Hpo Pathway Research Articles

Protein Phosphatase 2A with B' specificity subunits regulates the Hippo-Yorkie signaling axis in the Drosophila eye disc.

Hippo-Yorkie (Hpo-Yki) signaling is central to diverse developmental processes. Although its redeployment has been amply demonstrated, its context-specific regulation remains poorly understood. The Drosophila eye disc is a continuous epithelium folded into two layers, the peripodial epithelium (PE) and the retinal progenitor epithelium. Here, Yki acts in the PE, first to promote PE identity by suppressing retina fate, and subsequently to maintain proper disc morphology. In the latter process, loss of Yki results in the displacement of a portion of the differentiating retinal epithelium onto the PE side. We show that Protein Phosphatase 2A (PP2A) complexes comprising different substrate-specificity B-type subunits govern the Hpo-Yki axis in this context. These include holoenzymes containing the B‴ subunit Cka and those containing the B' subunits Wdb or Wrd. Whereas PP2A(Cka), as part of the STRIPAK complex, is known to regulate Hpo directly, PP2A(Wdb) acts genetically upstream of the antagonistic activities of the Hpo regulators Sav and Rassf. These in vivo data provide the first evidence of PP2A(B') heterotrimer function in Hpo pathway regulation and reveal pathway diversification at distinct developmental times in the same tissue.

Negative feedback couples Hippo pathway activation with Kibra degradation independent of Yorkie-mediated transcription.

The Hippo (Hpo) pathway regulates tissue growth in many animals. Multiple upstream components promote Hpo pathway activity, but the organization of these different inputs, the degree of crosstalk between them, and whether they are regulated in a distinct manner is not well understood. Kibra (Kib) activates the Hpo pathway by recruiting the core Hpo kinase cassette to the apical cortex. Here, we show that the Hpo pathway downregulates Drosophila Kib levels independently of Yorkie-mediated transcription. We find that Hpo signaling complex formation promotes Kib degradation via SCFSlimb-mediated ubiquitination, that this effect requires Merlin, Salvador, Hpo, and Warts, and that this mechanism functions independently of other upstream Hpo pathway activators. Moreover, Kib degradation appears patterned by differences in mechanical tension across the wing. We propose that Kib degradation mediated by Hpo pathway components and regulated by cytoskeletal tension serves to control Kib-driven Hpo pathway activation and ensure optimally scaled and patterned tissue growth.

Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K

Hippo (Hpo) signaling pathway controls tissue growth by regulating the subcellular localization of Yorkie (Yki)/Yap via a cytoplasmic kinase cassette containing an upstream kinase Hpo/MST1/2 and a downstream kinase Warts (Wts)/Lats1/2. Here we show that PRP4K, a kinase involved in mRNA splicing, phosphorylates Yki/Yap in the nucleus to prevent its nuclear accumulation and restrict Hpo pathway target gene expression. PRP4K inactivation accelerates whereas excessive PRP4K inhibits Yki-driven tissue overgrowth. PRP4K phosphorylates a subset of Wts/Lats1/2 sites on Yki/Yap to inhibit the binding of Yki/Yap to the Scalloped (Sd)/TEAD transcription factor and exclude Yki/Yap nuclear localization depending on nuclear export. Furthermore, PRP4K inhibits proliferation and invasiveness of cultured breast cancer cells and its high expression correlates with good prognosis in breast cancer patients. Our study unravels an unanticipated layer of Hpo pathway regulation and suggests that PRP4K-mediated Yki/Yap phosphorylation in the nucleus provides a fail-safe mechanism to restrict aberrant pathway activation.

Rap1 Negatively Regulates the Hippo Pathway to Polarize Directional Protrusions in Collective Cell Migration

In collective cell migration, directional protrusions orient cells in response to external cues, which requires coordinated polarity among the migrating cohort. However, the molecular mechanism has not been well defined. Drosophila border cells (BCs) migrate collectively and invade via the confined space between nurse cells, offering an invivo model to examine how group polarity is organized. Here, we show that the front/back polarity of BCs requires Rap1, hyperactivation of which disrupts cluster polarity and induces misoriented protrusions and loss of asymmetry in the actin network. Conversely, hypoactive Rap1 causes fewer protrusions and cluster spinning during migration. A forward genetic screen revealed that downregulation of the Hippo (Hpo) pathway core components hpo or mats enhances the Rap1V12-induced migration defect and misdirected protrusions. Mechanistically, association of Rap1V12 with the kinase domain of Hpo suppresses its activity, which releases Hpo signaling-mediated suppression of F-actin elongation, promoting cellular protrusions in collective cell migration.

Cloning and expression analysis of BmYki gene in silkworm, Bombyx mori.

The transcriptional coactivator Yorkie(Yki), is a critical downstream effector of the Hippo(Hpo) signaling pathway that controls organ size through the regulation of cell proliferation and apoptosis. During the past ten years the biological function of Yki has been studied extensively in Drosophila and a few other insects, however, little is known about it in the silkworm, Bombyx mori, a major research model of lepidopteran insect. Here, we describe the isolation, characterization and expression of the B. mori Yki ortholog, BmYki. The coding sequence of the BmYki was 1314 bp in length, encoding a protein of 437 amino acids containing two conserved WW domains. BmYki transcripts were ubiquitous but not abundant in all detected tissues and developmental stages. Comparatively, it was expressed at pretty high level in silk glands and at the stage of fifth-instar day-3 larvae. Overexpression of BmYki in cultured B. mori embryonic cells significantly promoted transcription of genes associated with cell proliferation and apoptosis, indicating that BmYki functions in the regulation of organ growth-related biological processes. Interestingly, transcription of silk protein-coding genes and transcription factors regulating the synthesis of silk proteins was downregulated remarkably, suggesting that BmYki was involved in the regulation of silk protein synthesis. This study provides new insights into the role of BmYki in Hpo pathway regulation in silkworm.

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster.

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

The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression.

Coordination of organ growth during development is required to generate fit individuals with fixed proportions. We recently identified Drosophila Dilp8 as a key hormone in coupling organ growth with animal maturation. In addition, dilp8 mutant flies exhibit elevated fluctuating asymmetry (FA) demonstrating a function for Dilp8 in ensuring developmental stability. The signals regulating Dilp8 activity during normal development are not yet known. Here, we show that the transcriptional co-activators of the Hippo (Hpo) pathway, Yorkie (Yki, YAP/TAZ) and its DNA-binding partner Scalloped (Sd), directly regulate dilp8 expression through a Hpo-responsive element (HRE) in the dilp8 promoter. We further demonstrate that mutation of the HRE by genome-editing results in animals with increased FA, thereby mimicking full dilp8 loss of function. Therefore, our results indicate that growth coordination of organs is connected to their growth status through a feedback loop involving Hpo and Dilp8 signalling pathways.

The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization.

Overlapping functions of the MAP4K family kinases Hppy and Msn in Hippo signaling.

The Hippo (Hpo) tumor suppressor pathway is an evolutionarily conserved signaling pathway that controls tissue growth and organ size in species ranging from Drosophila to human, and its malfunction has been implicated in many types of human cancer. In this study, we conducted a kinome screen and identified Happyhour (Hppy)/MAP4K3 as a novel player in the Hpo pathway. Our biochemical study showed that Hppy binds and phosphorylates Wts. Our genetic experiments suggest that Hppy acts in parallel and partial redundantly with Misshapen (Msn)/MAP4K4 to regulate Yki nuclear localization and Hpo target gene expression in Drosophila wing imaginal discs. Furthermore, we showed that cytoskeleton stress restricts Yki nuclear localization through Hppy and Msn when Hpo activity is compromised, thus providing an explanation for the Wts-dependent but Hpo-independent regulation of Yki in certain contexts. Our study has unraveled an additional layer of complexity in the Hpo signaling pathway and laid down a foundation for exploring how different upstream regulators feed into the core Hpo pathway.

Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system

The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie [Yki-Yes-activated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) in mammals]. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. Here, we examine Yki regulation in the Drosophila larval central nervous system and uncover a Hpo/Warts-independent function for the tumor suppressor kinase liver kinase B1 (LKB1) and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain/ventral nerve cord. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain/ventral nerve cord. Thus, we demonstrate a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system.

Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4.

Signalling through the Hippo (Hpo) pathway involves a kinase cascade, which leads to the phosphorylation and inactivation of the pro-growth transcriptional co-activator Yorkie (Yki). Despite the identification of a large number of pathway members and modulators, our understanding of the molecular events that lead to activation of Hpo and the downstream kinase Warts (Wts) remain incomplete. Recently, targeted degradation of several Hpo pathway components has been demonstrated as a means of regulating pathway activity. In particular, the stability of scaffold protein Salvador (Sav), which is believed to promote Hpo/Wts association, is crucially dependent on its binding partner Hpo. In a cell-based RNAi screen for ubiquitin regulators involved in Sav stability, we identify the HECT domain protein Herc4 (HECT and RLD domain containing E3 ligase) as a Sav E3 ligase. Herc4 expression promotes Sav ubiquitylation and degradation, while Herc4 depletion stabilises Sav. Interestingly, Hpo reduces Sav/Herc4 interaction in a kinase-dependent manner. This suggests the existence of a positive feedback loop, where Hpo stabilises its own positive regulator by antagonising Herc4-mediated degradation of Sav.

Emerging role of Hpo signaling and YAP in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related mortality worldwide. Due to the poor prognosis and limited therapeutic options, there is great interest in further understanding better the molecular underpinnings and potential molecular targets associated with HCC. The Hippo (Hpo) signaling pathway and YAP, its principal downstream effector, represent an innovative area of research in HCC. Pioneered in Drosophila melanogaster, the Hpo cascade controls tissue homeostasis including organ size, cell proliferation, apoptosis, as well as cell-cycle regulation and differentiation. This conserved kinase cascade in mammals depends on central control by the tumor suppressor mammalian sterile 20-like kinase 1/2 (Mst1/2). The Mst1/2 commences the downstream kinase cascade, ultimately activating the oncoprotein YAP and allowing its physical association with downstream targets to enhance the gene expression signatures that are involved in proliferation and survival. Alterations in YAP expression and defective regulation of other key Hpo pathway members, such as Mst1/2, Salvador, neurofibromatosis and Mer (Nf2/mer), large tumor suppressor homolog 1/2 (Lats1/2), and Mps one binder kinase activator-like 1A and 1B (Mob1) drive carcinogenesis in animal models. The dysregulation of the Hpo pathway – resulting in an unchecked activation of YAP – culminates in the development of a broad range of human tumor types, including HCC. The abrogation of Mst1/2-mediated YAP phosphorylation permits YAP entry into the nucleus in murine models and functions similarly in human HCCs. Chemoresistance mechanisms displayed by HCC tumors occur in a YAP-dependent manner. The HCC specimens exhibit YAP overexpression, and YAP serves as an independent prognostic marker for disease-free survival and overall survival in patients with HCC. Recently, the small molecule inhibitor, verteporfin has been shown to attenuate YAP activity in murine models, perhaps offering a novel therapeutic approach for patients with advanced HCC.

Cabut/dTIEG associates with the transcription factor Yorkie for growth control.

The Drosophila transcription factor Cabut/dTIEG (Cbt) is a growth regulator, whose expression is modulated by different stimuli. Here, we determine Cbt association with chromatin and identify Yorkie (Yki), the transcriptional co-activator of the Hippo (Hpo) pathway as its partner. Cbt and Yki co-localize on common gene promoters, and the expression of target genes varies according to changes in Cbt levels. Down-regulation of Cbt suppresses the overgrowth phenotypes caused by mutations in expanded (ex) and yki overexpression, whereas its up-regulation promotes cell proliferation. Our results imply that Cbt is a novel partner of Yki that is required as a transcriptional co-activator in growth control.

Drosophila Casein Kinase 2 (CK2) Promotes Warts Protein to Suppress Yorkie Protein Activity for Growth Control

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. In this study we report that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo-induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant-induced expanded (Ex) up-regulation and overgrowth phenotype, whereas it cannot affect wts mutant. Consistent with this, knockdown of CK2 up-regulates Hpo pathway target expression. We also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in the developing disc induces severe growth defects as well as caspase3 signals. Taken together, our results uncover a dual role of CK2; although its major role is promoting cell survive, it may potentially be a growth inhibitor as well.

Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo.

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. Here, we show that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. We find that the driving force behind such behavior is accelerated proliferation. Our results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process.

Crumbs promotes expanded recognition and degradation by the SCF(Slimb/β-TrCP) ubiquitin ligase.

In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. We show that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. We identify Skp/Cullin/F-box(Slimb/β-transducin repeats-containing protein) (SCF(Slimb/β-TrCP)) as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function.

Lgl Regulates the Hippo Pathway Independently of Fat/Dachs, Kibra/Expanded/Merlin and dRASSF/dSTRIPAK.

In both Drosophila and mammalian systems, the Hippo (Hpo) signalling pathway controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core pathway consists of a protein kinase Hpo (MST1/2 in mammals) that is regulated by a number of upstream inputs including Drosophila Ras Association Factor, dRASSF. We have previously shown in the developing Drosophila eye epithelium that loss of the apico-basal cell polarity regulator lethal-(2)-giant-larvae (lgl), and the concomitant increase in aPKC activity, results in ectopic proliferation and suppression of developmental cell death by blocking Hpo pathway signalling. Here, we further explore how Lgl/aPKC interacts with the Hpo pathway. Deregulation of the Hpo pathway by Lgl depletion is associated with the mislocalization of Hpo and dRASSF. We demonstrate that Lgl/aPKC regulate the Hpo pathway independently of upstream inputs from Fat/Dachs and the Kibra/Expanded/Merlin complex. We show depletion of Lgl also results in accumulation and mislocalization of components of the dSTRIPAK complex, a major phosphatase complex that directly binds to dRASSF and represses Hpo activity. However, depleting dSTRIPAK components, or removal of dRASSF did not rescue the lgl−/− or aPKC overexpression phenotypes. Thus, Lgl/aPKC regulate Hpo activity by a novel mechanism, independently of dRASSF and dSTRIPAK. Surprisingly, removal of dRASSF in tissue with increased aPKC activity results in mild tissue overgrowth, indicating that in this context dRASSF acts as a tumor suppressor. This effect was independent of the Hpo and Ras Mitogen Activated Protein Kinase (MAPK) pathways, suggesting that dRASSF regulates a novel pathway to control tissue growth.

VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex

Lung cancer is one of the most devastating diseases worldwide with high incidence and mortality. Hippo (Hpo) pathway is a conserved regulator of organ size in both Drosophila and mammals. Emerging evidence has suggested the significance of Hpo pathway in cancer development. In this study, we identify VGLL4 as a novel tumor suppressor in lung carcinogenesis through negatively regulating the formation of YAP-TEAD complex, the core component of Hpo pathway. Our data show that VGLL4 is frequently observed to be lowly expressed in both mouse and human lung cancer specimens. Ectopic expression of VGLL4 significantly suppresses the growth of lung cancer cells in vitro. More importantly, VGLL4 significantly inhibits lung cancer progression in de novo mouse model. We further find that VGLL4 inhibits the activity of the YAP-TEAD transcriptional complex. Our data show that VGLL4 directly competes with YAP in binding to TEADs and executes its growth-inhibitory function through two TDU domains. Collectively, our study demonstrates that VGLL4 is a novel tumor suppressor for lung cancer through negatively regulating the YAP-TEAD complex formation and thus the Hpo pathway.

Bantam is essential for Drosophila intestinal stem cell proliferation in response to Hippo signaling

The Drosophila midgut has emerged as an attractive model system to study stem cell biology. Extensive studies have been carried out to investigate the mechanisms of how the signaling pathways integrate to regulate intestinal stem cells (ISCs), yet, whether the microRNAs are involved in ISC self-renewal and maintenance is unknown. Here we demonstrate that the bantam microRNA is expressed specifically at high levels in Drosophila midgut precursor cells (including ISCs and enteroblasts) and secretory enteroendocrine cells while at extremely low levels in enterocytes. Furthermore, overexpression of bantam microRNA results in increase of the division of the midgut precursor cells, whereas loss of bantam microRNA decreases their proliferation. The mechanical studies show that bantam microRNA is essential for the Hpo pathway induced cell-autonomous ISC self-renewal, while it is disposable for EGFR and Notch pathways mediated ISC proliferation. More interestingly, we find that bantam microRNA is not required for the Hpo pathway mediated non-cell-autonomous ISC proliferation, revealing a novel mechanism by which the Hpo signaling pathway specifies its transcriptional targets in specific tissue to exhibit its biological functions.

A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity

The Hippo (Hpo) pathway controls tissue growth and organ size by regulating the activity of transcriptional co-activator Yorkie (Yki), which associates with transcription factor Scalloped (Sd) in the nucleus to promote downstream target gene expression. Here we identify a novel protein Sd-Binding-Protein (SdBP)/Tgi, which directly competes with Yki for binding to Sd through its TDU domains and inhibits the Sd-Yki transcriptional activity. We also find that SdBP retains Yki in the nucleus through the association with Yki WW domains via its PPXY motifs. Collectively, we identify SdBP as a novel component of the Hpo pathway, negatively regulating the transcriptional activity of Sd-Yki to restrict tissue growth.