self-renewal, presumably due to increased JAK/STAT signaling in these cells. Consistently, loss of JAK/STAT activity in Socs36E mutant clones severely impairs their self-renewal. Thus, these results enable the genetic separation of two essential processes that occur in stem cells. While some niche signals specify the intrinsic property of self-renewal, which is absolutely required in all stem cells for niche residence, additional signals control the ability of stem cells to compete with their neighbors. Socs36E is the node through which these processes are linked, demonstrating that negative feedback inhibition integrates multiple aspects of stem cell behavior (Amoyel, 2016).

Stem cell niches are complex environments that provide support for stem cells through molecular signals. Several well-characterized niches provide not just one but multiple signals which stem cells must integrate and interpret in order to remain at the niche and self-renew. How this integration is achieved is not well understood at present. Furthermore, in order to maintain the appropriate number of stem cells and the homeostatic balance between self-renewal and differentiation, it is necessary that self-renewal cues be present in limiting amounts or that their activity be dampened to prevent excessive accumulation of stem cells. One general feature of many signal transduction pathways is the presence of feedback inhibitors. These are dampeners of signaling, transcriptionally induced by the signaling itself, that prevent signal levels from being aberrantly high. One such family of feedback inhibitors is the Suppressor of Cytokine Signaling (SOCS) proteins, which were identified as inhibitors of JAK/STAT signal transduction, and are SH2- and E3-ligase domain-containing proteins. The SH2 domain binds phosphorylated (i.e., activated) signal transduction components and the E3-ligase targets them for degradation by Ubiquitin-dependent proteolysis. In mammals, SOCS proteins can thus inhibit several tyrosine kinase-dependent signaling pathways, including JAK/STAT and Mitogen-Activated Protein Kinase (MAPK) (Amoyel, 2016).

The Drosophila testis is an ideal model system to study questions of signal regulation and integration in stem cells. The testis niche, called the hub, supports two stem cell populations. The first, germ line stem cells (GSCs), gives rise to sperm after several transit-amplifying divisions leading up to meiosis. The second, somatic cyst stem cells (CySCs), gives rise to cyst cells, the essential support cells for germ line development. Many ligands for signaling pathways are produced by the hub, including the JAK/STAT pathway agonist, Unpaired (Upd), the Hedgehog (Hh) pathway ligand Hh and the Bone Morphogenetic Protein (BMP) homologs Decapentaplegic (Dpp) and Glass Bottom Boat (Gbb). The latter two signals are also produced by CySCs and are required in GSCs for self-renewal, indicating that CySCs constitute part of the niche for GSCs along with the hub. CySCs require JAK/STAT and Hh activity for self-renewal (Amoyel, 2016).

CySCs and GSCs compete for space at the niche, a phenomenon that was revealed by the analysis of testes lacking the JAK/STAT feedback inhibitor Socs36E(Issigonis, 2009; Singh, 2010). In these animals, excessive JAK/STAT activity was detected in CySCs, and Socs36E mutant CySCs displaced the resident wild type GSCs. Additionally, it has been shown that CySCs with sustained Hh signaling or sustained Yorkie (Yki) activity also outcompeted neighboring wild type GSCs, indicating that several signaling pathways can control niche competition (Amoyel, 2014). Moreover, prior to out-competing GSCs, mutant CySCs displaced neighboring wild type CySCs, indicating that both intra- (CySC-CySC) and inter-lineage (CySC-GSC) competition take place in the testis. While the two types of competition appear related, in that one precedes the other, there are instances in which only intra-lineage competition takes place. While the competitive phenotype of Socs36E mutant CySCs was ascribed to increased JAK/STAT signaling, it was surprising to find that clonal gain-of-function in JAK/STAT signaling in CySCs did not induce competitive behavior, and it was concluded that loss of Socs36E did not mimic increased JAK/STAT signaling in CySC (Amoyel, 2016).

This study addressed whether other mechanisms could account for the competitive behavior of Socs36E mutant CySCs. Because SOCS proteins can inhibit MAPK signaling in cultured cells and in Drosophila epithelial tissues (Kazi, 2014; Herranz, 2012; Almudi, 2009), this study examined if Socs36E repression of MAPK signaling underlied the Socs36E competitive phenotype. Indeed, it was found that Socs36E inhibits MAPK signaling in CySCs during self-renewal, and that gain of MAPK activity induces CySCs to outcompete wild type CySCs and GSCs at the niche. This study dissected the genetic relationship between Socs36E and the MAPK and JAK/STAT pathways and shows that loss of Socs36E can compensate for decreased self-renewal signaling within CySCs. Thus, CySCs integrate multiple self-renewal signals through the use of a feedback inhibitor that controls at least two signaling pathways regulating stem cell maintenance at the niche (Amoyel, 2016).

The data presented in this study implicate MAPK signaling as a major regulator of CySC competition for niche access and establish that the competitiveness of CySCs lacking Socs36E is derived primarily from their increased MAPK activity. The ability of a stem cell to self-renew reflects not only intrinsic properties but also extrinsic relationships with its neighbors. For instance, if a cell is unable to compete for space at the niche then it will be no longer able to receive short-range niche signals and will be more likely to differentiate. Conversely, if a cell is more competitive for niche space, this cell and its offspring will replace wild type neighbors and colonize the entire niche. (Amoyel, 2016).

These data show that CySCs with increased MAPK signaling out-compete neighboring stem cells in CySC-CySC as well as CySC-GSC competition and that CySCs with reduced MAPK activity are themselves out-competed. The interpretation is favored that MAPK regulates primarily competitiveness rather than self-renewal because while MAPK mutant clones are lost from the niche, lineage-wide inhibition of the pathway does not result in a complete loss of stem cells. This contrasts with the role of JAK/STAT signaling in CySCs. Stat92E mutant CySCs are lost and lineage-wide pathway inhibition results in pronounced and rapid stem cell loss. Based on these results, it is argued that JAK/STAT signaling in CySCs primarily controls their intrinsic self-renewal capability while MAPK signaling regulates their competitiveness. Interestingly, there are important similarities between Hh and MAPK function in CySCs in that CySCs lacking Hh signal transduction are out-competed and those with sustained Hh activity out-compete wild type neighbors. Lastly, it is noted that CySCs mutant for the tumor suppressor Hippo (Hpo) (which leads to sustained Yki activation) or Abelson kinase (Abl) also have increased competitiveness, suggesting the existence of multiple inputs controlling the ability of stem cells to stay in the niche at the expense of their neighbors. In the future, it would be interesting to determine if genetic hierarchies exist between competitive pathways or if they independently converge on similar targets. One outstanding question is how altering the competitiveness of CySCs affects the maintenance of the germ line. In the case of Socs36E, MAPK, Hh and Hpo, the competitive CySC displaces not only wild type CySCs but also wild type GSCs. While these observations suggest that out-competition of CySCs and GSCs is linked, the result that Abl mutant CySCs only compete with CySCs and not with GSCs indicates that these two competitive processes are separable genetically (Amoyel, 2016).

It is well established that Egfr/MAPK signaling is required in somatic cells for their proper differentiation and for their encystment of the developing germ line. In this study, an additional function for Egfr/MAPK was identified in the somatic stem cells, specifically that this pathway regulates competitiveness of CySCs, with each other and with GSCs. Regarding the latter, it is possible that the loss of GSCs when somatic cells have high MAPK signaling is linked to their possibly increased encystment by these cells. Indeed, recent work has shown that Egfr activity in CySCs regulates cytokinesis and maintenance stem cell fate in GSCs. It is tempting to speculate that increased somatic Egfr activity leads to increased encystment of GSCs and loss of stem cell fate in GSCs (Amoyel, 2016).

MAPK may play a conserved role in niche competitiveness as mouse intestinal stem cells that acquire activating mutations in Ras bias normal stem cell replacement dynamics and colonize the niche. Interestingly, the activating ligand Spi is produced by germ cells, suggesting that the germ line coordinates multiple behaviors in the somatic cell lineage. In addition to transducing signals from the germ line, CySCs also receive ligands from hub cells (including Hh and the JAK/STAT ligand Upd) and they have to integrate these various stimuli. If unmitigated, the combined effect of all of these signals could produce highly competitive CySCs, with overall negative effects on niche homeostasis. The data are consistent with a model in which the induction of Socs36E by the primary self-renewal pathway (JAK/STAT) results in the restraint of a competitive trigger (MAPK) in CySCs. In this way, Socs36E acts to integrate signals from different sources and maintain homeostatic balance between resident cell populations that share a common niche (Amoyel, 2016).

Socs36E limits STAT signaling via Cullin2 and a SOCS-box independent mechanism in the Drosophila egg chamber

The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. This study investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Jak/STAT activation during specification of motile border cells in Drosophila oogenesis. Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. Loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. These data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. It was also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo (Monahan, 2015).

The Jak/STAT pathway is a highly conserved cytokine signal transduction cascade, which transmits information from an extracellular cue to an intracellular response through transcriptional regulation. Briefly, ligand binding to a catalytically inert cytokine receptor (Domeless/Dome in Drosophila) induces a conformational change in the receptor and the cytoplasmically associated, non-receptor tyrosine kinase, Jak. This change stimulates phosphorylation of the cognate Jaks and the cytoplasmic domain of the receptor. Monomeric STAT proteins bind phosphotyrosines along the receptor, are phosphorylated by Jak, dissociate, homo-dimerize, and translocate into the nucleus as an active transcriptional regulator. The Jak/STAT pathway is essential for several developmental and cellular processes, including stem cell maintenance, immune response and regulation, cell proliferation, cell migration, and hematopoiesis. Drosophila utilizes a minimal, yet fully functional, Jak/ STAT signaling cascade that is required in many of the same cellular processes as in vertebrates, including the process of cell migration (Monahan, 2015).

During Drosophila oogenesis, a STAT-mediated collective cell migration occurs. The ovary is comprised of 16-18 ovariole chains, each with multiple developing eggs (called egg chambers). Each egg chamber consists of a monolayer of somatic epithelial cells (called follicle cells) that encase the germline (the oocyte and 15 nurse cells). At approximately mid-oogenesis, a subset of anterior follicle cells acquires invasive properties. These cells cluster around two immotile polar cells to form the border cell cluster. This cell collective later detaches from the anterior end of the egg chamber, invades the nurse cells, and migrates as a group to the oocyte. This process is essential for female fertility and proper patterning of the developing egg and future embryo (Monahan, 2015).

Border cell motility requires a precise level of STAT activity, which is tightly regulated by a genetic circuit that includes attenuation mediated by Suppressor of Cytokine Signaling at 36E (Socs36E) (Starz-Gaiano, 2008; Yoon, 2011; Montell, 2012; Monahan, 2013). The polar cells are the egg chamber's sole source of the Unpaired (Upd) family, which activates the Jak/STAT pathway in Drosophila. During stage 8, as Upd is released, surrounding follicle cells receive it and activate STAT in a spatial gradient. STAT promotes expression of the pro-migratory cue slow border cells (slbo) and the migratory inhibitor apontic (apt) in the anterior follicle cells. STAT activation is initially widespread, but must be dampened to produce an optimal number of invasive cells by stage 9. Apt feeds back to inhibit STAT activity, in part by regulating the expression of Socs36E and a Stat92E targeting microRNA (miR-279) to limit motility (Starz-Gaiano, 2008; Yoon, 2011; Monahan, 2013). The inability to shut off STAT signaling properly in anterior follicle cells enables an excessive number of cells to acquire invasive properties, which can impede border cell migration (Silver, 2005; Starz-Gaiano, 2008; Yoon, 2011; Monahan, 2013). However, while loss of Stat92E or slbo prevents border cell specification and migration, and significantly reduces female fertility, loss of apt, Socs36E, or miR-279 does not result in sterility, as not all egg chambers are equally affected (Starz-Gaiano, 2008; Yoon, 2011; Montell, 2012; Monahan, 2013). This reflects robust control of oogenesis, and underscores the complexity in the regulation of reproduction. While much is known about the genetic control of border cell migration, the molecular mechanisms that regulate signaling during border cell specification are less well understood (Monahan, 2015).

The SOCS family of proteins is a set of essential regulators of cytokine signaling that are conserved from humans to Drosophila. SOCS proteins possess two conserved domains: a Src Homology 2 (SH2) domain and a C-terminal SOCS box. However, the N-termini show no evidence of conserved domains, high sequence homology, or consistency in length between family members. Vertebrates contain eight SOCS proteins that are sub-divided into two groups: the vertebrate specific SOCS proteins (CIS and SOCS1-3) and the ancestral SOCS proteins (SOCS4-7). The vertebrate-specific SOCS proteins have strong phenotypes associated with their loss in vivo, which have led to extensive study both in vitro and in vivo. In contrast, ancestral SOCS members cause less severe loss of function phenotypes, likely due to genetic redundancies in mammals, and thereby there is a less clear understanding of how these proteins function. Since structural differences between the vertebrate-specific and ancestral SOCS proteins may mediate distinct mechanisms of action between the two groups (Alexander, 2002; Linossi, 2015), further characterization of the ancestral SOCS members is essential to elucidate their effects on signal transduction pathways. In contrast to mammals, Drosophila have only three SOCS proteins (Socs16D, Socs44A, and Socs36E) (Callus, 2002; Karsten, 2002; Rawlings, 2004). Socs36E, which is most similar to mammalian SOCS5, is the only one that appears to act in ovarian follicle cells (Rawlings, 2004; Monahan, 2013) (Monahan, 2015).

The SOCS box interacts with Elongin B and C adaptor proteins and a Cullin scaffolding protein,which incorporates SOCS members into an E3 ubiquitin ligase complex to promote protein turnover. Specifically, the SOCS protein acts as the substrate recognition component of some RING finger E3 ligase complexes, as the SH2 domain may target the complex to specific substrate(s) for ubiquitination. In vitro studies have shown that SOCS proteins bind members of the Cullin family of scaffolding proteins, particularly Cullin2 (Cul2) and Cullin5 (Cul5). Most studies investigating SOCS proteins support a SOCS-Cul5 interaction, although some have shown an interaction with Cul2 (Monahan, 2015).

This study utilized border cell motility as an in vivo system to study the mechanism b ywhich Socs36E attenuates STAT signaling in the Drosophila egg chamber. Socs36E was determined to genetically interact with Cul2, and that loss of Cul2 was shown to result in mis-specification of additional invasive cells. This study found a significant increase in activated, nuclear STAT protein levels during border cell specification, an expanded border cell precursor population, and an excessive number of invasive cells at stage 10 when Cul2 was reduced in the anterior follicle cells. These phenotypes are similar to those of Socs36E deficient egg chambers (Monahan, 2013). Importantly, it was determined that a reduction of Cul2 restored proper border cell migration when Stat92E was below endogenous levels, and that Cul2 genetically interacts with apt, another known STAT-regulator in the egg chamber. This study also discovered the SOCS box is not required for all functions of Socs36E in vivo. From this work, the STAT-regulatory genetic circuit in Drosophila egg chambers was refined by determining some modes of inhibition. It is proposed that Socs36E functions with Cul2 to restrict migratory fate to the border cell cluster through a ubiquitin-dependent mechanism, but that it can also regulate the Jak/STAT pathway in a SOCS-box independent manner (Monahan, 2015).

The involvement of SOCS proteins in RING Finger E3 ubiquitin ligases has been well-established, and is thought to be mediated by Cullin interaction. A previous report proposed that SOCS box proteins contain a Cullin5-box, while the highly related Von Hippel-Lindau (VHL) proteins have a Cullin2-box (Kamura, 2004). However, the only SOCS family members assayed were SOCS1 and SOCS3, both of which have been shown to bind Cul5. Several reports have shown that proteins with a SOCS box (including SOCS1) are able to interact with and bind Cul2. Analysis of the predicted Cul5-box of SOCS1, SOCS3, SOCS5, and Socs36E revealed low sequence similarity across the four proteins. Furthermore, the proposed key sequence of the Cul5-box (LPLP) (Kamura, 2004) is only found in SOCS5 (Monahan, 2015).

This study found that Socs36E attenuates the Jak/STAT pathway in a Cul2- dependent manner. Reducing Cul2 function in the anterior follicle cells of stage 8 egg chambers significantly expanded the border cell precursor population (Slbo+ anterior follicle cells) and heightened nuclear STAT (nSTAT) protein levels. These data show Cul2 acts to attenuate STAT in the egg chamber. There did not appear to be a similar requirement for other Cullin family members that were tested, although in the absence of amorphic alleles, it remains a possibility. The expanded Slbo-positive population observed in egg chambers deficient for Cul2 is likely due to higher than normal STAT activity in follicle cells far from the polar cells, which can explain the additional invasive cell phenotype (similar to when STAT signaling is increased in apt or Socs36E mutants (Starz-Gaiano, 2008; Monahan, 2013). In support of this, it was determined that lowering Cul2 rescues the delay in border cell migration that occurs when Stat92E is reduced. Collectively, these data strongly suggest that Cul2 limits STAT activity in the anterior follicle cells of the egg chamber (Monahan, 2015).

By finding that Cul2 genetically interacts with apt (a central component of STAT regulation in egg chambers), Cul2 can be incorporated into the previously described Jak/STAT regulatory circuit (Starz-Gaiano, 2008; Yoon, 2011; Monahan, 2013). It is postulated that Socs36E aids in the generation of a discrete boundary between migratory and non-migratory fates in the anterior follicle cells, by functioning as the substrate recognition component of a Cul2-E3 ubiquitin ligase complex. Given published biochemical data on SOCS family members (Kamizono, 2001; Boggio, 2007; Pozzebon, 2013) and the current genetic and alignment results, it is suggested that SOCS proteins may have the potential to bind both Cul2 and Cul5; however the binding preference may be tissue- or context-dependent. Analyses of these interactions are well-suited for future in vivo study (Monahan, 2015).

Previous work established Socs36E as a regulator of the Epidermal Growth Factor Receptor (EGFR) pathway in some Drosophila tissues. After border cell specification, EGFR works redundantly with the PDGF- and VEGF receptor related (PVR) pathway to promote protrusive activity and to guide the migration of the cluster to the oocyte. EGFR is also required to direct the border cell cluster dorsally to the oocyte nuclei at stage 10B. No defects were observed in the directed migration of the border cell cluster when Cul2 was reduced, similar to previous results with Socs36E (Monahan, 2013); thus it is not suspected that these genes are necessary for EGFR regulation in follicle cells. It is possible that the redundancy between EGFR and PVR in border cell chemotaxis masks Socs36E and Cul2 regulation of EGFR, therefore cannot be completely ruled out. However, the phenotypic results strongly suggest that the Jak/STAT pathway is the primary target of both Socs36E and Cul2 in the anterior follicle cells of the egg chamber (Monahan, 2015).

Previous work suggests that the Dome receptor is targeted for endocytosis after ligand binding, and that this event is required for proper migration of the border cells (Devergne, 2007; Vidal, 2010). While the mechanism has not been resolved in the egg chamber, a recent study using Drosophila Kc167 cells showed that Dome is degraded in the lysosome, in a ligand-dependent manner, and that loss of Socs36E delays receptor clearance (Stec, 2013). Knockout of Cul5 and Socs36E resulted in higher activation of Jak/STAT, relative to loss of Cul5 alone, although Cul2 was not assayed. These data may suggest a SOCS box-independent mechanism, but an additional interpretation could be that Socs36E also mediates receptor clearance in a Cul2-dependent manner. Stec also found that Socs36E can bind Dome, but only weakly interacts with Jak in a Dome-dependent manner (Stec, 2013). These data combined with the current work support a model in which Socs36E facilitates the degradation of an activated Dome-Jak complex in the anterior follicle cells of the egg chamber. This attenuates STAT signaling, which is essential to limit the acquisition of migratory fates (Monahan, 2015).

Other possible mechanisms of action were considered for Socs36E in the egg chamber. Several in vitro assays, including binding assays and crystallography, found the SOCS box directly interacts with Elongins B/C and Cullin. Many of these reports suggest that the SOCS box is essential for attenuation of cytokine signaling and SOCS protein stability. In contrast, several studies have found loss of the SOCS box impedes SOCS protein function, but does not eliminate it. These studies suggest that the SOCS box is necessary for complete SOCS-driven attenuation of cytokine signaling, but to a varying degree and possibly in a tissue specific manner. Because expression of UAS-Socs36EΔSB in a Socs36E deficient egg chamber restored approximately wild-type migration to Socs36E mutants, it is concluded that the SOCS box domain is largely dispensable for motile cell specification. However, it was not dispensable for normal cluster cohesion, as some invasive cells trailed behind the main cluster (Monahan, 2015).

Since SOCS proteins require the SOCS box to facilitate their incorporation into an E3 ligase complex, it is proposed that Socs36E can partially attenuate STAT activity independently of Cullin-E3 ligase activity in vivo. Consistent with vertebrate ancestral SOCS proteins, the current sequence analysis revealed the Socs36E N-terminus is intrinsically disordered. Intrinsically disordered proteins (IDPs) have a capacity to generate protein-protein interactions. Upon binding another protein, IDPs undergo an energetically favorable disordered to ordered transition. The intrinsic flexibility and ability to adopt several conformations and binding partners enables a single IDP to function in several signaling pathways and cellular processes (Monahan, 2015).

The long N-termini of SOCS5 and Socs36E have been proposed to play an essential role in the SOCS-receptor interaction and, in some cases, are critical for SOCS function. For example, the N-terminus of SOCS5 regulates T-cell differentiation by disrupting Jak1 association with IL4Rα, and a recent study proposed this region directly prevents Jak1/2 activity. Other studies have suggested that the N-termini of ancestral SOCS proteins play a critical role in SOCS-substrate interaction, including cell culture analysis of Socs36E. While this study did not locate a JIR consensus sequence in Socs36E, in vitro studies together with the current data suggest that the N-terminal region of Socs36E may be important for substrate binding. It is hypothesized that the N-terminus of Socs36E is intrinsically disordered and may play a role in limiting cytokine-activated signaling. It will be interesting to determine if and how the N-terminus functionally inhibits Jak/STAT signaling independently from a Cullin-E3-ligase complex. Many questions remain about IDPs, their interactions, structure, and other biochemical characteristics. New approaches will be required to fully study IDPs in a cellular context. It is suggested that the minimized pathway components, potent genetic tools, and high levels of genetic conservation make Drosophila an ideal system to study IDPs in an in vivo context. Thus, future work on Socs36E function in the ovary could provide further insight, not only into SOCS protein biology but also IDPs, in general (Monahan, 2015).

The SH2 domain of SOCS proteins is required for substrate binding, including the binding and turnover of Dome in Kc167 cells (Stec, 2013). The SH2 domain could also play an active role in cytokine attenuation. For instance, Socs36E may compete with STAT for the same or a proximal phosphotyrosine on the receptor, thereby preventing STAT phosphorylation, as has been proposed for CIS and SOCS2 (Croker, 2008; Linossi, 2015). These are not mutually exclusive ideas. Therefore, a model is favored in which Socs36E interacts with Cullin2 in an E3 ubiquitin ligase complex, but that can prevent Jak activity and/or block Dome/Jak access to STAT via its SH2 domain and/or N-terminus (Monahan, 2015).

The closest mammalian homolog of Socs36E, SOCS5, is a proposed tumor suppressor. Reduction of SOCS5 can result in loss of epithelial organization, increased tumor metastasis, and aggressive carcinomas. Thus, understanding its mechanism of action will enhance understanding of cancer progression, but analysis in mammals has proven challenging. This study shows that use of a genetic model organism enables in vivo assessment of SOCS proteins; this sheds light on how these proteins function (Monahan, 2015).

Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche

Adult stem cells reside in microenvironments called niches, where they are regulated by both extrinsic cues, such as signaling from neighboring cells, and intrinsic factors, such as chromatin structure. This study reports that in the Drosophila testis niche an H3K27me3-specific histone demethylase encoded by Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (dUTX) maintains active transcription of the Suppressor of cytokine signaling at 36E (Socs36E) gene by removing the repressive H3K27me3 modification near its transcription start site. Socs36E encodes an inhibitor of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Whereas much is known about niche-to-stem cell signaling, such as the JAK-STAT signaling that is crucial for stem cell identity and activity, comparatively little is known about signaling from stem cells to the niche. The results reveal that stem cells send feedback to niche cells to maintain the proper gene expression and architecture of the niche. dUTX acts in cyst stem cells (CySCs) to maintain gene expression in hub cells through activating Socs36E transcription and preventing hyperactivation of JAK-STAT signaling. dUTX also acts in germline stem cells to maintain hub structure through regulating DE-Cadherin levels. Therefore, these findings provide new insights into how an epigenetic factor regulates crosstalk among different cell types within an endogenous stem cell niche, and shed light on the biological functions of a histone demethylase in vivo (Tarayrah, 2013).

This study identified a new epigenetic mechanism that negatively regulates the JAK-STAT signaling pathway in the Drosophila testis niche: the H3K27me3-specific demethylase dUTX acts in CySCs to remove the repressive H3K27me3 histone modification near the TSS of Socs36E to allow its active transcription. Socs36E acts upstream to suppress Stat92E activity and to restrict CySCs from overpopulating the testis niche. In addition, dUTX acts in CySCs to prevent hyperactivation of Stat92E in hub cells, which would otherwise ectopically turn on Zfh1 expression. When zfh1 cDNA was ectopically droven in hub cells using the upd-Gal4 driver, no obvious defect could be identified. Therefore, the biological consequence of ectopic Zfh1 expression in hub cells remains unclear. However, ectopic Zfh1 expression in hub cells and the overpopulation of Zfh1-expressing cells around the hub are two connected phenomena because both phenotypes are caused by loss of dUTX in CySCs (Tarayrah, 2013).

UTX also acts in GSCs to regulate DE-Cadherin levels to maintain proper GSC-hub interaction and normal morphology of the hub. It has been reported that differential expression of different cadherins causes cells with similar cadherin types and levels to aggregate. In wt testes, hub cells express higher levels of DE-Cadherin and therefore tightly associate with each other. Loss of dUTX in germ cells leads to higher levels of DE-Cadherin in GSCs, which probably allows them to intermingle with hub cells and causes disrupted hub architecture. It has also been demonstrated that the major role of JAK-STAT in GSCs is for GSC-hub adhesion, suggesting that the expression and/or activity of cell-cell adhesion molecules, such as DE-Cadherin, depends on JAK-STAT signaling. Therefore, the abnormal DE-Cadherin activity in GSCs in dUTX testis could also result from misregulated JAK-STAT signaling in the testis niche. dUTX is a new negative epigenetic regulator of the JAK-STAT signaling pathway (Tarayrah, 2013).

The JAK-STAT signaling pathway plays crucial roles in stem cell maintenance in many different stem cell types across a wide range of species. These studies identify the histone demethylase dUTX as a new upstream regulator of the JAK-STAT pathway, which directly controls the transcription of Socs36E. In addition to acting as an antagonist of JAK-STAT signaling, Socs36E has been reported to be a direct target gene of the Stat92E transcription factor (Terry, 2006). Therefore, increased Stat92E would be expected to upregulate Socs36E expression, but this was not observed in dUTX mutant testes. Instead, the data revealed that Socs36E expression decreased, whereas Stat92E expression increased, in dUTX testes, consistent with the hypothesis that Socs36E is a direct target gene of dUTX and acts upstream of Stat92E (Tarayrah, 2013).

Sustained activity of the JAK-STAT pathway in cyst cells has been reported to activate BMP signaling, which leads to GSC self-renewal outside the niche and gives rise to a tumor-like phenotype in testis. To examine BMP pathway activity, immunostaining experiments were performed using antibodies against phospho-SMAD (pSMAD), a downstream target of BMP signaling. No obvious difference was detected in the pSMAD signal between the dUTX testes and wt control, nor were any germline tumors detected in dUTX testes. It is speculated that germline tumor formation upon activation of the JAK-STAT pathway is secondary to the overproliferation of Zfh1-expressing cells, which was not observed in dUTX testes (Tarayrah, 2013).

This study also provides an example of the multidimensional cell-cell communication that takes place within a stem cell niche. Many studies of the stem cell niche have focused on understanding niche-to-stem cell signaling in controlling stem cell identity and activity. For example, in the Drosophila female GSC niche, Upd secreted from terminal filaments activates the JAK-STAT pathway in cap cells and escort cells, which subsequently produce the BMP pathway ligand Decapentaplegic (Dpp) to activate BMP signaling and prevent transcription of the differentiation factor bag-of-marbles (bam) in GSCs. In the Drosophila intestinal stem cell (ISC) niche, the visceral muscle cells underlying the intestine secrete Wingless to activate Wnt signaling and Upd to activate JAK-STAT signaling in ISCs, which are required to maintain ISC identity and activity (Tarayrah, 2013).

More studies have now revealed the multidirectionality of signaling within the stem cell niche. For example, in the Drosophila female GSC niche, GSCs activate Epidermal growth factor receptor (Egfr) signaling in the neighboring somatic cells, which subsequently represses expression of the glypican Dally, a protein required for the stabilization and mobilization of the BMP pathway ligand Dpp. Through this communication between GSCs and the surrounding somatic cells, only GSCs maintain high BMP signaling. The current studies establish another example of the multidimensional cell-cell communications that occur within the testis stem cell niche, where CySCs and GSCs have distinct roles in regulating hub cell identity and morphology (Tarayrah, 2013).

The data identified new roles of a histone demethylase in regulating endogenous stem cell niche architecture and proper gene expression. Previous studies have reported in vivo functions of histone demethylases in several model organisms. For example, mammalian UTX has been shown to associate with the H3K4me3 histone methyltransferase MLL2, suggesting its potential antagonistic role to the PcG proteins. The PcG proteins play a crucial role in Hox gene silencing in both Drosophila and mammals. Consistently, mammalian UTX has been reported to directly bind and activate the HOXB1 gene locus. In addition to antagonizing PcG function, H3K27me3 demethylases play crucial roles during development. For example, in zebrafish, inactivating the UTX homolog (kdm6al) using morpholino oligonucleotides leads to defects in posterior development, and in C. elegans the dUTX homolog (UTX-1) is required for embryonic and postembryonic development, including gonad development. Furthermore, loss of UTX function in embryonic stem cells leads to defects in mesoderm differentiation, and somatic cells derived from UTX loss-of-function human or mouse tissue fail to return to the ground state of pluripotency. These reports demonstrate that UTX is not only required for proper cellular differentiation but also for successful reprogramming. However, despite multiple reports on the in vivo roles of H3K27me3-specific demethylases, little is known about their functions in any endogenous adult stem cell system (Tarayrah, 2013).

Whereas mammals have multiple H3K27me3 demethylases, dUTX is the sole H3K27me3-specific demethylase in Drosophila. This unique feature, plus the well-characterized nature of Drosophila adult stem cell systems, make interpretation of the endogenous functions of histone demethylases in Drosophila unambiguous. Because mammalian UTX has been reported as a tumor suppressor, understanding the endogenous functions of dUTX in an adult stem cell system might facilitate the use of histone demethylases for cancer treatment. In summary, these results demonstrate that stem cells send feedback to the niche cells to maintain their proper gene expression and morphology. Furthermore, this feedback is regulated through the JAK-STAT signaling pathway, the activity of which is controlled by a chromatin factor, providing an example of crosstalk between these two regulatory pathways (Tarayrah, 2013).

Drosophila SOCS36E negatively regulates JAK/STAT pathway signaling via two separable mechanisms

Conserved from humans to Drosophila, the Janus kinase/signal transducer and activators of transcription (JAK/STAT) signaling cascade is essential for multiple developmental and homeostatic processes, with regulatory molecules controlling pathway activity also highly conserved. This study has characterized the Drosophila JAK/STAT pathway regulator SOCS36E and shows that it functions via two independent mechanisms. First, Drosophila Elongin B/C and Cullin-5 were shown to act via the SOCS-box of SOCS36E to reduce pathway activity specifically in response to ligand stimulation--a process that involves endocytic trafficking and lysosomal degradation of the Domeless (Dome) receptor. Second, SOCS36E also suppresses both stimulated and basal pathway activity via an Elongin/Cullin-independent mechanism that is mediated by the N-terminus of SOCS36E, which is required for the physical interaction of SOCS36E with Dome. Although some human SOCS proteins contain N-terminal kinase-inhibitory domains, this study did not identify such a region in SOCS36E, and a model is proposed wherein the N-terminal of SOCS36E blocks access to tyrosine residues in Dome. This biochemical analysis of a SOCS-family regulator from Drosophila highlights the fundamental conserved roles played by regulatory mechanisms in signal transduction (Stec, 2013).

This study presents detailed molecular characterization of SOCS36E, a negative regulator of Drosophila JAK/STAT pathway signaling. Elongin B/C, Cullin-5, and SOCS36E negatively regulate the stability of Dome and act as negative regulators of the ligand-activated JAK/STAT pathway. This activity may be mediated by a sorting mechanism that directs receptor complexes to the lysosome and requires both the SOCS-box and an intact SH2 domain. SOCS36E was shown to be able to negatively regulate both basal and ligand-induced activity of the JAK/STAT pathway in a manner independent of SOCS-box, Elongin B/C, and Cullin-5 via its N-terminal region. Although the exact molecular mechanism by which the N-terminus of SOCS36E operates remains unresolved, it is likely that this activity is linked to the interactions of the N-terminal region of SOCS36E with Dome, which may act to prevent the phosphorylation of key tyrosine residues by Hop. Taking the results together, this study shows that SOCS36E negatively regulates both basal and activated activity of the Drosophila JAK/STAT pathway via two independent and separable mechanisms (Stec, 2013).

Mammalian SOCS proteins have been shown to affect internalization and endocytosis of cytokine receptors by more than one mechanism. For example, the granulocyte colony-stimulating factor receptor is targeted for degradation via ubiquitination by an Elongin-Cullin-SOCS3 complex. The current results indicate that, in Drosophila, Elongin B/C, Cullin-5, and SOCS36E are also involved in the regulation of Dome stability and pathway activity. In light of previous work identifying endocytosis as a negative regulator of the Drosophila JAK/STAT pathway (Vidal, 2010), it is proposed that the ECS complex represents an integral component of SOCS-mediated pathway regulation that functions via interactions with the endocytic machinery. By analogy to mammalian systems, it seems possible that the Drosophila ECS complex might mediate interactions with the endocytic machinery via its hypothesized E3 ubiquitin ligase activity and the subsequent ubiquitination of Dome (Stec, 2013).

A number of studies have focused on mammalian SOCS molecules, characterizing their molecular structure and function and their involvement in disease and cancer. However, only a handful of reports have identified a role for the relatively divergent N-terminal regions of SOCS-family members. The N-terminal of SOCS5 has been shown to interact with IL-4 receptor in a phosphotyrosine-independent manner, and SOCS5 has been reported to associate with EGFR in an N-terminal-dependent manner. A recent report has also identified a conserved motif in the N-terminus of SOCS4 and SOCS5 that has a potential role in protein interaction. Considering that SOCS36E is most closely related to mammalian SOCS4 and 5, the results provide further support for a conserved functional role of the long N-terminus present in these SOCS molecules. This study has shown that both the N-terminal of SOCS36E and its SH2 domain are required for efficient binding of SOCS36E to Dome, although it is not known whether the SOCS36E N-terminal itself is sufficient for this interaction or whether it stabilizes the interaction in a manner similar to the N-terminal of SOCS3. In the case of SOCS3, an N-terminally extended SH2 domain (N-ESS) has been described that is involved in orientating interactions with phosphorylated tyrosine residues. Truncation of the N-terminal of SOCS36E could have affected any N-ESS region that might be present and could thus destabilize the association of SOCS36E with Dome, explaining why higher levels of SOCS36EΔN overexpression were required to decrease receptor levels (Stec, 2013).

This study has also shown that, in addition to playing a role in protein:protein interactions, the N-terminal of SOCS36E is able to suppress JAK/STAT pathway activity irrespective of ligand stimulation. This function is independent of Elongin B/C, Cullin-5, or the SOCS-box domain and is consistent with previous genetic analysis showing that the N-terminal of SOCS36E is able to inhibit both the EGFR and JAK/STAT pathways in vivo. Endogenous SOCS36E was also shown to regulate the phosphorylation level of receptor complex components, a finding that correlates with its role in suppressing basal pathway activity under steady-state conditions, and full-length SOCS36E was also shown to reduce Dome phosphorylation following ligand binding. While not a line of enquiry followed in this study, the ability of SOCS36E to bind to receptors and modulate their tyrosine phosphorylation may also represent the mechanism via which SOCS36E is able to modulate EGFR activity in vivo (Stec, 2013).

Finally, the finding that overexpression of SOCS36E or its deletions were unable to significantly affect the phosphorylation of Hop and does not affect Hop kinase activity in vitro suggests SOCS36E functions as a steric inhibitor of Hop that prevents interactions with its substrate Dome. This proposed function for the N-terminal of SOCS36E also suggests that the unstructured N-terminal regions of other mammalian SOCS molecules, including the most closely homologous SOCS4 and SOCS5, might also act via similar mechanisms, possibly in a receptor-specific manner to suppress pathway activity under steady-state conditions. However, the possibility was not excluded that the N-terminal of SOCS36E mediates interaction with yet unknown cofactors that might directly or indirectly affect receptor phosphorylation levels. Irrespective of which hypothesis is true, further research into mammalian SOCS-family members and their homologues could provide insight into mechanisms of receptor:JAK complex inhibition, potentially laying the groundwork for novel drug development approaches in the future (Stec, 2013).

Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila epithelial transformation model

MicroRNAs (miRNAs) are emerging as cooperating factors that promote the activity of oncogenes in tumor formation and disease progression. This poses the challenge of identifying the miRNA targets responsible for these interactions. This study has identified the growth regulatory miRNA bantam and its target, Socs36E, as cooperating factors in EGFR-driven tumorigenesis and metastasis in a Drosophila model of epithelial transformation. bantam promotes growth by limiting expression of Socs36E, which functions as a negative growth regulator. Socs36E has only a modest effect on growth on its own, but behaves as a tumor suppressor in combination with EGFR activation. The human ortholog of SOCS36E, SOCS5, behaves as a candidate tumor suppressor in cellular transformation in cooperation with EGFR/RAS pathway activation (Herranz, 2012).

Socs36E attenuates STAT signaling to optimize motile cell specification in the Drosophila ovary

The JAK/STAT) pathway determines cell fates by regulating gene expression. One example is the specification of the motile cells called border cells during Drosophila oogenesis. It has been established that too much or too little STAT activity disrupts follicle cell identity and cell motility, which suggests the signaling must be precisely regulated. This study found that Socs36E is a necessary negative regulator of JAK/STAT signaling during border cell specification. When STAT signaling is too low to induce migration in the presumptive border cell population, nearby follicle cells uncharacteristically become invasive to enable efficient migration of the cluster. A genetic null allele was generated that reveals Socs36E is required in the anterior follicle cells to limit invasive behavior to an optimal number of cells. Socs36E was further shown to genetically interact with the required STAT feedback inhibitor apontic (apt) and APT's downstream target, mir-279, and evidence is provided that suggests APT directly regulates Socs36E transcriptionally. This work shows Socs36E plays a critical role in a genetic circuit that establishes a boundary between the motile border cell cluster and its non-invasive epithelial neighbors through STAT attenuation (Monahan, 2013).

The Maf factor Traffic jam both enables and inhibits collective cell migration in Drosophila oogenesis

Border cell cluster (BCC) migration in the Drosophila ovary is an excellent system to study the gene regulatory network that enables collective cell migration. This study identified the large Maf transcription factor Traffic jam (Tj) as an important regulator of BCC migration. Tj has a multifaceted impact on the known core cascade that enables BCC motility, consisting of the Jak/Stat signaling pathway, the C/EBP factor Slow border cells (Slbo), and the downstream effector DE-cadherin (DEcad). The initiation of BCC migration coincides with a Slbo-dependent decrease in Tj expression. This reduction of Tj is required for normal BCC motility, as high Tj expression strongly impedes migration. At high concentration, Tj has a tripartite negative effect on the core pathway: a decrease in Slbo, an increase in the Jak/Stat inhibitor Socs36E, and a Slbo-independent reduction of DEcad. However, maintenance of a low expression level of Tj in the BCC during migration is equally important, as loss of tj function also results in a significant delay in migration concomitant with a reduction of Slbo and consequently of DEcad. Taken together, it is concluded that the regulatory feedback loop between Tj and Slbo is necessary for achieving the correct activity levels of migration-regulating factors to ensure proper BCC motility (Gunawan, 2013).

Requirement for JAK/STAT signaling throughout border cell migration in Drosophila

The evolutionarily conserved JAK/STAT signaling pathway is essential for the proliferation, survival and differentiation of many cells, including cancer cells. Recent studies have implicated this transcriptional pathway in the process of cell migration in humans, mice, Drosophila and Dictyostelium. In the Drosophila ovary, JAK/STAT signaling is necessary and sufficient for the specification and migration of a group of cells called the border cells; however, it is not clear to what extent the requirement for cell fate is distinct from that for cell migration. It was found that STAT protein is enriched in the migrating border cells throughout their migration and is an indicator of cells with highest JAK/STAT activity. In addition, stat^ts mutants exhibit border cell migration defects after just 30 minutes at the non-permissive temperature, prior to any detectable change in the expression of cell fate markers. At later times, cell fate changes became evident, indicating that border cell fate is labile. JAK/STAT signaling was also required for organization of the border cell cluster. Finally, it is shown that both the accumulation of STAT protein and nuclear accumulation are positively regulated by JAK/STAT activity. The activity of the pathway is negatively regulated by overexpression of a suppressor of cytokine signaling (SOCS) protein and by blocking endocytosis. Together, these findings suggest that the requirement for STAT in border cells extends beyond the initial specification and delamination of cells from the epithelium (Silver, 2005).

Extra and ectopic border cells can be induced in at least two different ways. When ectopic polar cells form, for example in eyes absent or costal 2 (costa) mutant clones, or following mis-expresssion of activated Notch, they recruit surrounding cells into a cluster and these are capable of migration. Alternatively, ectopic expression of UPD, HOP or HOP^Tum is sufficient to induce large numbers of ectopic migrating border cells. These cells migrate in a variety of sizes of clusters, which lack polar cells, and can even migrate as individual cells. These findings indicate that UPD might be the only factor produced by polar cells that functions to recruit border cells and sustain their motility. To test this hypothesis, ectopic expression of UPD was induced in single anterior follicle cells, or in pairs of cells, to see whether UPD expression alone is as effective as polar cells in recruiting border cells. For comparison, the same method was also used to express a form of UPD that contains a transmembrane domain (UPD^TM). Expression of the wild-type form of UPD results in the recruitment of neighboring cells into a cluster, and these cells express high levels of STAT protein, like normal border cells. However, UPD alone is not as effective as a normal polar cell because, normally, two polar cells recruit four to eight cells to surround them, whereas UPD alone results in the recruitment of an average of only 1.1 border cell per UPD-expressing cell. Cells expressing the UAS-UPD^TM are actually more effective at recruiting border cells than cells expressing wild-type UPD. A single cell expressing the membrane-tethered form of UPD recruits an average of 3.25 cells to surround it, similar to normal or ectopic polar cells. In the case of the membrane-tethered form of UPD, ectopic migratory cells are observed only adjacent to the UPD-expressing cells. These findings suggested that JAK/STAT signaling contributes to the organization of the migrating border cell cluster. This was investigated further by analyzing the organization of migrating border cells following the reduction of JAK/STAT function (Silver, 2005).

Suppressors of cytokine signaling are thought to inhibit the JAK/STAT pathway, either by blocking JAK or STAT function via a SOCS SH2 domain, or by causing the destruction of these proteins by ubiquitination. There are three SOCS genes in Drosophila but, to date, no loss-of-function mutants have been reported. Using both slbo-GAL4,1310 and c306-GAL4 drivers, it was found that overexpression of wild-type SOCS36E inhibits border cell migration and recruitment, and overexpression of SOCS36E lacking the SOCS domain weakly inhibits migration. By contrast, overexpression of a SOCS36E protein that lacks the SH2 domain, or of slbo-GAL4,1310 or c306-GAL4 alone failed to disrupt border cell migration. Compared with an average border cell number of six for wild-type egg chambers, slbo-GAL4,1310;UAS-SOCS and c306-GAL4;UAS-SOCS egg chambers have an average number of border cells of 3.3 and 4.9, respectively. This is consistent with findings that reduction of JAK/STAT activity inhibits border cell recruitment. Those border cells that do form and migrate, frequently do so as single cells rather than as a cluster. In addition, the border cells had reduced levels of STAT (Silver, 2005).

Consistent with a requirement in cluster organization, when JAK/STAT signaling is reduced by overexpression of a dominant-negative form of dome lacking the cytoplasmic domain, border cells migrate slower than wild-type cells, and frequently as single cells. This is consistent with the finding that in dome mosaic egg chambers, border cells often fail to migrate in a cluster. Taken together, these results support the idea that signaling through the JAK/STAT pathway is responsible for the organization of the border cell cluster (Silver, 2005).

Normally border cells migrate as a cohesive cluster with the non-migratory, UPD-expressing cells in the center and the migratory cells surrounding them. These two cell types are dependent upon each other, since the central cells cannot migrate and are carried by the surrounding cells, and the migratory cells cannot move in the absence of the UPD signal from the central cells. Thus, the organization of the border cell cluster is crucial for normal migration. In addition to its function in border cell specification and motility, several lines of evidence demonstrate the role of UPD/JAK/STAT in organizing the border cell cluster. Ectopic expression of UPD in single anterior follicle cells, for example, is sufficient to recruit adjacent cells to form a cluster capable of migration. In addition, a variety of treatments that reduce STAT activity (dome mosaic clones, overexpression of dominant-negative Dome, and overexpression of SOCS) leads to disruptions of cluster formation. Disruption of the cluster is likely to affect migration through the egg chamber. For example, PAR6, an epithelial protein required for polarity and the migration of border cells, is disrupted in border cells in which dominant-negative Dome is overexpressed, lending support to the idea that JAK/STAT signaling helps to regulate the organization of cells within the cluster. Once the cluster is disrupted, the migratory cells become separated from the polar cells, presumably reducing STAT activity further and aggravating the migration defect. Thus, STAT activity promotes cluster organization, which feeds back to promote efficient UPD/DOME/JAK/STAT signaling (Silver, 2005).

Taken together, the results presented here demonstrate several inter-related properties of JAK/STAT signaling in the control of border cell migration and function. Both anatomical and biochemical mechanisms feed back upon each other to regulate the level of STAT activity precisely throughout the six hours of border cell migration. Positive-feedback mechanisms include maintaining close contact between UPD-expressing cells and the migratory cells, as well as stabilization and nuclear enrichment of STAT protein in response to signaling. One negative regulatory mechanism is the expression of SOCS36E (Silver, 2005).

The findings described here may also have relevance for understanding the requirement of STAT signaling in the progression of cancer. Constitutively activated STAT3 is associated with the aggressive clinical behavior of a number of cancers, including ovarian and renal cancers. Blocking STAT3 in pancreatic cancer cells inhibits tumor growth and metastases in mice, whereas expression of activated STAT3 promotes metastasis. Inhibiting STAT3 expression or activation in ovarian carcinoma cells impedes their motility in vitro. Thus, cancer cells too appear to require sustained activation of this pathway to survive, proliferate and migrate. The finding that JAK/STAT signaling appears to be tightly regulated by its own activity, by that of SOCS inhibitors and by endocytic processes suggests that these may provide points of clinical intervention in the treatment of STAT-dependent cancers (Silver, 2005).

Competitiveness for the niche and mutual dependence of the germline and somatic stem cells in the Drosophila testis are regulated by the JAK/STAT signaling

In many tissues, two or more types of stem cells share a niche, and how the stem cells coordinate their self-renewal and differentiation is poorly understood. In the Drosophila testis, germ line stem cells (GSCs) and somatic cyst progenitor cells (CPCs) contact each other and share a niche (the hub). The hub expresses a growth factor unpaired (Upd) that activates the JAK/STAT pathway in GSCs to regulate the stem cell self-renewal. This study demonstrates that the JAK/STAT signaling also regulates CPCs self-renewal. A negative regulator, the suppressor of cytokine signaling 36E (SOCS36E), suppresses JAK/STAT signaling in somatic cells, preventing them from out-competing the GSCs. Furthermore, through selectively manipulating the JAK/STAT signaling level in either CPCs or GSCs, it was demonstrated that the somatic JAK/STAT signaling is essential for self-renewal and maintenance of both CPCs and GSCs. These data suggest that a single JAK/STAT signal from the niche orchestrate the competitive and dependent co-existence of GSCs and CPCs in the Drosophila testis niche (Singh, 2010).

JAK-STAT signal inhibition regulates competition in the Drosophila testis stem cell niche

Adult stem cells often reside in local microenvironments, or niches. Although niches can contain multiple types of stem cells, the coordinate regulation of stem cell behavior is poorly understood. In the Drosophila testis, JAK-STAT signaling is directly required for maintenance of the resident germline and somatic stem cells. JAK-STAT signaling target and inhibitor SOCS36E is required for germline stem cell maintenance. SOCS36E suppresses JAK-STAT signaling specifically in the somatic stem cells, preventing them from displacing neighboring germline stem cells in a manner that depends on the adhesion protein integrin. Thus, in niches housing multiple stem cell types, negative feedback loops can modulate signaling, preventing one stem cell population from outcompeting the other (Issigonis, 2009).

Competition between SOCS36E and Drk modulates Sevenless receptor tyrosine kinase activity

Modulation of signalling pathways can trigger different cellular responses, including differences in cell fate. This modulation can be achieved by controlling the pathway activity with great precision to ensure robustness and reproducibility of the specification of cell fate. The development of the photoreceptor R7 in the Drosophila melanogaster retina has become a model in which to investigate the control of cell signalling. During R7 specification, a burst of Ras small GTPase (Ras) and mitogen-activated protein kinase (MAPK) controlled by Sevenless receptor tyrosine kinase (Sev) is required. Several cells in each ommatidium express sev. However, the spatiotemporal expression of the boss ligand and the action of negative regulators of the Sev pathway will restrict the R7 fate to a single cell. The Drosophila SOCS36E protein contains an SH2 domain and acts as a Sev signalling attenuator. By contrast, downstream of receptor kinase (Drk), the fly homolog of the mammalian Grb2 adaptor protein, which also contains an SH2 domain, acts as a positive activator of the pathway. This study applied the Forster resonance energy transfer (FRET) assay to transfected Drosophila S2 cells and demonstrated that Sev binds directly to either the suppressor protein SOCS36E or the adaptor protein Drk. A mechanistic model is proposed in which the competition between these two proteins for binding to the same docking site results in either attenuation of the Sev transduction in cells that should not develop R7 photoreceptors or amplification of the Ras-MAPK signal only in the R7 precursor (Almudi, 2010).

Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of notch signaling by the JAK/STAT pathway

Although the JAK/STAT pathway regulates numerous processes in vertebrates and invertebrates through modulating transcription, its functionally relevant transcriptional targets remain largely unknown. With one jak (hopscotch) and one stat (stat92E), Drosophila provides a powerful system for finding new JAK/STAT target genes. Genome-wide expression profiling on eye discs in which Stat92E is hyperactivated, revealed 584 differentially regulated genes, including known targets domeless, socs36E, and wingless. Other differentially regulated genes (chinmo, lama, Mo25, Imp-L2, Serrate, Delta) were validated and may represent new Stat92E targets. Genetic experiments revealed that Stat92E cell-autonomously represses Serrate, which encodes a Notch ligand. Loss of Stat92E led to de-repression of Serrate in the dorsal eye, resulting in ectopic Notch signaling and aberrant eye growth there. Thus, this micro-array study documents a new Stat92E target gene and a previously unidentified inhibitory action of Stat92E on Notch signaling. These data suggest that this study will be a useful resource for the identification of additional Stat92E targets (Flaherty, 2009).

SOCS36E specifically interferes with Sevenless signaling during Drosophila eye development

During the development of multicellular organisms the fate of individual cells is specified with great precision and reproducibility. Although classical genetic approaches led to the identification of many of the signaling pathways contributing to cell fate specification, they have provided little insight into the mechanisms that ensure robustness and reproducibility. This study used the specification of the R7 photoreceptor cells controlled by the Sevenless receptor tyrosine kinase (Sev) pathway to screen for modulators of pathway activity and to uncover the mechanisms underlying the robustness of cell fate decisions. This study provides genetic evidence that the Drosophila SOCS36E adaptor protein containing an SH2 domain and a SOCS box acts as an attenuator of Sev signaling. Overexpression of Socs36E strongly suppresses the specification of extra R7 photoreceptor cells in response to constitutive activation of Sev, and loss of Socs36E function suppresses the loss of R7 cells when Sev activity is impaired. In a wild-type background, however, loss and gain of Socs36E function exhibits little effect on R7 specification. The SH2 domain of SOCS36E is essential for this function in inhibiting Sev action and Socs36E expression is suppressed by high Sev pathway activity. In this model, only the cell able to activate high levels of receptor tyrosine kinase signaling will repress SOCS36E expression, reduce the negative effect on Sev signaling and allow this cell to differentiate into R7. In contrast, the remaining cells fail to receive high signaling, and thus maintain high levels of SOCS36E. This represses residual Sev activity and blocks R7 development. Therefore, Socs36E constitutes a novel partially redundant feedback mechanism that contributes to the robustness of R7 specification. The SOCS family of adaptor proteins may have evolved as modulators of specific signaling pathways that contribute to the robustness and precision of cell fate specification (Almudi, 2009).

GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo

JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, three constructs were generated that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. These reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand Unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, this study has generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila (Bach, 2007).

Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities

The Janus kinase (JAK) cascade is an essential and well-conserved pathway required to transduce signals for a variety of ligands in both vertebrates and invertebrates. While activation of the pathway is essential to many processes, mutations from mammals and Drosophila demonstrate that regulation is also critical. The SOCS (Suppressor Of Cytokine Signaling) proteins in mammals are regulators of the JAK pathway that participate in a negative feedback loop, as they are transcriptionally activated by JAK signaling. Examination of one Drosophila SOCS homologue, Socs36E, demonstrated that its expression is responsive to JAK pathway activity and it is capable of downregulating JAK signaling, similar to the well characterized mammalian SOCS. Based on sequence analysis of the Drosophila genome, there are three identifiable SOCS homologues in flies. All three are most similar to mammalian SOCS that have not been extensively characterized: Socs36E is most similar to mammalian SOCS5, while Socs44A and Socs16D are most similar to mammalian SOCS6 and 7. Although Socs44A is capable of repressing JAK activity in some tissues, its expression is not regulated by the pathway. Furthermore, Socs44A can enhance the activity of the EGFR/MAPK signaling cascade, in contrast to Socs36E. It is concluded that two Drosophila SOCS proteins have some overlapping and some distinct capabilities. While Socs36E behaves similarly to the canonical vertebrate SOCS, Socs44A is not part of a JAK pathway negative feedback loop. Nonetheless, both SOCS regulate JAK and EGFR signaling pathways, albeit differently. The non-canonical properties of Socs44A may be representative of the class of less characterized vertebrate SOCS with which it shares greatest similarity (Rawlings, 2004).

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1; see Drosophila Cas). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. This study shows that suppressor of cytokine signaling 6 (SOCS6; see Drosophila Socs36E) and Cullin 5 (see Drosophila Cullin 5), two components of the CRL5SOCS6 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5SOCS6 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration (Teckchandani, 2016).

Search PubMed for articles about Drosophila Socs36E

Alexander, W. S. (2002). Suppressors of cytokine signalling (SOCS) in the immune system. Nat Rev Immunol 2: 410-416. PubMed ID: 12093007

Almudi, I., Stocker, H., Hafen, E., Corominas, M. and Serras, F. (2009). SOCS36E specifically interferes with Sevenless signaling during Drosophila eye development. Dev Biol 326: 212-223. PubMed ID: 19083999

Almudi, I., Corominas, M. and Serras, F. (2010). Competition between SOCS36E and Drk modulates Sevenless receptor tyrosine kinase activity. J Cell Sci 123: 3857-3862. PubMed ID: 20980384

Amoyel, M., Simons, B. D. and Bach, E. A. (2014). Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo. EMBO J 33: 2295-2313. PubMed ID: 25092766

Amoyel, M., Anderson, J., Suisse, A., Glasner, J. and Bach, E.A. (2016). Socs36E controls niche competition by repressing MAPK signaling in the Drosophila testis. PLoS Genet 12: e1005815. PubMed ID: 26807580

Bach, E. A., Ekas, L. A., Ayala-Camargo, A., Flaherty, M. S., Lee, H., Perrimon, N. and Baeg, G. H. (2007). GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo. Gene Expr Patterns 7: 323-331. PubMed ID: 17008134

Boggio, R., Passafaro, A. and Chiocca, S. (2007). Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation. J Biol Chem 282: 15376-15382. PubMed ID: 17392274

Callus, B. A. and Mathey-Prevot, B. (2002). SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signalling in the imaginal wing disc. Oncogene 21: 4812-4821. PubMed ID: 12101419

Croker, B. A., Kiu, H. and Nicholson, S. E. (2008). SOCS regulation of the JAK/STAT signalling pathway. Semin Cell Dev Biol 19: 414-422. PubMed ID: 18708154

Devergne, O., Ghiglione, C. and Noselli, S. (2007). The endocytic control of JAK/STAT signalling in Drosophila. J Cell Sci 120: 3457-3464. PubMed ID: 17855388

Flaherty, M. S., Zavadil, J., Ekas, L. A. and Bach, E. A. (2009). Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of notch signaling by the JAK/STAT pathway. Dev Dyn 238: 2235-2253. PubMed ID: 19504457

Gunawan, F., Arandjelovic, M. and Godt, D. (2013). The Maf factor Traffic jam both enables and inhibits collective cell migration in Drosophila oogenesis. Development 140: 2808-2817. PubMed ID: 23720044

Herranz, H., Hong, X., Hung, N. T., Voorhoeve, P. M. and Cohen, S. M. (2012). Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila epithelial transformation model. Genes Dev 26: 1602-1611. PubMed ID: 22802531

Issigonis, M., Tulina, N., de Cuevas, M., Brawley, C., Sandler, L. and Matunis, E. (2009). JAK-STAT signal inhibition regulates competition in the Drosophila testis stem cell niche. Science 326: 153-156. PubMed ID: 19797664

Kario, E., Marmor, M. D., Adamsky, K., Citri, A., Amit, I., Amariglio, N., Rechavi, G. and Yarden, Y. (2005). Suppressors of cytokine signaling 4 and 5 regulate epidermal growth factor receptor signaling. J Biol Chem 280: 7038-7048. PubMed ID: 15590694

Karsten, P., Hader, S. and Zeidler, M. P. (2002). Cloning and expression of Drosophila SOCS36E and its potential regulation by the JAK/STAT pathway. Mech Dev 117: 343-346. PubMed ID: 12204282

Kazi, J. U., Kabir, N. N., Flores-Morales, A. and Ronnstrand, L. (2014). SOCS proteins in regulation of receptor tyrosine kinase signaling. Cell Mol Life Sci 71: 3297-3310. PubMed ID: 24705897

Kamizono, S., Hanada, T., Yasukawa, H., Minoguchi, S., Kato, R., Minoguchi, M., Hattori, K., Hatakeyama, S., Yada, M., Morita, S., Kitamura, T., Kato, H., Nakayama, K. and Yoshimura, A. (2001). The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J Biol Chem 276: 12530-12538. PubMed ID: 11278610

Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W. and Nakayama, K. I. (2004). VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18: 3055-3065. PubMed ID: 15601820

Linossi, E. M. and Nicholson, S. E. (2015). Kinase inhibition, competitive binding and proteasomal degradation: resolving the molecular function of the suppressor of cytokine signaling (SOCS) proteins. Immunol Rev 266: 123-133. PubMed ID: 26085211

Monahan, A. J., M. (2013). Socs36E attenuates STAT signaling to optimize motile cell specification in the Drosophila ovary. Dev Biol 379: 152-166. PubMed ID: 23583584

Monahan, A. J., M. (2015). Socs36E limits STAT signaling via Cullin2 and a SOCS-box independent mechanism in the Drosophila egg chamber. Mech Dev 138 Pt 3:313-27. PubMed ID: 26277564

Montell, D. J., Yoon, W. H. and Starz-Gaiano, M. (2012). Group choreography: mechanisms orchestrating the collective movement of border cells. Nat Rev Mol Cell Biol 13: 631-645. PubMed ID: 23000794

Pozzebon, M. E., Varadaraj, A., Mattoscio, D., Jaffray, E. G., Miccolo, C., Galimberti, V., Tommasino, M., Hay, R. T. and Chiocca, S. (2013). BC-box protein domain-related mechanism for VHL protein degradation. Proc Natl Acad Sci U S A 110: 18168-18173. PubMed ID: 24145437

Rawlings, J. S., Rennebeck, G., Harrison, S. M., Xi, R. and Harrison, D. A. (2004). Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities. BMC Cell Biol 5: 38. PubMed ID: 15488148

Singh, S. R., Zheng, Z., Wang, H., Oh, S. W., Chen, X. and Hou, S. X. (2010). Competitiveness for the niche and mutual dependence of the germline and somatic stem cells in the Drosophila testis are regulated by the JAK/STAT signaling. J Cell Physiol 223: 500-510. PubMed ID: 20143337

Silver, D. L., Geisbrecht, E. R. and Montell, D. J. (2005). Requirement for JAK/STAT signaling throughout border cell migration in Drosophila. Development 132(15): 3483-92. PubMed ID: 16000386

Starz-Gaiano, M., Melani, M., Wang, X., Meinhardt, H. and Montell, D. J. (2008). Feedback inhibition of Jak/STAT signaling by apontic is required to limit an invasive cell population. Dev Cell 14: 726-738. PubMed ID: 18477455

Stec, W., Vidal, O. and Zeidler, M. P. (2013). Drosophila SOCS36E negatively regulates JAK/STAT pathway signaling via two separable mechanisms. Mol Biol Cell 24: 3000-3009. PubMed ID: 23885117

Tarayrah, L., Herz, H. M., Shilatifard, A. and Chen, X. (2013). Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche. Development 140: 1014-1023. PubMed ID: 23364332

Teckchandani, A. and Cooper, J. A. (2016). The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells. Elife 5 [Epub ahead of print]. PubMed ID: 27656905

Terry, N. A., Tulina, N., Matunis, E. and DiNardo, S. (2006). Novel regulators revealed by profiling Drosophila testis stem cells within their niche. Dev Biol 294: 246-257. PubMed ID: 16616121

Vidal, O. M., Stec, W., Bausek, N., Smythe, E. and Zeidler, M. P. (2010). Negative regulation of Drosophila JAK-STAT signalling by endocytic trafficking. J Cell Sci 123: 3457-3466. PubMed ID: 20841381

Yoon, W. H., Meinhardt, H. and Montell, D. J. (2011). miRNA-mediated feedback inhibition of JAK/STAT morphogen signalling establishes a cell fate threshold. Nat Cell Biol 13: 1062-1069. PubMed ID: 21857668

date revised: 5 December 2023

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