Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S phase and its relief at G2 phase.

The cell cycle depends on a sequence of steps that are triggered and terminated via the synthesis and degradation of phase-specific transcripts and proteins. Although much is known about how stage-specific transcription is activated, less is understood about how inappropriate gene expression is suppressed. Here, we demonstrate that Groucho, the Drosophila orthologue of TLE1 and other related human transcriptional corepressors, regulates normal cell cycle progression in vivo. We show that, although Groucho is expressed throughout the cell cycle, its activity is selectively inactivated by phosphorylation, except in S phase when it negatively regulates E2F1. Constitutive Groucho activity, as well as its depletion and the consequent derepression of e2f1, cause cell cycle phenotypes. Our results suggest that Cdk1 contributes to phase-specific phosphorylation of Groucho in vivo. We propose that Groucho and its orthologues play a role in the metazoan cell cycle that may explain the links between TLE corepressors and several types of human cancer.

An Activating Mutation in ERK Causes Hyperplastic Tumors in a scribble Mutant Tissue in Drosophila.

Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf, or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo To determine if a point mutation could render ERK intrinsically active and oncogenic, we have assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog and has also been identified in a few human tumors. Our analyses indicate that a fly ERK ortholog harboring this mutation alone (RolledR80S), and more so in conjunction with the known sevenmaker mutation (RolledR80S+D334N), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of RolledR80S and RolledR80S+D334N induces tissue overgrowth in an established Drosophila cancer model. Our findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.

Capicua controls Toll/IL-1 signaling targets independently of RTK regulation.

The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here, we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-κB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.

Novel interplay between JNK and Egfr signaling in Drosophila dorsal closure.

Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the Drosophila embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of decapentaplegic (dpp). Second, Dpp, a secreted TGF-ß homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. Here we uncover a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained dpp expression in the LE. Specifically, we demonstrate that Egfr pathway activity in the lateral epidermis prevents expression of the gene scarface (scaf), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. Our results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway.

High-Throughput In Vitro Identification of Direct MAPK/Erk Substrates.

Phosphorylation mediated by cellular protein kinases is an effective mechanism employed by an organism to regulate central processes such as cell-cycle progression, metabolic pathways, cytoskeletal function, cell migration and differentiation. Thus, for example, various signaling pathways utilize sequential phosphorylation events to relay external cues from the cell surface to the nucleus, where eventually gene expression profiles are altered and, consequently, changes in cell fates and function are induced. Accordingly, recognizing the direct targets of key effector kinases is of utmost importance for understanding the cellular responses to pathway activity. Here we describe a high-throughput genome-wide proteomics approach aimed at uncovering novel nuclear targets for the single Drosophila MAPK/Erk. Briefly, pools of cDNA are transcribed and translated in vitro in the presence of [35S]Methionine, generating a library of radiolabeled protein pools which are subsequently subjected to biochemical kinase assays using recombinant, active Erk2. Phosphorylated proteins representing potential MAPK/Erk substrates are then detected due to their shifted mobility on SDS-PAGE gels. This protocol can be easily adjusted and applied toward identifying targets of other kinases for which in vitro phosphorylation assays are available.

Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche.

In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. We have found that the phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period.

Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes.

Origins of context-dependent gene repression by capicua.

Receptor Tyrosine Kinase (RTK) signaling pathways induce multiple biological responses, often by regulating the expression of downstream genes. The HMG-box protein Capicua (Cic) is a transcriptional repressor that is downregulated in response to RTK signaling, thereby enabling RTK-dependent induction of Cic targets. In both Drosophila and mammals, Cic is expressed as two isoforms, long (Cic-L) and short (Cic-S), whose functional significance and mechanism of action are not well understood. Here we show that Drosophila Cic relies on the Groucho (Gro) corepressor during its function in the early embryo, but not during other stages of development. This Gro-dependent mechanism requires a short peptide motif, unique to Cic-S and designated N2, which is distinct from other previously defined Gro-interacting motifs and functions as an autonomous, transferable repressor element. Unexpectedly, our data indicate that the N2 motif is an evolutionary innovation that originated within dipteran insects, as the Cic-S isoform evolved from an ancestral Cic-L-type form. Accordingly, the Cic-L isoform lacking the N2 motif is completely inactive in early Drosophila embryos, indicating that the N2 motif endowed Cic-S with a novel Gro-dependent activity that is obligatory at this stage. We suggest that Cic-S and Gro coregulatory functions have facilitated the evolution of the complex transcriptional network regulated by Torso RTK signaling in modern fly embryos. Notably, our results also imply that mammalian Cic proteins are unlikely to act via Gro and that their Cic-S isoform must have evolved independently of fly Cic-S. Thus, Cic proteins employ distinct repressor mechanisms that are associated with discrete structural changes in the evolutionary history of this protein family.

RTK signaling modulates the Dorsal gradient.

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. Here, we demonstrate that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, we show that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, our results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo.

Phosphorylation of the Drosophila melanogaster RNA-binding protein HOW by MAPK/ERK enhances its dimerization and activity.

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW-mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles.

The Capicua repressor--a general sensor of RTK signaling in development and disease.

Receptor tyrosine kinase (RTK) signaling pathways control multiple cellular decisions in metazoans, often by regulating the expression of downstream genes. In Drosophila melanogaster and other systems, E-twenty-six (ETS) transcription factors are considered to be the predominant nuclear effectors of RTK pathways. Here, we highlight recent progress in identifying the HMG-box protein Capicua (CIC) as a key sensor of RTK signaling in both Drosophila and mammals. Several studies have shown that CIC functions as a repressor of RTK-responsive genes, keeping them silent in the absence of signaling. Following the activation of RTK signaling, CIC repression is relieved, and this allows the expression of the targeted gene in response to local or ubiquitous activators. This regulatory switch is essential for several RTK responses in Drosophila, from the determination of cell fate to cell proliferation. Furthermore, increasing evidence supports the notion that this mechanism is conserved in mammals, where CIC has been implicated in cancer and neurodegeneration. In addition to summarizing our current knowledge on CIC, we also discuss the implications of these findings for our understanding of RTK signaling specificity in different biological processes.

Phosphorylation of Ind by MAP kinase enhances Ind-dependent transcriptional repression.

The Drosophila neuroectoderm is initially subdivided into three longitudinal domains that give rise to columns of neuroblasts. This subdivision is coordinately accomplished by the action of the signaling pathways, Dorsal and Epidermal Growth Factor Receptor (EGFR), in conjunction with the homeodomain proteins, Ventral nervous system defective, Intermediate neuroblasts defective (Ind) and Muscle Segment Homeobox. We previously demonstrated that Ind expression is activated in response to the EGFR pathway. Here we show that EGF signaling subsequently mediates the direct phosphorylation of Ind by MAP kinase, which enhances the capacity of Ind to repress target genes, such as achaete. Specifically, we show that reduced EGF signaling results in diminished repression of achaete in the intermediate column, despite the presence of high levels of Ind protein. We also demonstrate that ectopic activation of MAP kinase results in the lateral expansion of the Ind expression domain with a corresponding reduction in achaete expression. This regulation is also dependent on the co-repressor, Dichaete. Our data indicate that EGF signaling, acting through MAP kinase, impinges on multiple aspects of Ind regulatory activity. While it has been often demonstrated that MAP kinase phosphorylation of transcriptional repressors attenuates their repressor activity, here we provide an example of phosphorylation enhancing repressor activity.

Phosphorylation of Groucho mediates RTK feedback inhibition and prolonged pathway target gene expression.

BACKGROUND: Signaling by receptor tyrosine kinase (RTK) pathways plays fundamental roles in processes of cell-fate determination, often through the induction of specific transcriptional responses. Yet it is not fully understood how continuous target gene expression, required for irreversible cell-fate specification, is preserved after RTK signaling has ended. Here we address this question using the Drosophila embryo, a model system that has been instrumental in elucidating the developmental functions of RTK signal transduction. RESULTS: The Groucho corepressor is phosphorylated and downregulated in response to RTK signaling. Here we show that RTK pathways use Groucho phosphorylation as a general mechanism for inducing expression of pathway target genes encoding cell-fate determinants as well as feedback antagonists, indicating that relief of Groucho-dependent repression is an integral element of RTK signaling networks. We further demonstrate that after mitogen-activated protein kinase (MAPK) has been deactivated, sustained phosphorylation of Groucho is essential for persistent RTK-induced target gene expression and cell-fate determination in several developmental contexts. CONCLUSIONS: Phosphorylation of Groucho by MAPK plays a dual role in the regulation of RTK responses: (1) it mediates rapid feedback inhibition, and (2) it provides a stable memory mechanism of past MAPK activity. We propose that, in this manner, phosphorylation of Groucho enables transiently active RTK pathways to fix the spatiotemporal expression profiles of downstream targets over time.

Substrate-dependent control of MAPK phosphorylation in vivo.

Phosphorylation of the mitogen-activated protein kinase (MAPK) is essential for its enzymatic activity and ability to control multiple substrates inside a cell. According to the current models, control of MAPK phosphorylation is independent of its substrates, which are viewed as mere sensors of MAPK activity. Contrary to this modular view of MAPK signaling, our studies in the Drosophila embryo demonstrate that substrates can regulate the level of MAPK phosphorylation in vivo. We demonstrate that a twofold change in the gene dosage of a single substrate can induce a significant change in the phosphorylation level of MAPK and in the conversion of other substrates. Our results support a model where substrates of MAPK counteract its dephosphorylation by phosphatases. Substrate-dependent control of MAPK phosphorylation is a manifestation of a more general retroactive effect that should be intrinsic to all networks with covalent modification cycles.

Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila.

RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

Detection of RTK pathway activation in Drosophila using anti-dpERK immunofluorescence staining.

In Drosophila, like in other metazoans, receptor tyrosine kinase (RTK) signaling pathways control diverse cellular processes such as migration, growth, fate determination, and differentiation (Shilo, Development 132:4017-4027, 2005). Activation of RTKs by their extracellular ligands triggers a signal transduction cascade, mediated by the Ras/Raf/MEK cassette, which ultimately leads to dual phosphorylation and activation of the mitogen-activated protein kinase/extracellularly regulated kinase (MAPK/Erk). Once active, MAPK/Erk phosphorylates its cytoplasmic and nuclear substrates, consequently modulating (i.e., stimulating or inhibiting) their biological function (Murphy and Blenis, Trends in Biochemical Sciences 31:268-275, 2006). The currently available antibody specific for the doubly phosphorylated form of MAPK/Erk (dpERK) (Yung et al., FEBS Letters 408:292-296, 1997) provides a valuable readout for RTK signaling: it enables the spatiotemporal detection of RTK pathway activity in the developing organism, in situ (Gabay et al., Development 124:3535-3541, 1997; Gabay et al., Science 277:1103-1106, 1997). Here, we present a detailed protocol for anti-dpERK immunofluorescent staining that can be applied to the analysis of MAPK/Erk signaling in Drosophila embryogenesis.

MAPK substrate competition integrates patterning signals in the Drosophila embryo.

Terminal regions of the Drosophila embryo are patterned by the localized activation of the mitogen-activated protein kinase (MAPK) pathway. This depends on the MAPK-mediated downregulation of Capicua (Cic), a repressor of the terminal gap genes. We establish that downregulation of Cic is antagonized by the anterior patterning morphogen Bicoid (Bcd). We demonstrate that this effect does not depend on transcriptional activity of Bcd and provide evidence suggesting that Bcd, a direct substrate of MAPK, decreases the availability of MAPK for its other substrates, such as Cic. Based on the quantitative analysis of MAPK signaling in multiple mutants, we propose that MAPK substrate competition coordinates the actions of the anterior and terminal patterning systems. In addition, we identify Hunchback as a novel target of MAPK phosphorylation that can account for the previously described genetic interaction between the posterior and terminal systems. Thus, a common enzyme-substrate competition mechanism can integrate the actions of the anterior, posterior, and terminal patterning signals. Substrate competition can be a general signal integration strategy in networks where enzymes, such as MAPK, interact with their multiple regulators and targets.

Context-dependent regulation of Groucho/TLE-mediated repression.

Groucho/TLE proteins are global corepressors that are recruited to target promoters by different families of DNA-binding repressors. As these corepressors are widely expressed, the long-standing view had been that Groucho/TLE-mediated repression is regulated solely by the spatial and temporal distribution of partner repressors. It has recently emerged, however, that Groucho/TLE repressor activity is itself regulated, in a signal induced, context-dependent manner. Here we review the essential roles played by Groucho/TLE factors in different cell-signalling processes that illustrate different modes for regulating Groucho/TLE-mediated repression: (i) via the expression of partner repressors; (ii) by competition with coactivators and (iii) through post-translational modifications of Groucho/TLE. We also discuss how the intrinsic properties of repressors can result in differential responses to Groucho/TLE regulation.

Drosophila CK2 phosphorylates Hairy and regulates its activity in vivo.

Hairy is a repressor that regulates bristle patterning, and its loss elicits ectopic bristles (neural hyperplasia). However, it has remained unknown whether Hairy is regulated by phosphorylation. We describe here the interaction of protein kinase CK2 and Hairy. Hairy is robustly phosphorylated by the CK2-holoenzyme (CK2-HoloE) purified from Drosophila embryos, but weakly by the catalytic CK2alpha-subunit alone, suggesting that this interaction requires the regulatory CK2beta-subunit. Consistent with this, Hairy preferentially forms a direct complex with CK2-HoloE. Importantly, we demonstrate genetic interactions between CK2 and hairy (h). Thus, flies trans-heterozygous for alleles of CK2alpha and h display neural hyperplasia akin to homozygous hypomorphic h alleles. In addition, we show that similar phenotypes are elicited in wild-type flies upon expression of RNAi constructs against CK2alpha/beta, and that these defects are sensitive to h gene dosage. Together, these studies suggest that CK2 contributes to repression by Hairy.

Multiple RTK pathways downregulate Groucho-mediated repression in Drosophila embryogenesis.

RTK pathways establish cell fates in a wide range of developmental processes. However, how the pathway effector MAPK coordinately regulates the expression of multiple target genes is not fully understood. We have previously shown that the EGFR RTK pathway causes phosphorylation and downregulation of Groucho, a global co-repressor that is widely used by many developmentally important repressors for silencing their various targets. Here, we use specific antibodies that reveal the dynamics of Groucho phosphorylation by MAPK, and show that Groucho is phosphorylated in response to several RTK pathways during Drosophila embryogenesis. Focusing on the regulation of terminal patterning by the Torso RTK pathway, we demonstrate that attenuation of Groucho's repressor function via phosphorylation is essential for the transcriptional output of the pathway and for terminal cell specification. Importantly, Groucho is phosphorylated by an efficient mechanism that does not alter its subcellular localisation or decrease its stability; rather, modified Groucho endures long after MAPK activation has terminated. We propose that phosphorylation of Groucho provides a widespread, long-term mechanism by which RTK signals control target gene expression.