Mechanisms of Hippo pathway regulationgenesdev.cshlp.org/content/30/1/1.full.pdfREVIEW Mechanisms of...

REVIEW

Mechanisms of Hippo pathway regulationZhipeng Meng, Toshiro Moroishi, and Kun-Liang Guan

Department of Pharmacology, Moores Cancer Center, University of California at San Diego, La Jolla, California 92093, USA

The Hippo pathway was initially identified inDrosophilamelanogaster screens for tissue growth two decades agoand has been a subject extensively studied in bothDroso-phila and mammals in the last several years. The core ofthe Hippo pathway consists of a kinase cascade, transcrip-tion coactivators, and DNA-binding partners. Recentstudies have expanded the Hippo pathway as a complexsignaling network with >30 components. This pathwayis regulated by intrinsic cell machineries, such as cell–cell contact, cell polarity, and actin cytoskeleton, aswell as a wide range of signals, including cellular energystatus, mechanical cues, and hormonal signals that actthrough G-protein-coupled receptors. The major func-tions of the Hippo pathway have been defined to restricttissue growth in adults and modulate cell proliferation,differentiation, and migration in developing organs. Fur-thermore, dysregulation of the Hippo pathway leads to ab-errant cell growth and neoplasia. In this review, we focuson recent developments in our understanding of the mo-lecular actions of the core Hippo kinase cascade and dis-cuss key open questions in the regulation and functionof the Hippo pathway.

TheHippo pathwaywas initially identified inDrosophila;however, most of the recent studies focus on its functionand regulation in mammalian cells. Many new regulatorsof the Hippo pathway have been identified and character-ized. We first discuss recent discoveries in the mammali-an Hippo pathway and then introduce the Drosophilacounterparts to provide a brief history of research in theHippo pathway. This reviewmainly focuses on themolec-ular regulation and function of the core Hippo pathwaycomponents.

The core kinase cascade of the Hippo pathway

Core components of the mammalian Hippo pathway

In a classical view, the core of the Hippo pathway inmam-mals is a kinase cascade in which the mammalian Ste20-like kinases 1/2 (MST1/2; homologs of Drosophila Hippo[Hpo]) phosphorylate and activate large tumor suppressor

1/2 (LATS1/2; homologs of Drosophila Warts [Wts]) (Fig.1A). The physiological output of this kinase cascade isto restrict the activities of two transcriptional coactiva-tors, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ; two homologsof Drosophila Yorkie [Yki]). When YAP and TAZ are ac-tive, they translocate into the nucleus to bind the TEADtranscription factor family (homologs of Drosophila Scal-loped [Sd]) and induce expression of a wide range of genesthat are involved in cell proliferation, survival, andmigration.Mechanistically, the Hippo kinase cascade can be initi-

ated by TAO kinases (TAOK1/2/3), which phosphorylatethe activation loop of MST1/2 (Thr183 for MST1 andThr180 for MST2; hereafter, all residues refer to humanproteins) and thereby lead toMST1/2 activation (Boggianoet al. 2011; Poon et al. 2011). There is also evidenceshowing that the activation loop phosphorylation can beachieved by MST1/2 autophosphorylation (Praskovaet al. 2004). Consistent with this model, the activationloop phosphorylation is enhanced by MST1/2 dimeriza-tion (Glantschnig et al. 2002). Therefore, it is possiblethat MST1/2 activation can be initiated by dimerizationand does not neccesarily require upstream kinases. ActiveMST1/2 phosphorylate SAV1 (homolog of DrosophilaSalvador [Sav]) and MOB1A/B (homologs of DrosophilaMats) (Callus et al. 2006; Praskova et al. 2008), two scaf-fold proteins that assist MST1/2 in the recruitment andphosphorylation of LATS1/2 at their hydrophobic motifs(T1079 for LATS1 and T1041 for LATS2) (Hergovichet al. 2006; Yin et al. 2013). Another key player in this ac-tion is NF2/Merlin, which directly interacts with LATS1/2 and facilitates LATS1/2 phosphorylation by the MST1/2–SAV1 complex (Yin et al. 2013). LATS1/2 subsequentlyundergo autophosphorylation and are activated (Chanet al. 2005) and in turn phosphorylate and inactivateYAP and TAZ (Zhao et al. 2007). In parallel to MST1/2,two groups of MAP4Ks (mitogen-activated protein kinasekinase kinase kinase), MAP4K1/2/3/5 (homologs ofDrosophila Happyhour [Hppy]) and MAP4K4/6/7 (homo-logs of Drosophila Misshapen [Msn]), can also directlyphosphorylate LATS1/2 at their hydrophobic motifs and

[Keywords: LATS; MAP4K; MST; TAZ; TEAD; YAP]Corresponding author: [email protected] is online at http://www.genesdev.org/cgi/doi/10.1101/gad.274027.115.

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result in LATS1/2 activation (Meng et al. 2015; Zhenget al. 2015). In HEK293A cells, triple knockout ofMAP4K4/6/7 reduces the phosphorylation of YAP/TAZmore dramatically than MST1/2 double knockout underserum deprivation, indicating that MAP4Ks may play amore prominent role than MST in Hippo pathway regula-tion under certain conditions (Meng et al. 2015). However,deletion of both MST1/2 and MAP4Ks is required to abol-ish YAP phosphorylation in response to LATS-activatingsignals, such as contact inhibition, energy stress, serum

deprivation, and F-actin disassembly (Meng et al. 2015).Therefore,MST1/2 andMAP4Ks have partially redundantroles in LATS1/2 regulation. Phosphorylation of YAP andTAZ leads to their binding with 14-3-3, and the 14-3-3binding causes cytoplasmic sequestration of YAP/TAZ(Zhao et al. 2007). Moreover, LATS-induced phosphoryla-tion triggers subsequent phosphorylation of YAP/TAZby Casein kinase 1δ/ε and recruitment of the SCF E3 ubiq-uitin ligase, leading to eventual YAP/TAZ ubiquitinationand degradation (Liu et al. 2010; Zhao et al. 2010). In

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Figure 1. The core Hippo pathway in mammals andDrosophila. (A) The mammalian Hippo pathway. When the Hippo pathway is inac-tive, YAP and TAZ are unphosphorylated and localized in the nucleus to compete with VGLL4 for TEAD binding and activation of genetranscription. The Hippo pathway can be activated by TAO kinases, which phosphorylate MST1/2 at its activation loop. MST1/2 in turnphosphorylate LATS1/2, facilitated by scaffold proteins SAV1, MOB1A/B, and NF2.MAP4K4/6/7 andMAP4K1/2/3/5 also phosphorylateand activate LATS1/2. Phosphorylation of LATS1/2 byMAP4K4/6/7 requiresNF2 (also known asMer). Activated LATS1/2 phosphorylateYAP and TAZ, leading to 14-3-3-mediated YAP and TAZ cytoplasmic retention and SCF-mediated YAP and TAZ degradation. (B) TheDrosophilaHippo pathway. Active Yki competes Tgi to interact with Sd in the nucleus and activates the transcription of Sd target genes.When Hpo is activated by Tao kinase or dimerization, it phosphorylates and activates Wts with the assistance of the scaffold proteins Savand Mats as well as Mer. It is unclear whether Msn and Hppy require Mer and Sav to phosphorylate and activate Wts. Active Wts phos-phorylates and inactivates Yki, leading to 14-3-3-mediated Yki cytoplasmic retention.

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addition, YAP protein can also be degraded by autophagy(Liang et al. 2014).YAP and TAZ are transcriptional coactivators and do

not haveDNA-binding domains. Rather, when translocat-ed into the nucleus, they regulate gene expression throughinteraction with TEAD1–4, which are sequence-specifictranscription factors that mediate the main transcription-al output of the Hippo pathway in mammlian cells (Zhaoet al. 2008). TEAD1–4 can also bind to VGLL4 in the nu-cleus and thus function as transcriptional repressors.The interaction between YAP/TAZ and TEAD1–4 dis-sociates VGLL4 from TEAD1–4 and thereby activatesTEAD-mediated gene transcription to promote tissuegrowth and inhibit apoptosis (Koontz et al. 2013). Mousemodels with deletion of MST1/2, SAV1, MOB1A/B,NF2, or LATS1/2 or YAP overexpression all exhibit up-regulation of TEAD target gene expression, increasedexpansion of progenitor cells, and tissue overgrowth(Camargo et al. 2007; Dong et al. 2007; Zhou et al. 2009;Cai et al. 2010; Lee et al. 2010; Lu et al. 2010; Song et al.2010; Zhang et al. 2010; Nishio et al. 2012; Chen et al.2015b), supporting the functional roles of these genes inthe Hippo pathway.

The Drosophila Hippo core components

TheHippo pathwaywas named after hpo, aDrosophila ki-nase gene that was independently identified to restrict tis-sue growth by several groups more than a decade ago (Fig.1B; Harvey et al. 2003; Jia et al. 2003; Pantalacci et al.2003; Udan et al. 2003; Wu et al. 2003). hpo mutants ex-hibit uncontrolled growth in multiple tissues due to ex-cessive cell proliferation and reduced apoptosis. Thesephenotypes, together with the elevated transcription ofcycE and diap1, are very similar to those previously ob-served inmutants of salvador (sav) andwarts (wts) (Justiceet al. 1995; Xu et al. 1995; Kango-Singh et al. 2002; Taponet al. 2002). Hpo, Sav, and Wts show genetic interactions,and Hpo directly phosphorylates and activates Wts. Infact, the Hippo pathway is also known as the Salvador/Warts/Hippo (SWH) pathway (Harvey and Tapon 2007).Sav serves as an adaptor protein for Hpo to phosphorylateWts and can also be phosphorylated by Hpo (Pantalacciet al. 2003; Wu et al. 2003). Sav phosphorylation by Hpopromotes its interaction with Hpo, which promotes phos-phorylation of Wts and transcriptional repression of cycEand diap1. In addition, the physical binding of Hpo to Savpromotes protein stability of Sav by preventing interac-tion between Sav and the HECT domain protein Herc4(HECT and RLD domain-containing E3 ligase), whichfunctions as a Sav E3 ligase and induces Sav ubiquitinyla-tion and degradation (Aerne et al. 2015). Another corecomponent, Mats (Mob as tumor suppressor), was lateridentified as a Wts-interacting protein that potentiatesWts kinase activity (Lai et al. 2005). Loss of mats also re-sults in uncontrolled tissue growth similar to hpo or wtsmutation in Drosophila.The Hippo pathway effector Yki, which serves as the

key link between Wts and the transcriptional regulationof cycE and diap1, was discovered by a yeast two-hybrid

screen in 2005 (Huang et al. 2005). Overexpression ofYki recapitulates the hpo, wts, or sav mutant phenotypein cell proliferation, apoptosis, and tissue growth. The un-derlying biochemical mechanism is that Wts phosphory-lates Yki and leads to Yki’s interaction with 14-3-3 andcytoplasmic retention (Dong et al. 2007). Yki regulatesgene transcription through interacting with the Scalloped(Sd) transcription factor (Goulev et al. 2008; Wu et al.2008; Zhang et al. 2008). In the absence of Yki binding,Sd binds to Tondu domain-containing growth inhibitor(Tgi) by default and actually represses gene expression.Yki replaces Tgi and converts Sd into a transcriptionalactivator (Koontz et al. 2013). Therefore, a common mo-lecular mechanism of Hippo pathway regulation is highlyconserved between Drosophila and mammals.The Hippo pathway is regulated by a variety of intrinsic

and extrinsic signals. In most scenarios, the central eventof the Hippo pathway appears to be phosphorylation-dependent Wts activation and Yki inhibition. The majorkinase for Wts is Hpo, which can be phosphorylated andactivated by the Tao kinase (Boggiano et al. 2011; Poonet al. 2011). Phosphorylation of Wts by Hpo also requiresadaptor proteins such as Mats and Merlin (Mer) to recruitWts to the plasma membrane (Wei et al. 2007; Yin et al.2013). Recent studies show that two other kinases, Mis-shapen (Msn) and Happyhour (Hppy), can activate Wtsand repress Yki independently of Hpo (Li et al. 2014a;Zheng et al. 2015). There is evidence that, like Hpo,Hppy also phosphorylates the hydrophobic motif of Wts(Zheng et al. 2015). This study also shows thatMsn cannotdirectly phosphorylate Wts. However, it is worth notingthat human MAP4K4/6/7 (the Msn homologs) candirectly phosphorylate and activate LATS (Meng et al.2015). Therefore, future studies are needed to clarifywhether Msn can directly phosphorylate and activateWts. Identifications of Msn, Hppy, and their mammalianhomologs, MAP4Ks, have greatly broadened the scope ofthe Hippo pathway and also revealed the molecular basisof how various signals can activate LATS in MST1/2knockout cells (Kim et al. 2011; Yu et al. 2012, 2013;Zhao et al. 2012; Meng et al. 2015).

Upstream signals that regulate the Hippo pathway

Studies in the last decade have cemented YAP and TAZ asthemajor effectors of the Hippo pathway, which regulatesthe phosphorylation-induced cytoplasmic retention andprotein degradation of YAP and TAZ in response to amyr-iad of intrinsic and extrinsic signals. These signals, inmost scenarios, modulate phosphorylation events of thecore kinase cascade through peripheral components ofthe Hippo pathway. In addition, there are a number of pro-teins that directly regulate YAP localization or transacti-vation without affecting LATS kinase activity (Yu andGuan 2013). Moreover, the Hippo pathway cross-talkswith Wingless/Ints (Wnt), bone morphogenetic proteins(BMPs), Notch, and Hedgehog (Hh), as these signals alsomodulate the activities of YAP and TAZ (Hansen et al.2015). In this section, we summarize upstream signals

Mechanisms of Hippo pathway regulation

GENES & DEVELOPMENT 3




and the peripheral Hippo pathway components that relaysignals to the core kinase cascade (Fig. 2).

Physical cues: cell contact and mechanical signal

Organ growth and development involve many coordinat-ed actions of cells to adapt to physical restraints and extra-cellular mechanical cues. Tissue architecture physicallyrestricts cell growth and proliferation and in many casesleads to cell quiescence. For example, cell–cell contactat high cell density produces a growth inhibitory signalthat is in large part mediated by the Hippo pathway(Zhao et al. 2007; Ota and Sasaki 2008; Nishioka et al.2009). As a result, LATS kinase is activated at high celldensity, whereas LATS is inactive at low cell density.YAP inactivation is critically important for cell contactinhibition in cell culture. The regulation of the YAP–TEAD transcription program by contact inhibition isalso crucial for embryo development (Ota and Sasaki2008; Nishioka et al. 2009; Gumbiner and Kim 2014).The increased adherens junctions and tight junctions inconfluent cells contribute to activation of LATS and inac-tivation of YAP and TAZ (Zhao et al. 2007; Silvis et al.2011). Furthermore, loss of cell spreading or decrease ofcell size may also be involved, as extracellular matrix(ECM) stiffness regulates YAP and TAZ subcellular local-ization through changes in cell geometry and cytoskele-ton tension (Dupont et al. 2011; Driscoll et al. 2015).Physical attachment of cells to ECM is essential forcells to survive and grow. Attachment of cells to ECM in-duces YAP nuclear localization through activation ofRho-GTPases or the FAK–Src–PI3K pathway (Zhao et al.2012; Kim and Gumbiner 2015). Disruption of F-actin

blocks the effect of attachment on YAP phosphorylationand nuclear localization. In contrast, detachment of cellsinactivates YAP and TAZ and triggers anoikis in a LATS-dependentmanner (Zhao et al. 2012). The cell attachmentcertainly providesmechanical signal to the cell, as the cul-ture plate surface has high stiffness. In addition, YAP andTAZ activities are also modulated by stretching and edge/curvature contouring an epithelial sheet (Aragona et al.2013). This regulation by mechanical forces similarly re-quires Rho-GTPases and F-actin capping/severing pro-teins as mediators and is proposed to function as aphysical checkpoint of cell growth and a cell fate determi-nation during stem cell differentiation (Aragona et al.2013). In fact, activation of YAP and TAZ by increasingsubstrate rigidity greatly enhances differentiation of hu-man pluripotent stem cells into motor neuron cells, sug-gesting a potential application of engineered substratesto produce particular types of differentiated cells (Sunet al. 2014).

Recent studies also show that YAP and TAZ are activat-ed by shear stress from fluid flow, indicating a physio-logical and disease-relevant role of YAP and TAZ inendothelial cell differentiation and vascular homeostasis(Kim et al. 2014; Sabine et al. 2015). The physiological rel-evance of mechanical forces and cell growth has also beenestablished in a Drosophila study (Rauskolb et al. 2014).Cytoskeleton tension inhibits Wts and subsequently acti-vates Yki and promotes wing growth through recruitingWts to adherens junctions by α-catenin and Jub. Upon tis-sue injury, anatomic alternations and emerging space inorgans promote cells to exit quiescence and re-enter thecell cycle to expand cell populations and thus maintaintissue homeostasis. From Drosophila to rodent models,

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Figure 2. Regulation of the Hippo pathway by up-stream signals. Cyclic stretch or high extracellularmatrix stiffness inhibits LATS1/2 phosphorylationthrough Rho-GTPases and JNK1/2. G-protein-cou-pled receptors (GPCRs) can either activate or sup-press LATS1/2 depending on the types of the Gαproteins involved. The LATS1/2 activation is alsocontrolled by cell polarity and architecture throughKIBRA/NF2, adherens junctions (AJ), and tight junc-tions (TJ). Energy status modulates YAP and TAZ ac-tivity via AMPK. Cell cycle affects YAP and TAZthrough either LATS1/2- or CDK1-mediated proteinphosphorylation.

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genetic inactivation of theHippo pathway consistently re-sults in overgrowth phenotypes in a variety of organs,whereas inactivation of YAP/TAZ impairs wound healing(Yu et al. 2015).Most studies have indicated that Rho-GTPases and the

actin cytoskeleton play an essential role in regulation ofYAP and TAZ by mechanotransduction; however, the in-volvement of the Hippo core kinase cascade (MST–LATS)is still under debate. Earlier studies exclude MST1/2 andLATS1/2 in the regulation of YAP/TAZ nuclear transloca-tion and transcriptional activation because RNAi target-ing LATS1/2 does not block YAP and TAZ regulation byECM stiffness (Dupont et al. 2011). However, it was re-cently reported that mechanical strain suppresses YAPphosphorylation and promotes YAP nuclear translocationby inactivating LATS1/2 in a JNK-dependent manner(Codelia et al. 2014). Future studies are required to delin-eate the mechanosensor/receptor as well as the role ofthe core Hippo kinase in mechanical signal-inducedYAP/TAZ regulation.

Soluble factors and G-protein-coupled receptors (GPCRs)

Tissue growth requires nutrients as well as hormonal sig-nals via autocrine, paracrine, and endocrine mechanisms.Moreover, nutrient uptake is also under the control ofgrowth-stimulating signals. It had long been speculatedthat extracellular molecules, such as hormones or growthfactors, might regulate the Hippo pathway in order to con-trol tissue growth and homeostasis. Amajor breakthroughin the Hippo pathway came with the discovery that diffu-sive molecules, such as lysophosphatidic acid (LPA) andsphingosine-1-phosphate (S1P), activate and stabilizeYAP and TAZ through their GPCRs, LPA receptor(LPAR) and S1P receptor (S1PR) (Yu et al. 2012). A seriesof studies further demonstrate that regulation of theHippo pathway by GPCRs is indeed a universal responseof cells to hormonal cues (Miller et al. 2012; Mo et al.2012; Yu et al. 2013; Gong et al. 2015; Zhou et al.2015a). Mechanistically, Rho-GTPases mediate the ac-tions ofGPCRs onYAPandTAZ.Gα12/13- andGαq/11-cou-pled GPCRs activate Rho-GTPases, which in turninactivate LATS1/2 by a yet unknown mechanism thatis dependent on F-actin assembly (Yu et al. 2012). In con-trast, activation of GαS-coupled GPCRs by epinephrineand glucagon increases LATS kinase activities and inacti-vates YAP and TAZ in a manner dependent on proteinkinase A (PKA) (Yu et al. 2013). Therefore, depending onthe nature of downstream G proteins, GPCRs can eitheractivate or inhibit the LATS kinase to stimulate or sup-press YAP activity. Elevated GPCR expression or muta-tion of Gα proteins leads to aberrant YAP activation andexhibits strong disease implications (Feng et al. 2014; Yuet al. 2014; Liu et al. 2015; Zhou et al. 2015a). For example,estrogen acts throughG protein-coupled estrogen receptor(GPER) to inhibit LATS and activate YAP/TAZ, indicat-ing a possible role of YAP/TAZ activation by estrogen inbreast cancer (Zhou et al. 2015a). GPCRs are the largestfamily of the plasma membrane receptors and mediatethe actions of hundreds of extracellular molecules (Lap-

pano and Maggiolini 2011). The regulation of YAP andTAZ by GPCRs implies that the Hippo pathway notonly is modulated by a large number of hormonal signalsbut also contributes to a wide range of physiological regu-lation and may be targeted for disease intervention withGPCR agonists or antagonists.Among GPCR ligands, the Wnt proteins, such as

Wnt5a/b, are particularly noteworthy. Wnt5a/b activatenoncanonical Wnt signaling by binding to the Frizzled re-ceptors, the class F GPCRs (Anastas and Moon 2013). Asboth the Hippo pathway and canonical Wnt signalingaremaster regulators of tissue growth andmorphogenesis,cross-talk between the two pathways has been extensivelystudied and thoroughly summarized by recent reviews(Piccolo et al. 2014; Hansen et al. 2015). It was recently re-ported that the noncanonical Wnt ligands Wnt5a/b acti-vate YAP/TAZ through the Gα12/13–Rho–LATS signalingaxis by binding to the Frizzled receptors (Park et al.2015). This regulation of YAP/TAZ by Wnt5a/b is indeedrequired for noncanonical Wnt signaling to function incell differentiation and migration as well as antagonizingthe canonical Wnt/β-catenin activation.

Stress signals

Themost recognized functional output of YAPandTAZ isto promote cell survival and proliferation (Huang et al.2005; Camargo et al. 2007; Dong et al. 2007). Therefore,it is not surprising that several stress signals canmodulateYAP and TAZ activities. However, the regulation of YAPand TAZ by stress signals, such as energy stress, endoplas-mic reticulum stress, and hypoxia, has been characterizedonly in the last couple of years, although activation ofMST1/2 by a high concentration of sodium arsenite orheat shock was observed a long time ago (Taylor et al.1996). MST1/2 are also activated by hydrogen peroxideand are involved in cellular oxidative stress responses(Lehtinen et al. 2006; Geng et al. 2015). On the otherhand, YAP physically interacts with FOXO1 and activatesFoxO1-mediated transcription of catalase and MnSODgenes and subsequently reduces oxidative stress and is-chaemia/reperfusion (I/R)-induced injury in the heart(Shao et al. 2014), implicating a physiological role ofYAP in reactive oxygen species (ROS) scavenging.Cells rely on carbohydrates as their main energy source.

Energy stress caused by glucose deprivation rapidly induc-es YAP and TAZ phosphorylation due to LATS1/2 activa-tion, which is enhanced by phosphorylation of AMOTL1at Ser793 by AMPK (DeRan et al. 2014). Furthermore, en-ergy stress-activated AMPK directly phosphorylates YAPat multiple sites, and this phosphorylation interfereswith the interaction between YAP and TEAD, thus inhib-iting TEAD-mediated gene transcription (Mo et al. 2015;Wang et al. 2015). The additional layer of YAP andTAZ regulation by AMPK is physiologically importantin the central brain/ventral nerve cord development inDrosophila neural systems (Gailite et al. 2015). Accessi-bility of nutrients other than glucose also affects the Hip-po pathway. For instance, the nutrient-sensing kinasessalt-induced kinase 2 and kinase 3 phosphorylate Sav at






Ser413 to promote Yki target gene expression (Wehr et al.2013). Both mTORC1 and mTORC2 are reported to posi-tively regulateYAP in perivascular epithelioid cell tumorsand glioblastomas (Liang et al. 2014; Artinian et al. 2015;Sciarretta et al. 2015). It is worth noting that mTORC1is highly sensitive to nutrient availability and cellular en-ergy status. In Drosophila, the Tor pathway can regulateYki’s ability to access its target genes in the nucleus(Parker and Struhl 2015). Tor inhibition by nutrient depri-vation prevents nuclear Yki from activating its targetgenes. Besides nutrient stress, inhibition of cholesterolsynthesis indirectly inhibits YAP, possibly due to inhibi-tion of the Rho familyGTPases, which require C-terminalisoprenylation andmembrane localization for their properbiological functions (Sorrentino et al. 2014). Therefore,the Hippo pathway is subjected to regulation by cellularnutrient status.

In contrast to oxidative stress and energy stress, hypoxiaseems to induce YAP and TAZ activation by inhibitingLATS. Hypoxia activates an E3 ubiquitin ligase, SIAH2,which destabilizes LATS2. Targeting SIAH2 in tumorcells restores the tumor suppressor function of LATS2 ina xenograft animal model (Ma et al. 2015). The regulationof YAP by the unfolded protein response (UPR) remainsconvoluted and seems to be more complicated than otherstresses. In the initial stage of UPR, YAP is activated byPERK–eIF2α. However, prolonged ER stress suppressesYAP (Wu et al. 2015). In fact, deletion ofMST1/2 inmouselivers triggers UPR and induces hepatocellular carcino-genesis, while attenuation of UPR by tauroursodeoxy-cholic acid causes degradation of YAP and reducestumor burden.

Cell polarity and architecture

In Drosophila, apical–basal polarity and planar cell polar-ity provide the intrinsic cues to restrict Yki activity in theepithelium. Many types of polarity machineries, such asadherens junctions, tight junctions, the Mer/Ex/Kibracomplex, Crumbs (Crb), the Par complex, and Fat/Dachs-ous, are involved in this action to maintain the differenti-ation and morphology of the epithelium in a partiallyredundantmanner. This subject has been comprehensive-ly reviewed elsewhere (Schroeder andHalder 2012; Yu andGuan 2013). Recent studies show that loss of Spectrin, acontractile protein at the cytoskeleton–membrane inter-face, also generates Hpo mutant-like tissue overgrowthphenotypes inDrosophilawings and eyes, which are like-ly due to dysregulated cytoskeleton tension (Deng et al.2015; Fletcher et al. 2015; Wong et al. 2015).

The restriction of Yki activity by cell polarity is causedby either the increased activity and the availability ofHpo/Wts toward Yki or sequestration of Yki at cell junc-tions (Yin et al. 2013; Yu and Guan 2013; Sun et al.2015). Mammalian cells have a very similar polarity ma-chinery for YAP/TAZ regulation. For example, PARD3regulates TAZ activity by promoting the LATS1 and pro-tein phosphatase 1 (PP1) interaction (Lv et al. 2015).Therefore, YAP/TAZ activity is low in terminally differ-entiated cells in the epithelium and preferentially present

in tissue progenitor cells in mammals (Camargo et al.2007; Cai et al. 2010). In addition, YAP/Yki activity is reg-ulated both autonomously and nonautonomously by api-cal–basal cell polarity proteins and adherens junctions,respectively (Yang et al. 2015a), indicating that differentsignal inputs may use different cell polarity complexesand junction proteins to regulate the Hippo pathway.

Cell cycle

LATS1/2 have been considered as regulators of G1/S, G2/M, and mitosis checkpoints and are phosphorylated in acell cycle-dependent manner in HeLa cells (Tao et al.1999). However, the intrinsic mechanisms by whichLATS1/2 are activated during the cell cycle are still un-clear, although a few kinases, such as CDK1 and AuroraA, have been shown to directly phosphorylate LATS1and LATS2, respectively, during mitosis (Morisaki et al.2002; Toji et al. 2004; Yabuta et al. 2011; Zhang et al.2012). It was recently reported that extra centrosomescaused by cytokinesis failure activate LATS2, which inturn stabilizes p53 and inhibits YAP/TAZ transcription-al activity (Ganem et al. 2014). LATS1 also interactswith CDK2 in response to genotoxic stress to restrictCDK2-mediated phosphorylation of BRCA2 and supportRAD51 nucleofilaments, thereby maintaining genome fi-delity during replication stalling (Pefani et al. 2014). YAP,in complex with the transcription factor PKNOX1, hasbeen shown to control S-phase temporal progression andgenomic stability of retinal stem cells (Cabochette et al.2015).

YAP and TAZ are phosphorylated at multiple sites byCDK1 during the G2/M phase of the cell cycle (Yanget al. 2013, 2015b,c; Zhao et al. 2014; Dent et al. 2015;Zhao and Yang 2015). However, the physiological out-comes of these phosphorylation events are rather perplex-ing, as their effects on cell growth and migration are notentirely consistent among reports from different groups.While some studies show that CDK1-mediated YAP phos-phorylation during the G2–M phase may promote neo-plastic transformation via enhancing cell migration andinvasion (Yang et al. 2013, 2015c), others suggest thatanti-tubulin drugs require YAP phosphorylation byCDK1 to induce cancer cell death (Zhao et al. 2014).This inconsistency may be due to different experimentalconditions that differentially affect the coordination be-tween CDK1 and LATS1/2 in the modulation of YAPactivity.

Mechanisms of Hippo kinase cascade activation

LATS1/2 belong to theNDR (nuclear Dbf2-related) familyof kinases, a subgroup of the protein kinase A/G/C (AGC)family (Pearce et al. 2010). The other two membersof the NDR family are NDR1 (STK38) and NDR2(STK38L). Several recent proteomic studies of the Hippopathway interactome consistently place NDR1/2 in theHippo pathway network (Couzens et al. 2013; Kwonet al. 2013; Wang et al. 2014). NDR1/2 might function

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as a YAP kinase to inhibit YAP-driven tumorigenesis inthe intestinal epithelium (Zhang et al. 2015). It is worthnoting that NDR1/2 and LATS1/2 share similar phos-phorylation motifs. However, the exact role of NDR1/2in Hippo pathway regulation still needs to be furtherdefined, as deletion of LATS1/2 is sufficient to abolishYAP phosphorylation and cause constitutive YAP nuclearlocalization under most conditions examined (Menget al. 2015).The NDR family kinases require phosphorylation of a

conserved Ser/Thr residue within the activation loopand the hydrophobic motif regulatory site for activation.Phosphorylation of the hydrophobic motif is mediatedby upstream Ste20-like kinases: MST1/2 for LATS1/2and NDR1/2 (Chan et al. 2005; Vichalkovski et al. 2008;Hergovich et al. 2009; Tang et al. 2015), and MST3 forNDR1/2 (Stegert et al. 2005). Recent studies have shownthat MAP4Ks, also members of the Ste20 family, canphosphorylate the LATS1/2 hydrophobic motif (Menget al. 2015; Zheng et al. 2015). The Ste20-like kinase-me-diated phosphorylation of the hydrophobic motif pro-motes LATS autophosphorylation in the activation loop,therefore leading to an increase of kinase activity (Tama-skovic et al. 2003; Stegert et al. 2004). Interestingly, the in-teraction between MOB proteins (the human genomeencodes six MOBs: MOB1A/B, MOB2, and MOB3A/B/C)and the N-terminal regulatory domain of the kinasesis a common feature of NDR family kinases, althoughLATS1/2 and NDR1/2 appear to use distinct subsets ofMOB proteins. MOB1A/B associate with both LATS1/2andNDR1/2,whileMOB2mediates an inhibitory interac-tion with NDR1/2 but not with LATS1/2 (Bichsel et al.2004; Bothos et al. 2005; Hergovich et al. 2005; Kohleret al. 2010).A recent crystal structure study provides newmolecular

insights into MOB1’s roles in LATS1/2 phosphorylation,and activation byMST1/2 as a sequential phosphorylationmodel is proposed (Fig. 3; Ni et al. 2015). MST2 autophos-phorylates its long linker between the kinase domainand the SARA domain to generate a phosphor-dockingmotif, which can recruit MOB1. The structure of theMOB1–phosphoMST2 complex shows that the bindingof MOB1 to the phosphorylated MST2 relieves MOB1from its autoinhibitory conformation and makes MOB1accessible to LATS1. Next, LATS1 binds to the MOB1–phosphoMST2 complex to form the MST2–MOB1–LATS1 ternary complex, thereby enhancing the phosphor-ylation of MOB1 at its N-terminal tail (T35 and T12) andLATS1 at its hydrophobic motif (T1079) by MST2. Phos-phorylation of T1079 in LATS1 by MST2 directly con-tributes to LATS1 activation, while phosphorylation ofMOB1 actually triggers the dissociation of phosphory-lated LATS1 and MOB1 from MST2. The structure ofthe phosphoMOB1 and LATS1 complex further revealsthat, in addition to mediating the actions of MST2 onLATS1, the phosphorylated MOB1 allosterically pro-motes LATS1 autophosphorylation at its activation loop(S909), which is required for LATS1 activation after its hy-drophobic motif, T1079, has been phosphorylated byMST2. This study reveals structural insights into the mo-

lecularmechanism of LATS1/2 activation byMST1/2 andthe critical role of MOB1 in this process.Moreover, another recent study using the crystal struc-

ture of the budding yeast homologs of NDR and MOB,Cbk1 and Mob2, shows that Mob2 binding to Cbk1 notonly promotes enzymatic activity of Cbk1 but also createsa docking motif for Cbk1 substrates (Gogl et al. 2015).This docking is crucial for robustness and substrate selec-tivity of Cbk1, which is unique among AGC family kinas-es, indicating a role of MOB in not only kinase activationbut also substrate specificity. One may speculate that asimilar mechanism is used in the activation of mammali-an MOB and NDR/LATS kinases.

The regulation of LATS-activating kinases MST1/2and MAP4Ks

The LATS1/2-dependent phosphorylation appears to bethe most important event in YAP/TAZ regulation inmammals, as LATS1/2 knockout cells abolish most, ifnot all, YAP/TAZ phosphorylation in response to manyknown regulatory signals of the Hippo pathway (Menget al. 2015).We and others have recently reported that MST1/2 and

MAP4Ks act in parallel to phosphorylate and activateLATS1/2, and deletion of bothMST1/2 andMAP4Ks is re-quired to abolish LATS1/2 hydrophobic motif phosphory-lation and activation (Li et al. 2014a; Meng et al. 2015;Zheng et al. 2015). Regulation of MST1/2 kinase activityhas been extensively studied. MST1/2 requires phosphor-ylation of the activation loop to be fully active, which canbe achieved by transphosphorylation by TAOK1/2/3 orautophosphorylation by MST dimerization (Glantschniget al. 2002; Praskova et al. 2004; Boggiano et al. 2011;Poon et al. 2011). A few other kinases, such as AKT,ABL, and mTOR, may phosphorylate MST1/2 at multiplesites and modulate kinase activity of MST1/2 by differentmechanisms (Jang et al. 2007; Yuan et al. 2010; Collaket al. 2012). However, the role of MST1/2 regulation bythese kinases in the Hippo pathway has not been impli-cated. The STRIPAK complex, the core of which isPP2A, interacts with MST1/2 and may contribute toMST1/2 dephosphorylation in some contexts (Couzenset al. 2013). However, this regulation by STRIPAK still re-quires further studies because it is unknown whether theinteraction or activity of STRIPAK is regulated by signalsthat are known to control the Hippo pathway.In addition to interacting with MOB1, MST1/2 also

directly interact with SAV1 and RASSFs. SAV1 is also asubstrate of MST1/2 and is stabilized by MST1/2 phos-phorylation (Callus et al. 2006). It mainly works as a scaf-fold protein to bridgeMST1/2 to LATS1/2. It has not beenshown whether SAV1 directly affects MST1/2 kinase ac-tivity. The functional role of RASSFs in MST1/2 regula-tion can be either positive or negative and may dependon the status of MST1/2 (Khokhlatchev et al. 2002; Pras-kova et al. 2004; Guo et al. 2007, 2011). Nevertheless, itis evident that RASSFs disrupt MST1/2 dimerizationand prevent their autophosphorylation. However, interac-tion of RASSFs with already activated MST1/2 may






prevent MST1/2 dephosphorylation and therefore sustainMST1/2 kinase activity (Guo et al. 2011; Ni et al. 2013).

In Drosophila, the Rho-type guanine nucleotide ex-change factor Pix (PAK-interacting exchange factor) andGPCR kinase-interacting protein (Git) have also been sug-gested to influenceHpo kinase activity by facilitatingHpodimerization and autophosphorylation (Dent et al. 2015).However, a mammalian homolog of Pix, ARHGEF7, mayfunction as a scaffold protein between LATS1/2 and YAP/TAZ to facilitate actions of theHippo kinase cascade (Hei-daryArash et al. 2014). Therefore, the regulation ofMST israther complex, and future studies are needed to provide aclear biochemical understanding of MST activation in re-sponse to various upstream signals.

On the other hand, the regulation of MAP4Ks has notbeen extensively studied. MAP4Ks as well as MST1/2can be cleaved byCaspase 3/6/7 upon Fas-induced apopto-

sis. The cleaved kinase domain is active and may activatethe JNK pathway (MEKK1–MKK4/7–JNK1/2) or thep38 pathway (MAP3K–MKK3/6–p38MAPK) (Dan et al.2001). However, the cleaved MAP4Ks and especiallyMST1/2 have lost certain domains—such as the coiled-coil and SARAH domains—that are essential for their in-teraction with SAV1 or LATS1/2 (Dan et al. 2001). There-fore, the caspase-dependent MAP4Ks and MST1/2activation may not be relevant to the Hippo pathway. AfewMAP4Ks, includingMAP4K1/4/6, are known to inter-act with NCK1 (noncatalytic region of tyrosine kinaseadaptor protein 1), which is an adaptor protein containingSrc homology 2 (SH2) and SH3 domains (Su et al. 1997;Ling et al. 1999, 2001; Hu et al. 2004). NCK1 is locatedin the cytoplasm and is involved in Ras-GTPase activa-tion by receptor tyrosine kinases (Ger et al. 2011) aswell as Rho-GTPase activation and actin cytoskeleton

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Figure 3. A proposed sequential phosphorylation model of LATS activation byMST. First, upon activationMST1/2 autophosphorylatesits linker region (which is between the catalytic domain and the SARAH domain) at multiple sites to create a phosphor-docking site forMOB1. Second, phosphorylated MST binds to MOB1 and changes MOB1 from an autoinhibitory state to a conformation that is open forLATS binding. Third, LATS binds toMOB1 potentially through recruitment byNF2 to theMST–SAV1 complex at the plasmamembrane.Fourth, the formation of the MST–MOB1–LATS complex enables MST to phosphorylate MOB1’s N-terminal tail at Thr12 (T12) andThr35 (T35) and phosphorylate LATS at its hydrophobic motif (HM). Fifth, the phosphorylated N-terminal tail of MOB1 competeswith the phosphoMST linker for the same binding site on MOB1 and thus releases MOB1 and LATS from MST. Sixth, phosphoMOB1allosterically enhances LATS autophosphorylation at its activation loop (AL) and thus promotes LATS activation.

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remodeling (Ruusala et al. 2008; Buvall et al. 2013). Giventhe important role of Rho-GTPases and actin cytoskele-ton in Hippo regulation, it would be important to investi-gate whether NCK1 relays the signals of growth factors,cytoskeleton, and mechanotransduction to LATS1/2through MAP4Ks.

Spatial regulation of MST and LATS

Unlike the large changes in kinase activity of LATS1/2upon stimulation, kinase activity of MST1/2 does not ap-pear to be dramatically altered under conditions that areknown to affect the Hippo pathway. Instead, the accessi-bility of MST1/2 to LATS1/2 may be a key factor forLATS1/2 activation by MST1/2.Models of spatial regulation of LATS1/2 kinase activi-

ties were proposed a long time ago and have been furtherrefined in the last few years. Early studies have shownthat Hpo/Sav interact with Mer/Ex and that Wts associ-ates with Kibra (Genevet et al. 2010; Yu et al. 2010).This interaction suggests that the Mer/Ex/Kibra complexmay recruit Hpo and Wts to the apical plasma membraneand results in Wts phosphorylation by Hpo. This model isalso supported by evidence that membrane targetingMST1/2 or MOB1 greatly elevates the kinase activitiesof LATS1/2 in mammalian cells (Khokhlatchev et al.2002; Hergovich et al. 2006). An updated model proposesthat LATS1/2 and MST1/2 are cytoplasmic in theirinactive state and are recruited by NF2 and SAV1, respec-tively, to plasma membrane, where LATS1/2 are phos-phorylated and activated by MST1/2 (Yin et al. 2013).However, a recent study inDrosophila suggests that inac-tive Wts is localized at adherens junctions through Jub,and Wts and Hpo are relocated to Crb–Ex apical junctionsto induce Wts phosphorylation and activation (Sun et al.2015). It is noteworthy that Crb3, a mammalian Crumbsisoform that determines epithelial apical domain identity,also promotes the interaction between YAP and LATS1/2at apical cell junctions to induce YAP phosphorylationand thereby control airway cell differentiation (Szyma-niak et al. 2015). Therefore, an appealingmodel is that spa-tial regulation by NF2-dependent recruitment plays a keyrole in LATS1/2 activation.

Regulation of LATS1/2 protein levels by ubiquitinationand beyond

LATS1/2 activities are regulated through various meansbeyond phosphorylation (Fig. 4). One notable post-tran-slational regulation of LATS1/2 is ubiquitination. AWW domain-containing HECT class E3 ubiquitin ligase,ITCH, ubiquitinates LATS1 and promotes cell growthand survival (Ho et al. 2011; Salah et al. 2011). AnotherE3 ubiquitin ligase, CRL4 (DCAF1), which is activatedin NF2-deficient tumor cells, inhibits LATS1 and LATS2by ubiquitination in the nucleus (Li et al. 2014b). Ubiqui-tination of LATS1/2 also plays a role in stress responseand differentiation. For instance, a hypoxia-activated E3ubiquitin ligase, SIAH2, destabilizes LATS2 and promotesYAP activation and tumorigenesis (Ma et al. 2015).

NEDD4, another E3 HECT ubiquitin ligase, ubiquitinatesand destabilizes both LATS2 and SAV1 and thus activatesYAP to enhance intestinal stem cell self-renewal (Baeet al. 2015). In fact, ubiquitination of other Hippo pathwaycomponents has also been reported (Wang et al. 2012; Lig-nitto et al. 2013; Rodrigues-Campos andThompson 2014),indicating that ubiquitination is a common regulatorymechanism often resulting in degradation of the Hippopathway components and hyperactivation of YAP andTAZ.LATS is also regulated at the transcriptional level.

LATS2 is a direct target gene of YAP/TAZ, and LATS2mRNA levels are increased upon YAP/TAZ activation(Chen et al. 2015b; Moroishi et al. 2015b). This LATS2up-regulation constitutes a negative feedback loop tomaintain the homeostasis of the Hippo pathway and pre-vent overactivation of YAP/TAZ. In addition, LATS1/2are regulated by aurora kinase-mediated or PKA-mediatedphosphorylation (Toji et al. 2004; Kim et al. 2013) or phys-ical interaction with ARHGEF7, ZYXIN, AMOT, andLIMD1 (Hirota et al. 2000; Sun and Irvine 2013; HeidaryArash et al. 2014; Li et al. 2015). These differential regula-tions of LATS1/2 serve as additional layers of control, inconcurrencewithMST1/2-mediated andMAP4K-mediat-ed protein phosphorylation, to modulate cell survival andgrowth.

The YAP/TAZ transcriptional programsand their functional output

Studies ofDrosophila andmousemodels have establishedthe role of YAP/Yki in regulating tissue progenitor cellself-renewal and expansion, especially in gastrointestinaltissues (Cai et al. 2010; Barry et al. 2013; Gregorieff et al.2015; Imajo et al. 2015; Taniguchi et al. 2015; Yimlamaiet al. 2015). Although earlier transgenic mouse studieshave shown striking phenotypes and established a roleof the Hippo pathway in development and carcinogenesis(Morin-Kensicki et al. 2006; Camargo et al. 2007; Donget al. 2007; Yabuta et al. 2007), many more refined trans-genic mouse models with tissue-specific deletion and in-ducible overexpression have been generated in the lastfew years. These mouse model studies allow for more de-tailed charaterizations of the physiological contributionof individual Hippo pathway components to tissuegrowth, cell differentiation, cell competition, and malig-nant transformation (Zhou et al. 2009; Cai et al. 2010; Bar-ry et al. 2013; Chen et al. 2014, 2015b; Yimlamai et al.2014; Imajo et al. 2015; Mamada et al. 2015; Shen et al.2015).An important function of the Hippo pathway seems to

be the inactivation of YAP and TAZ in differentiated cellsto maintain cell quiescence (Zhou et al. 2009; Yu et al.2015). Upon tissue injury, the Hippo pathway is sup-pressed, and YAP and TAZ are activated to promotestem/progenitor cell self-renewal and tissue repair (Fig.5; Cai et al. 2010; Schlegelmilch et al. 2011; Gregorieffet al. 2015; Taniguchi et al. 2015). Consistently,woundingof in vitro cultured cells dramatically activates YAP, and






the highly nuclear YAP drives cell migration and prolifer-ation to promote wound healing (Zhao et al. 2007, 2008;Lee et al. 2014). Both basic and clinical cancer researchhas implicated a role of YAP and TAZ in cancer initiationand development through suppressing cell apoptosis andpromoting cell prolifieration (Moroishi et al. 2015a). Con-cordantly, YAP and TAZ have been considered as thera-peutic targets for a number of cancers (Liu-Chittendenet al. 2012; Jiao et al. 2014; Yu et al. 2014; Zhou et al.2015b) as well as several other diseases (Plouffe et al.2015). Notably, the R331W missense mutation of YAPhas recently been linked to a germline risk in lung adeno-carcinoma (Chen et al. 2015a). Moreover, almost all epi-thelioid hemangioendotheliomas contain gene fusions ofTAZ–CAMTA1, TAZ–FOSB, or YAP–TFE3 (Tanas et al.2011; Antonescu et al. 2013; Patel et al. 2015), stronglysupporting a role of YAP/TAZ in human tumorigenesis.

The function of YAP and TAZ is believed to be mainlymediated through TEAD1–4, as YAP and TAZ do not bindto DNA directly and act as transcriptional coactivators of

TEAD1–4 (Zhao et al. 2008), although YAP and TAZ havebeen also reported to associate with several other tran-scription factors (Hansen et al. 2015).Many genes are tran-scriptionally activated by TEAD complexed with YAPand/or TAZ (Zhu et al. 2015). Mechanistically, YAP andTAZ stimulate TEAD transcriptional activity by recurit-ing components of the SWI/SNF chromatin remodelingcomplex or NCOA6 histone methyltransferase complex(Oh et al. 2013, 2014; Qing et al. 2014; Skibinski et al.2014). Interestingly, the YAP/TAZ–TEAD complex canalso operate as transcriptional corepressors by recruitingthe NuRD histone deacetylate complex for additional tar-get genes, such as DDIT4 and Trail (Kim et al. 2015) orΔNp63 (Valencia-Sama et al. 2015). Further studies areneeded to show the generality of YAP/TAZ as transcripi-tional repressors. Nevertheless, nuclear YAP/TAZ can ei-ther induce or repress gene expression.

Recent efforts to elucidate the genome-wide action ofYAP/TAZ through deep sequencing have led to some un-anticipated features of YAP and TAZ in transcriptional

PUBA PPxYPPxY PBD HM

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Figure 4. Domain structure and proteininteraction of LATS1/2. Kinase activityof LATS1/2 is primarily regulated byMST1/2 and MAP4Ks through the hydro-phobic motif phosphorylation followed byautophosphorylation of the activationloop of LATS1/2. There is a putative ubiq-uitin-associated domain (UBA) in the Ntermini of the kinases, and several E3ubiquitination ligases, such as ITCH,SIAH-2, NEDD4, and DCAF1, are knownto regulate LATS1/2 protein stability.The binding of MOB, ZYXIN, LIMD1with the protein-binding domain (PBD)can also regulate LATS1/2 kinase activityor availability. YAP and TAZ interactwith LATS1/2 through the PPxY motifsof LATS1/2. Aurora kinases A/B phos-

phorylate LATS2 and affect its subcellular locations. (PA repeat) Proline–alanine residue repeat; (AL) activation loop; (HM) hydropho-bic motif.

Survivale.g. Survivin, Bcl2

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Figure 5. The transcription programof theHippo pathway. YAP and TAZ bind toTEAD at promoters or enhancers of the tar-get genes to regulate transcription activa-tion or pause release. The TEAD targetgenes are involved in a variety of physio-logical processes, such as cell survival, pro-liferation, differentiation, migration, andinvasion.

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regulation. In Drosophila, Yki binds to promoter regionsto mediate transcriptional activation (Oh et al. 2013;Ikmi et al. 2014). Similary, the transcriptional functionsof YAP/TAZ have been previously associated with thebinding of TEADs at the promoters of target genes (Lianet al. 2010). However, ChIP-seq (chromatin immunopre-cipitation [ChIP] combined with deep sequencing) studiesin cancer cells (breast cancer, glioblastoma, cholangiocar-cinoma, and malignant mesothelioma) as well as non-transformed cells (IMR90) have revealed that themajority of YAP/TAZ and TEAD binds to distal enhancerregions to induce gene transcription (Galli et al. 2015;Stein et al. 2015; Zanconato et al. 2015). Through denovo motif analyses at YAP/TAZ peaks, Stein et al.(2015) and Zanconato et al. (2015) confirmed early obser-vations that YAP/TAZ mainly interact with TEAD tobind DNA (Zhao et al. 2008). However, these two inde-pendent studies also identified that the consensus motiffor AP-1 transcription factors is significantly enriched inthe YAP-binding regions (Stein et al. 2015; Zanconatoet al. 2015), suggesting that YAP/TAZ–TEAD cooperatewith AP-1 to synergistically activate target genes. AP-1is a heterodimeric protein complex composed of JUNand FOS families of leucine zipper proteins (Eferl andWag-ner 2003). TEADs appear to mediate the interaction withAP-1 (Zanconato et al. 2015). Supporting the role of AP-1in YAP/TAZ/TEAD-mediated transcription, YAP/TAZ-induced MCF10A mammary epithelial cell growth is en-hanced by AP-1, while AP-1-driven skin tumorigenesisis blunted by YAP/TAZ depletion (Zanconato et al.2015). Galli et al. (2015) found that YAP associates withtheMediator complex to recruit CDK9 elongating kinase,mediating transcriptional pause release. Consistently, ad-ministration of the CDK9 kinase inhibitor flavopiridolprevented YAP-driven hepatomegary in mice (Galli et al.2015).A model for YAP/TAZ in gene expression has emerged.

YAP/TAZ bind to DNA mainly via TEAD. However, themajority of YAP/TAZ proteins appear to bind to distal en-hancers, although some YAP/TAZ proteins bind to pro-moters to induce target gene expression. YAP/TAZcoorporate with AP-1 and/or recruit additional regulatorsto stimulate de novo transcription initiation and enhancetranscription elongation, thereby increasing target geneexpression. Given the role of YAP and TAZ in controllingstem/progenitor cells in development and tissue homeo-stasis, it would be informative to perform ChIP-seq analy-ses of YAP/TAZ and TEAD in stem cells or primary tissueprogenitor cells in order to gain new insights into how theYAP/TAZ-mediated transcription program coordinatesthe expression of multiple downstream genes to controltissue development and homeostasis.

Redefining the Hippo pathway

TheHippo pathway is named after theDrosophilaHpo ki-nase. The Hpo–Wts kinase cascade constitutes the axis ofthe Hippo pathway inDrosophila, and the functional out-puts are exclusively mediated by Yki. To date, other thanWts, Sav, and Mats, there have been few reports on Hpo

substrates in Drosophila. However, in mammalian cells,the functional outputs of MST1/2 are not limited toYAP/TAZ.MST1/2 are known to phosphorylate a numberof other “non-Hippo” proteins in addition to LATS1/2,SAV1, and MOB1A/B. For instance, NDR1/2 are reportedto be MST1/2 substrates that regulate thymocyte egressand T-cell migration (Vichalkovski et al. 2008; Tanget al. 2015). MST1/2 can phosphorylate FOXO1 to pro-mote its nuclear localization and transcription of genespromoting apoptosis in mammalian neurons (Lehtinenet al. 2006). The apoptotic and functional roles of MST1in pancreatic β cells also appear to be independent ofLATS1/2 but rely on PDX1 phosphorylation by MST1and JNK (Ardestani et al. 2014). Similarly, LATS1/2 arenot involved in PRDX1 phosphorylation and inactivationby MST1 in hydrogen peroxide-treated cells (Rawat et al.2013). In addition to their roles in cell death and stressresponses,MST1/2 affect autophagy by directly phosphor-ylating Beclin 1 and LC3, although it is still unclearwhether MST1 promotes or inhibits autophagy (Maejimaet al. 2013; Wilkinson et al. 2015). A few other proteins,such as H2B histone proteins and VASP, are reported asMST1/2 substrates (Cheung et al. 2003; Nishikimi et al.2014). Therefore, MST1/2 have broad functions in addi-tion to regulating core Hippo pathway components ofLATS1/2 and YAP/TAZ.Conversely, LATS1/2 and YAP/TAZ can be regulated

even in the absence of MST1/2. In Drosophila, Hppy canphosphorylate Wts at the hydrophobic motif of Wts. Infact, both Hppy and Msn activate Wts kinase activitiesand inhibit Yki transcriptional activity, as does Hpo (Liet al. 2014a;Meng et al. 2015; Zheng et al. 2015). Hppy ho-mologs (MAP4K1/2/3/5) and Msn homologs (MAP4K4/6/7) can directly phosphorylate and activate LATS1/2 andappear to have a more important function than MST1/2in the regulation of LATS1/2 and YAP/TAZ in mammali-an cells in response to several upstream signals (Menget al. 2015). On the contrary, LATS1/2 are essential for reg-ulation of YAP and TAZ under most conditions tested,whereas MST1/2 are not. Therefore, the definition of theHippo pathway may need to be redefined. AlthoughMST1/2 are the mammalian homologs of the DrosophilaHpo, not all proteins and functions that are regulated byMST1/2 should be defined as the Hippo pathway. Onthe other hand, the functional output of YAP/TAZ andproteins that specifically regulate LATS1/2 kinase activi-ty and/or YAP/TAZ transcriptional activity should beconsidered as the Hippo pathway. This definition willmore accurately describe the actions of the Hippo path-way in response to extracellular and intracellular stimuliand their physiological outcomes.

Acknowledgments

Weapologize for those primaryworks that are not cited due to thescopeof this reviewand space constraints.We thankKimberlyLinand Steven Plouffe for critical reading of this manuscript. Re-search in the laboratory of K.-L.G. is supported by grants fromthe National Institutes of Health (CA196878, GM51586, andEY22611).






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Zhipeng Meng, Toshiro Moroishi and Kun-Liang Guan Mechanisms of Hippo pathway regulation

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