Gene name - yorkie
Synonyms - CG4005
Cytological map position - 60B7--8
Function - transcriptional coactivator
Symbol - yki
FlyBase ID: FBgn0034970
Genetic map position - 2R
Classification - WW domain, PDZ binding motif
Cellular location - presumably nuclear and cytoplasmic
|Recent literature||Barron, D.A. and Moberg, K. (2016). Inverse
regulation of two classic Hippo pathway target genes in Drosophila
by the dimerization hub protein Ctp. Sci Rep 6: 22726. PubMed ID:
The LC8 family of small ~8 kD proteins are highly conserved and interact with multiple protein partners in eukaryotic cells. LC8-binding modulates target protein activity, often through induced dimerization via LC8:LC8 homodimers. Although many LC8-interactors have roles in signaling cascades, LC8's role in developing epithelia is poorly understood. Using the Drosophila wing as a developmental model, this study found that the LC8 family member Cut up (Ctp) is primarily required to promote epithelial growth, which correlates with effects on the pro-growth factor dMyc and two genes, diap1 and bantam, that are classic targets of the Hippo pathway coactivator Yorkie. Genetic tests confirm that Ctp supports Yorkie-driven tissue overgrowth and indicate that Ctp acts through Yorkie to control bantam (ban) and diap1 transcription. Quite unexpectedly however, Ctp loss has inverse effects on ban and diap1: it elevates ban expression but reduces diap1 expression. In both cases these transcriptional changes map to small segments of these promoters that recruit Yorkie. Although LC8 complexes with Yap1, a Yorkie homolog, in human cells, an orthologous interaction was not detected in Drosophila cells. Collectively these findings reveal that that Drosophila Ctp is a required regulator of Yorkie-target genes in vivo and suggest that Ctp may interact with a Hippo pathway protein(s) to exert inverse transcriptional effects on Yorkie-target genes.
|Chan, P., Han, X., Zheng, B., DeRan, M., Yu, J., Jarugumilli, G. K., Deng, H., Pan, D., Luo, X. and Wu, X. (2016). Autopalmitoylation of TEAD proteins regulates transcriptional output of the Hippo pathway. Nat Chem Biol [Epub ahead of print]. PubMed ID: 26900866
TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ (homologs of Drosophila Yorkie) and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, this study discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. The crystal structures of lipid-bound TEADs were determined, and the lipid chain of palmitate was found to insert into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. This study study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.
|Atkins, M., Potier, D., Romanelli, L., Jacobs, J., Mach, J., Hamaratoglu, F., Aerts, S. and Halder, G. (2016). An ectopic network of transcription factors regulated by Hippo signaling drives growth and invasion of a malignant tumor model. Curr Biol [Epub ahead of print]. PubMed ID: 27476594
Cancer cells have abnormal gene expression profiles; however, to what degree these are chaotic or driven by structured gene regulatory networks is often not known. This study focused on a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with next-generation sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, it was discovered that the bulk of the tumor-specific gene expression is controlled by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/SRC3, and Mef2. Notably, many of these transcription factors also are hyperactivated in human tumors. Bioinformatic analysis predicted that these factors directly regulate the majority of the tumor-specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness, and knockdown of several factors caused a reversion of the tumor-specific expression profile but had no observable effect on normal tissues. It was further found that the Hippo pathway effector Yorkie is strongly activated in tumor cells and initiates cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identifies an ectopic and ordered network of master regulators that control a large part of tumor cell-specific gene expression.
|Wang, S., Lu, Y., Yin, M. X., Wang, C., Wu, W., Li, J., Wu, W., Ge, L., Hu, L., Zhao, Y. and Zhang, L. (2016). Importin α1 mediates Yorkie nuclear import via an N-terminal non-canonical nuclear localization signal. J Biol Chem 291: 7926-7937. PubMed ID: 26887950
The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway is inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. This study provides evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, it was found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, this study has identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin alpha1 in Yorkie nuclear import.
|Liu, S., Sun, J., Wang, D., Pflugfelder, G. O. and Shen, J. (2016). Fold formation at the compartment boundary of Drosophila wing requires Yki signaling to suppress JNK dependent apoptosis. Sci Rep 6: 38003. PubMed ID: 27897227
Compartment boundaries prevent cell populations of different lineage from intermingling. In many cases, compartment boundaries are associated with morphological folds. However, in the Drosophila wing imaginal disc, fold formation at the anterior/posterior (A/P) compartment boundary is suppressed, probably as a prerequisite for the formation of a flat wing surface. Fold suppression depends on optomotor-blind (omb). Omb mutant animals develop a deep apical fold at the A/P boundary of the larval wing disc and an A/P cleft in the adult wing. A/P fold formation is controlled by different signaling pathways. Jun N-terminal kinase (JNK) and Yorkie (Yki) signaling are activated in cells along the fold and are necessary for the A/P fold to develop. While JNK promotes cell shape changes and cell death, Yki target genes are required to antagonize apoptosis, explaining why both pathways need to be active for the formation of a stable fold.
|Dubey, S.K. and Tapadia, M.G. (2017). Yorkie regulates neurodegeneration through canonical pathway and innate immune response. Mol Neurobiol [Epub ahead of print]. PubMed ID: 28102471
Expansion of CAG repeats in certain genes has long been known to be associated with neurodegeneration, but the quest to identity the underlying mechanisms is still on. This study analyzes the role of Yorkie, the coactivator of the Hippo pathway, and provides evidence that it is a robust genetic modifier of polyglutamine (PolyQ)-mediated neurodegeneration. Yorkie reduces the pathogenicity of inclusion bodies in the cell by activating cyclin E and bantam, rather than by preventing apoptosis through DIAP1. PolyQ aggregates inhibit Yorkie functioning at the protein, rather than the transcript level, and this is probably accomplished by the interaction between PolyQ and Yorkie. PolyQ aggregates upregulate expression of antimicrobial peptides (AMPs) and Yorkie negatively regulates immune deficiency (IMD) and Toll pathways through relish and cactus, respectively, thus reducing AMPs and mitigating PolyQ affects. These studies strongly suggest a novel mechanism of suppression of PolyQ-mediated neurotoxicity by Yorkie through multiple channels.
|Li, D., Liu, Y., Pei, C., Zhang, P., Pan, L., Xiao, J., Meng, S., Yuan, Z. and Bi, X. (2017). miR-285-Yki/Mask double-negative feedback loop mediates blood-brain barrier integrity in Drosophila. Proc Natl Acad Sci U S A 114(12):E2365-E2374. PubMed ID: 28265104
The blood-brain barrier (BBB) physiologically isolates the brain from circulating blood or the hemolymph system, and its integrity is strictly maintained to perform sophisticated neuronal functions. The underlying mechanisms of subperineurial glia (SPG) growth and BBB maintenance during development are not clear. This study reports an miR-285-Yorkie (Yki)/Multiple Ankyrin repeats Single KH domain (Mask) double-negative feedback loop that regulates SPG growth and BBB integrity. Flies with a loss of miR-285 have a defective BBB with increased SPG ploidy and disruptive septate junctions. Mechanistically, miR-285 directly targets the Yki cofactor Mask to suppress Yki activity and down-regulates the expression of its downstream target cyclin E, a key regulator of cell cycle. Disturbance of cyclin E expression in SPG causes abnormal endoreplication, which leads to aberrant DNA ploidy and defective septate junctions. Moreover, the expression of miR-285 is increased by knockdown of yki or mask and is decreased with yki overexpression, thus forming a double-negative feedback loop. This regulatory loop is crucial for sustaining an appropriate Yki/Mask activity and cyclin E level to maintain SPG ploidy and BBB integrity. Perturbation of this signaling loop, either by dysregulated miR-285 expression or Yki activity, causes irregular SPG ploidy and BBB disruption. Furthermore, ectopic expression of miR-285 promotes canonical Hippo pathway-mediated apoptosis independent of the p53 or JNK pathway. Collectively, these results reveal an exquisite regulatory mechanism for BBB maintenance through an miR-285-Yki/Mask regulatory circuit.
|Umegawachi, T., Yoshida, H., Koshida, H., Yamada, M., Ohkawa, Y., Sato, T., Suyama, M., Krause, H. M. and Yamaguchi, M. (2017). Control of tissue size and development by a regulatory element in the yorkie 3'UTR. Am J Cancer Res 7(3): 673-687. PubMed ID: 28401020
Regulation of the Hippo pathway via phosphorylation of Yorkie (Yki), the Drosophila homolog of human Yes-associated protein 1, is conserved from Drosophila to humans. Overexpression of a non-phosphorylatable form of Yki induces severe overgrowth in adult fly eyes. This study shows that yki mRNA associates with microsomal fractions and forms foci that partially colocalize to processing bodies in the vicinity of endoplasmic reticulum. This localization is dependent on a stem-loop (SL) structure in the 3' untranslated region of yki. Surprisingly, expression of SL deleted yki in eye imaginal discs also results in severe overgrowth phenotypes. When the structure of the SL is disrupted, Yki protein levels increase without a significant effect on RNA levels. When the SL is completely removed, protein levels drastically increase, but in this case, due to increased RNA stability. In the latter case, it was shown that the increased RNA accumulation is due to removal of a putative miR-8 seed sequence in the SL. These data demonstrate the function of two novel regulatory mechanisms, both controlled by the yki SL element, that are essential for proper Hippo pathway mediated growth regulation.
Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), has been identified as a missing link between Wts and transcriptional regulation. Yki (named for its loss-of-function phenotype after a very small breed of dog, the Yorkshire Terrier) is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang, 2005).
The increase in cell number that accompanies the growth of an organ or organism results from the balanced coordination of three simultaneous processes, including cell growth, cell proliferation, and cell death. Cell growth is a prerequisite for cell proliferation during normal organ growth, and sustained cell proliferation must be coupled to appropriate cell growth. With appropriate cell growth, a net increase in cell number in a growing organ depends on the rate at which they are generated via cell proliferation, as well as the rate at which they are eliminated by cell death (apoptosis). How cell proliferation and cell death are coordinated during tissue growth and homeostasis is yet to be completely understood, and this mechanism must be intact throughout life to prevent diseases such as cancer (Huang, 2005).
Recent studies in mice and fruit flies have revealed two distinct modes in which cell proliferation and cell death could be coupled. In the first mode, increased proliferation, such as that resulting from activation of the Myc oncogene, is coupled in an obligatory fashion to increased cell death. Such coupling between proliferation and apoptosis provides an important failsafe mechanism to prevent inappropriate proliferation of somatic cells. In the second mode, increased proliferation, such as that resulting from activation of the microRNA bantam, or inactivation of the tumor suppressors hippo (hpo), salvador (sav), and warts (wts), is accompanied by an inhibition of cell death. Here, suppression of cell death might allow the overproliferating cells to overcome proliferation-induced apoptosis, thus resulting in a robust increase in organ size. In many aspects, these circumstances resemble certain cancer cells, which display both increased cell proliferation and suppressed cell death (Huang, 2005 and references therein).
hpo, sav, and wts (also called lats) were identified from genetic screens in Drosophila for negative regulators of tissue growth. Inactivation of any of these genes results in increased cell proliferation and reduced apoptosis. hpo encodes a Ste20 family protein kinase, sav encodes a protein containing WW and coiled-coil domains, and wts encodes an NDR (nuclear Dbf-2-related) family protein kinase. Studies have suggested that these genes function in a common pathway that coordinately regulates cell proliferation and apoptosis by targeting the cell-cycle regulator CycE and the cell-death inhibitor DIAP1. Using a combination of genetic and biochemical assays, it has been shown that Hpo, Sav, and Wts define a novel protein kinase cascade wherein Hpo, facilitated by Sav, phosphorylates Wts (Wu, 2003). It was further demonstrated that this pathway, hereafter referred to as the Hpo pathway, negatively regulates the transcription of diap1 (Wu, 2003). It is worth noting that this model differs significantly from an alternative model by others that suggests that this pathway regulates DIAP1 posttranscriptionally through phosphorylation of DIAP1 by Hpo. Another unresolved issue in Hpo signaling concerns the molecular mechanism of the Wts/Lats kinase. While previous studies have identified a number of putative targets for this tumor suppressor, including the G2/M regulator cdc2 and the actin regulators zyxin and LIMK1, none of them could account for the excessive overgrowth associated with wts mutant clones. Thus, the most critical target of the Wts/Lats kinase has remained elusive (Huang, 2005 and references therein).
yorkie (yki) has now been identified as the elusive target of the Wts/Lats tumor suppressor. yki encodes the Drosophila ortholog of yes-associated protein (YAP), a transcriptional coactivator in mammalian cells (Yagi, 1999; Strano, 2001; Vassilev, 2001). Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo, sav, or wts. Taken together, these studies identify a missing link between Hpo signaling and transcriptional control and provide further support for the model implicating the Hpo signaling pathway in transcriptional regulation of diap1. These studies further reveal a functional conservation between YAP and Yki and implicate YAP as a potential oncogene in mammals (Huang, 2005).
Activation of yki leads to massive tissue overgrowth that resembles the loss-of-function phenotype of hpo, sav, or wts. To probe the physiological function of yki, the “flip-out” technique was used to generate clones of cells in which yki is overexpressed during development. yki-overexpressing clones lead to marked overgrowth in adult epithelial structures. Wing imaginal discs containing multiple yki-overexpressing clones reach up to eight times the area of control wing discs raised under identical conditions. Besides the overgrowth phenotype, adult cuticles secreted by yki-overexpressing cells display an unusual texture. In yki-overexpressing clones on the notum, the apical surface of the epidermal cells is domed such that cell-cell boundaries are visible between adjacent cells, whereas cell boundaries are not visible in the neighboring wild-type tissues. Both the overgrowth and the abnormal cell morphology caused by yki overexpression closely resemble those shown previously for hpo and wts mutant cells, suggesting that these genes might function in a common pathway (Huang, 2005).
Cell-doubling time for control and yki-overexpressing cells in the wing imaginal disc was determined by analyzing well-separated flip-out clones 48 hr post clone induction. The cell-doubling time for wild-type and yki-overexpressing clones (30 pairs of clones analyzed) was 16.1 hr and 12.0 hr, respectively. Thus, like mutant clones of hpo or wts, yki-overexpressing cells multiply faster. Notably, while cells in the control clones intermingle with their neighbors and form wiggly borders, yki-overexpressing cells minimize their contacts with their neighbors and form round smooth borders. This phenotype indicates distinct adhesive properties of the yki-overexpressing cells and resembles that seen with loss-of-function wts clones. FACS analysis shows that yki-overexpressing cells have a similar cell-cycle profile and cell size distribution as compared to wild-type cells. Thus, like loss-of-function of hpo (Wu, 2003), activation of yki does not accelerate a particular phase of the cell cycle. Rather, each phase of the cell cycle is proportionally accelerated (Huang, 2005).
Activation of yki in the eye imaginal disc leads to increased number of interommatidial cells without affecting photoreceptor differentiation. Focus was placed on the eye imaginal disc, a pseudostratified epithelium in which cell differentiation, proliferation, and apoptosis occur in a highly stereotyped manner. In the third instar, the morphogenetic furrow (MF) traverses the eye disc from posterior to anterior. Cells anterior to the MF are undifferentiated and divide asynchronously, whereas cells in the MF are synchronized in the G1 phase of the cell cycle. Posterior to the MF, cells either exit the cell cycle and differentiate or undergo one round of synchronous division (second mitotic wave, SMW) before differentiation. These cells assemble into approximately 750 ommatidia, leaving behind approximately 2000 superfluous cells that are eliminated by a wave of apoptosis ~36 hr after puparium formation (APF) (Huang, 2005).
To investigate whether activation of yki perturbs photoreceptor differentiation, the neuronal marker Elav was examined. yki-overexpressing ommatidial clusters have the normal complement of photoreceptor cells. The spacing between adjacent ommatidial clusters is increased due to the presence of extra interommatidial cells. The formation of extra interommatidial cells is most evident in pupal eye discs, when yki-overexpressing clones contain many additional cells between photoreceptor clusters. Thus, like loss-of-function of hpo, sav, or wts, yki overexpression results in an increased number of uncommitted, interommatidial cells without affecting early retina patterning (Huang, 2005).
Activation of yki leads to increased cell proliferation and decreased apoptosis. To pinpoint the developmental cause of yki-induced overgrowth, cell proliferation and apoptosis were monitored in eye imaginal discs. In wild-type eye discs, cells posterior to the MF undergo a synchronous second mitotic wave (SMW) that can be revealed as a band of BrdU-positive cells. Few BrdU-positive cells are found posterior to the SMW. In yki-overexpressing clones, cells fail to undergo cell-cycle arrest posterior to the SMW and continue cell cycles as shown by BrdU incorporation as well as M phase marker phospho-histone H3 (PH3). Thus, yki overexpression results in increased cell proliferation (Huang, 2005).
Using the TUNEL assay, cell death was monitored in the pupal retina at a point when a wave of apoptosis normally removes excess interommatidial cells around 36 hr APF. Strikingly, cell death was significantly suppressed in yki-overexpressing clones, even though abundant apoptosis was detected in the neighboring wild-type cells. Thus, normal developmental cell death is largely inhibited by yki overexpression (Huang, 2005).
The mechanisms of how body and organ size are regulated are just beginning to be understood. Recent studies in Drosophila have implicated a number of pathways in the coordinate control of cell growth, proliferation, and apoptosis, which ultimately regulate body and organ size. The insulin/Tsc/TOR signaling network, for example, plays a major role in coordinating organ growth with environmental cues such as nutrients. The Hpo signaling pathway, in contrast, might contribute to an intrinsic size 'checkpoint' that normally stops growth when a given organ reaches its characteristic size. Thus, molecular elucidation of the Hpo signaling pathway should provide important insights into size-control mechanisms in development (Huang, 2005).
Previous studies of the Wts/Lats tumor suppressor have failed to identify any target of this kinase that could account for its potent growth-regulatory activity. This study has provided genetic and biochemical evidence implicating Yki, the Drosophila ortholog of the mammalian coactivator protein YAP, as a direct, critical target of Wts/Lats in the Hpo pathway. Yki associates with and is phosphorylated by Wts. Moreover, Wts-mediated phosphorylation of Yki is stimulated by upstream components of the Hpo pathway, and the extent of Yki phosphorylation induced by Hpo pathway components in vitro correlates with the severity of the overexpression phenotype caused by the respective transgenes in vivo. Most importantly, overexpression of yki phenocopies loss of hpo, sav, or wts, while loss of yki results in the opposite phenotype, and epistasis analyses unambiguously places yki downstream of hpo, sav, and wts. Taken together, these results provide compelling evidence that Yki is a critical target of Wts/Lats in the Hpo pathway. It is further speculated that the relationship between Yki and Hpo signaling is likely conserved during evolution since overexpression of mammalian YAP is able to rescue the lethality associated with hyperactivation of the Hpo pathway in Drosophila. The functional conservation between Yki and YAP further suggests that YAP might function as an oncogene in mammals (Huang, 2005).
Yki is the first substrate identified for NDR family kinases, which include, besides Wts/Lats, Cbk1, Dbf2, and Dbf20 in budding yeast, Sid2 and Orb6 in fission yeast, Cot-1 in Neurospora, Sax-1 in C. elegans, Trc in Drosophila, and NDR1 and NDR2 in mammals (reviewed by Tamaskovic, 2003). The NDR family kinases are involved in diverse events in cell-cycle and cell morphogenesis, such as maintaining cell polarity (Cbk1 and Orb6), coordinating CDK inactivation and cytokinesis (Dbf2, Dbf20, and Sid2), and neuronal morphogenesis (Sax-1). Despite their diverse cellular functions, all NDR family kinases share similar structural features, such as the insertion of 30-60 amino acids between kinase subdomains VII and VIII, the presence of conserved activation loop and hydrophobic motif, and the presence of N-terminal noncatalytic domain (Tamaskovic, 2003). These common features suggest that NDR family kinases may employ similar mechanisms to interact with their substrates and regulators. Along this line, it is suggested that the approach described in this study, which uses the N-terminal noncatalytic domain of Wts as yeast two-hybrid bait, might provide a general method to discover substrates for other NDR family kinases (Huang, 2005).
A model has been proposed whereby Hpo, somehow facilitated by Sav, phosphorylates Wts (Wu, 2003). While this model implied that phosphorylation of Wts leads to activation of its kinase activity, it was not possible to directly test this due to the lack of an appropriate assay that measures pathway activity downstream of Wts. The identification of Yki as a Wts substrate provides a new tool to evaluate the earlier model. Consistent with the previous model implicating Hpo as an activating kinase of Wts, it has been shown that in S2 cells, the phosphorylation of Yki induced by transfected Wts is dependent on the endogenous Hpo protein. Furthermore, the in vitro kinase activity of Wts toward Yki is strongly stimulated when Wts is coexpressed with Hpo-Sav. It is suggested that such a relationship between Hpo and Wts is likely conserved during evolution. Indeed, a recent study (Chan, 2005) has demonstrated the activation of the mammalian Lats1 kinase by the mammalian Hpo homologs Mst1/Mst2 (Huang, 2005).
It is worth noting that several Ste20-like kinases have been implicated in the activation of NDR kinases. Such examples include the activation of Wts by Hpo (Wu, 2003; Chan, 2005), the activation of Dbf2 by Cdc15, the regulation of Orb6 by Pak1, and the regulation of Sid2 by Sid1. Thus, activation by Ste20-like kinases might represent a general mechanism for regulating NDR kinases. In retrospect, the difficulties in identifying substrates for NDR kinases might be due to their substrate specificity in conjunction with a requirement for activation by upstream kinases. Another emerging feature of the NDR kinases concerns their regulation by the Mob family of small regulatory proteins, which have been found to associate with multiple NDR family kinases, such as Dbf2, Orb6, Sid2, Cbk1, NDR1, and NDR2 (Tamaskovic, 2003). In Drosophila, Mats, a Mob family protein, has recently been identified as a tumor suppressor gene that likely regulates Wts in the Hpo signaling pathway (Lai, 2005). Thus, regulation by Mob family proteins likely represents an important and shared feature of modulating NDR family kinases (Huang, 2005 and references therein).
Previous studies of Hpo signaling have suggested two contrasting models on how this pathway regulates the cell-death regulator DIAP1. Using a diap1-lacZ reporter to follow diap1 transcription, elevated diap1 transcription was observed in mutant clones of hpo, sav, or wts that closely matches the increase in DIAP1 protein levels. Based on these results, it was proposed that the Hpo pathway negatively regulates diap1 at the level of transcription (Wu, 2003). However, an alternative model suggested that Hpo regulates DIAP1 posttranscriptionally by directly phosphorylating DIAP1, thus promoting its degradation. This model was largely based on two lines of evidence, including in situ hybridization showing unchanged diap1 mRNA level in mutant clones and the ability of Hpo to phosphorylate DIAP1 in vitro. It is noted, however, that in situ hybridization used in the latter studies did not involve the marking of mutant clones and thus may be less definitive than the diap1-lacZ reporter. A major drawback of the posttranscriptional model is that it cannot easily account for the involvement of Wts in the Hpo pathway. A direct link between Hpo and DIAP1 inevitably implies Wts as acting upstream or in parallel with Hpo, which is contradictory to other studies of the NDR kinases that generally place them downstream of the Ste20-like kinases (Huang, 2005 and references therein).
If the Hpo signaling pathway regulates diap1 via a transcriptional mechanism, then there should exist transcriptional regulator(s) that control diap1 transcription and whose activity may be regulated by the Hpo signaling pathway. Furthermore, such transcriptional regulator(s) must account for the mutant phenotypes resulting from deregulation of the Hpo pathway, such as changes in diap1 transcription and overgrowth. This current study demonstrates that Yki represents such a regulator, thus further supporting the previous model implicating the Hpo pathway in regulating diap1 transcription (Huang, 2005).
Understanding the molecular mechanisms by which the Hpo pathway regulates diap1 transcription will provide important insights into the developmental coordination of tissue growth and apoptosis. Like other coactivators, Yki presumably functions by interacting with DNA binding transcription factors. YAP, the mammalian homolog of Yki, is known to function as coactivator for a number of transcription factors, such as the p53 family member p73 (Strano, 2001), the Runt family member PEBP2α (Yagi, 1999), and the four TEAD/TEF transcription factors (Vassilev, 2001). This interaction is generally mediated by WW domains of YAP and PPxY motifs of the cognate transcription factors. At present, it is unclear whether any of the reported mammalian proteins represents the physiological partner for YAP. Along this line, it is worth noting that while the reported ability of YAP to transactivate p73 in cultured mammalian cells is more suggestive of a tumor suppressor function for YAP (Basu, 2003), these studies clearly implicate Yki and YAP as potential oncogenes. One interesting possibility (Lowe, 2004) is that the reported coupling of mammalian YAP to p73 might represent a fail-safe mechanism to limit the oncogenic potential of YAP in much the same way as cell death is obligatorily linked to oncogene activation (Huang, 2005).
An important direction in the future is to identify the DNA binding transcription factor that partners with Yki to regulate gene transcription; identifying the factor should provide critical insights into how Yki (and likely YAP as well) could function as a potent oncogene. This effort should be facilitated by the dissection of the diap1 promoter and the identification of a minimal Hpo-responsive element that confers transcriptional regulation of diap1 by the Hpo pathway. With such a DNA element, one should be able to identify the DNA binding transcription factor that partners with Yki to regulate the transcription of diap1 and other Hpo-pathway-responsive genes (Huang, 2005).
Many components of the Hpo pathway are conserved throughout evolution, suggesting that this emerging pathway might play a similar role in mammals. Indeed, previous studies have shown that human homologs of wts, hpo, and mats could rescue the respective Drosophila mutants. Moreover, mice lacking a wts homolog are prone to tumor formation, and the human orthologs of sav and mats are mutated in several cancer cell lines. Such conservation is further extended in the current study, showing that Yki and YAP have similar biological activity when assayed in Drosophila. These results suggest that the Hpo signaling pathway might play a conserved role in mammalian growth control. Furthermore, inactivation of growth suppressors of the Hpo pathway, such as Hpo, Sav, Wts, and Mats, and hyperactivation of growth promoters of the pathway, such as YAP, are likely to contribute to mammalian tumorigenesis (Huang, 2005).
Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), homologs of Drosophila Yorkie. However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. This study dissects how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, these results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. (Yang, 2014).
This report addresses the effects of AJs and basolateral cell polarity determinants on the activity of the Hippo pathway in Drosophila imaginal discs. Knockdown of AJs and basolateral components both induced ectopic activation of Yki. However, knockdown of AJs and basolateral proteins had strikingly different effects on Yki. Disruption of the basolateral module induced mainly a cell-autonomous increase in Yki activity, whereas knockdown of AJs caused non-autonomous induction of Yki reporters. Therefore, these data identify and genetically uncouple multiple different molecular pathways from AJs and the basolateral module that regulate Yki activity (Yang, 2014).
These studies further show that knockdown of AJs induces cell-autonomous reduction of Yki activity and causes cell death and decreased size of Drosophila imaginal discs. Likewise, E-cad and :alpha;-cat mutant clones do not survive in imaginal discs. This effect may be mediated by LIM domain proteins of the Zyxin and Ajuba subfamilies, which regulate Hippo signaling by directly inhibiting Wts/Lats kinases and by interacting with Salvador (Sav), an adaptor protein that binds to the Hpo/MST kinases. A recent report shows that α-Cat recruits Ajuba and indirectly Wts to AJs and loss of Ajuba leads to activation of Wts and hence phosphorylation and inhibition of Yki and diminished tissue size. Thus, α-cat mutant cells may inactivate Yki because they lose Ajuba function (Yang, 2014).
In contrast, in mammalian systems, several in vivo and in vitro studies have shown the opposite effect on Hippo signaling upon AJ disruption; knockdown of E-cad or α-cat caused an increase in cell proliferation and nuclear accumulation of YAP, and conditional knockout of α-cat in mouse skin cells caused tumor formation and elevated nuclear YAP staining. This suggests that AJ components have a tumor suppressor function in mammals. The observation that Scrib is mislocalized upon disruption of AJs in several different mammalian cell lines suggested that YAP activation could be due to the concomitant disruption of the basolateral module. However, the finding that acute disruption of AJs can cause YAP activation without disrupting Scrib localization and vice versa indicates that AJs and the basolateral module also act independently on the Hippo pathway in mammalian cells. In mammalian cells, α-Cat forms a complex with YAP and 14-3-3 proteins, thereby sequestering phosphorylated YAP at the plasma membrane. However, α-Cat may function as a tumor suppressor only in epidermal stem cells, as conditional deletion of α-cat in differentiated cells only caused a mild phenotype with no overgrowth and tumor formation. Therefore, it is possible that the negative regulation of YAP by α-Cat is cell type-specific, although further testing is required to fully address this issue (Yang, 2014).
The non-cell-autonomous effect of AJ knockdown on the Hippo pathway is an intriguing phenomenon. Several groups reported non-autonomous effects on the Hippo pathway in Drosophila in other mutant conditions. Disrupting the expression gradients of the atypical Cadherin Dachsous or that of its regulator Four-jointed, clones of cells mutant for the tumor suppressor genes vps25 or hyperplastic discs (hyd) , clones of cells overexpressing Src64, or overexpression of the proapoptotic gene reaper or the JNK signaling ligand eiger all cause non-autonomous activation of Yki. This non-autonomous activation of Yki may be part of a regenerative response that stimulates cell proliferation in cells neighboring tissue defects. The signals that activate Yki in these situations are not known, nor is it known whether these mutant conditions activate the same or different signaling mechanisms. The non-autonomous activation of Yki around cells with AJ knockdown may be mediated by changes in mechanical forces. AJs are important for maintaining tension between cells across epithelia, and disruption of AJs leads to an imbalance of apical tension. Mechanical forces are known to regulate the Hippo pathway, and YAP/TAZ act as mediators of mechanical cues from the cellular microenvironment such as matrix stiffness. In particular, the Zyxin and Ajuba family LIM domain proteins can act as sensors of mechanical forces and may be involved in the non-autonomous activation of Yki. The effects on Hippo signaling of solely changing Zyxin and Ajuba may not be as strong as those described here, and these proteins may thus cooperate with other molecular conduits to regulate the activity of the Hippo pathway in response to changes in AJ strength. Unraveling these mechanisms will provide important new insights into understanding how cells interact with neighboring cells to regulate proliferation, apoptosis, and the Hippo pathway (Yang, 2014).
It is currently unknown whether AJs also exert non-autonomous effects on the Hippo pathway in mammalian tissues. Amphiregulin, an EGF ligand, is a downstream target of YAP and can induce non-cell-autonomous cell proliferation through EGFR signaling. However, it is not known whether YAP itself is activated non-cell-autonomously to contribute to the hyper-proliferation phenotypes observed upon disruption of AJs in vivo and in vitro. It will be interesting to determine whether AJs and other cell-cell signaling mechanisms also have non-cell-autonomous effects on the activity of YAP in mammalian tissues, for example during regeneration (Yang, 2014).
Finally, the apical proteins aPKC and Crb modulate the activity of the Hippo pathway, and many Hippo pathway components are apically localized, which is important for their activity. The data presented in this study add to these findings. Disruption of AJs causes reduced Yki activity, despite the fact that Crb and Mer are mislocalized. Thus, AJs and cell polarity components regulate Yki activity through multiple, genetically separable inputs. It will be interesting to decipher all of the different underlying molecular mechanisms of how AJs and basolateral proteins regulate the Hippo pathway and how these mechanisms evolved in Drosophila and in mammals (Yang, 2014).
Yki is most closely related to the human yes-associated protein (YAP, also called YAP65) (Sudol, 1994), with 31% identity between the two proteins. Both proteins contain two WW domains, protein-protein interaction modules of 35-40 amino acids that are known to interact with PPXY-containing polypeptides. The similarity between Yki and YAP extends beyond the WW domains and includes a stretch of sequence similarity at the N-terminal part of the proteins. The WW domains of Yki are required for its interaction with Wts. While initially isolated as a protein that interacts with the SH3 domain of the Yes proto-oncogene, the involvement of YAP in Yes signaling has not been validated (Sudol, 1994). Notably, the corresponding SH3 binding region (Sudol, 1994) is absent in the Drosophila Yki protein. In contrast, YAP has been implicated as a transcriptional coactivator, a class of transcriptional regulators that do not bind to DNA themselves but associate with DNA binding transcription factors and supply or stimulate transcriptional activation of the cognate transcription factors. Specifically, YAP has been shown to function as a coactivator for a number of transcription factors, such as the p53 family transcription factor p73 (Strano, 2001), the Runt family protein PEBP2α (Yagi, 1999), and the TEAD/TEF family transcription factors (Vassilev, 2001). However, these studies have been performed exclusively in cultured mammalian cells and little is known about the physiological function of YAP (Huang, 2005).
date revised: 20 November 2005
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