Acta Biochim Biophys Sin, 2015, 47(1), 29–38 doi: 10.1093/abbs/gmu107 Advance Access Publication Date: 4 December 2014 Review

Review

Structural dissection of Hippo signaling Zhubing Shi1,2, Shi Jiao1, and Zhaocai Zhou1,* 1

*Correspondence address. Tel/Fax: +86-21-54921291; E-mail: [email protected] Received 16 September 2014; Accepted 26 October 2014

Abstract The Hippo pathway controls cell number and organ size by restricting cell proliferation and promoting apoptosis, and thus is a key regulator in development and homeostasis. Dysfunction of the Hippo pathway correlates with many pathological conditions, especially cancer. Hippo signaling also plays important roles in tissue regeneration and stem cell biology. Therefore, the Hippo pathway is recognized as a crucial target for cancer therapy and regeneration medicine. To date, structures of several key components in Hippo signaling have been determined. In this review, we summarize current available structural studies of the Hippo pathway, which may help to improve our understanding of its regulatory mechanisms, as well as to facilitate further functional studies and potential therapeutic interventions. Key words: Hippo pathway, three-dimensional structure, protein–protein/DNA interaction, regulatory mechanism

Introduction Multiple signaling pathways participate in the development and tissue homeostasis, and their aberrant regulation can cause various diseases such as cancer. Among them, the Hippo pathway is an emerging regulator of tissue growth and cell fate [1–6]. The Hippo pathway regulates cell number and organ size through inhibiting cell growth and proliferation, as well as promoting cell death. Core components of the Hippo pathway were firstly identified in Drosophila by genetic screen. The activation of Hippo (Hpo), Salvador (Sav), Warts (Wts), or Mps one binder kinase activator-like 1 (Mob1; also known as Mob as tumor suppressor protein 1 or Mats) causes overgrowth of Drosophila tissues [7–16]. Hpo, Sav, Wts, and Mob1 form a kinase cascade, in which the Hpo–Sav complex phosphorylates Wts and Mob1, and then activated Wts–Mob1 complex further phosphorylates and inactivates transcriptional coactivator Yorkie (Yki) [11,16–22]. The Hippo pathway is highly conserved in mammals, and its corresponding components are mammalian sterile 20 (STE20)-like protein kinase 1/2 (MST1/2), Salvador homolog 1 (SAV1, also known as 45 kDa WW domain protein or WW45), large tumor suppressor homolog 1/2 (LATS1/2), MOB kinase activator 1A/B (MOB1A/B), and Yesassociated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ, also known as WW domain-containing

transcription regulator protein 1 or WWTR1) [19,23,24]. Phosphorylated Yki, YAP, or TAZ is recruited by 14-3-3 proteins and therefore sequestered in cytoplasm [23–25]. Then phosphorylated YAP/TAZ by LATS1/2 are further phosphorylated by casein kinase I isoforms δ/ɛ (CK1δ/ɛ) and degraded by proteasome dependent on F-box/WD repeat-containing protein (β-TrCP) [26,27]. In the absence of Hippo signaling, unphosphorylated Yki, YAP, or TAZ translocates to nucleus and acts as transcriptional coactivator to activate Scalloped (Sd in Drosophila)-dependent or TEA domain family member 1 to 4 (TEAD1–4 in mammal, also known as TEF-1, TEF-4, TEF-5, and TEF-3, respectively)-dependent transcription of pro-proliferative and antiapoptotic genes. Many regulators of the Hippo pathway have been identified [28– 31]. The protein components involved in the apical-basal polarity, such as neurofibromin-2, kidney and brain protein (KIBRA), crumbs homolog (CRB), angiomotin (AMOT), scribble homolog (SCRIB), and disks large homolog (DLG), regulate the Hippo pathway via modulating subcellular localization of Hippo components. Planar cell polarity can activate or suppress Hippo signaling. In Drosophila, Fat (Ft) and Dachsous (Ds) affect Wts and Yki activity. Mechanical cues sensed by extracellular matrix and cytoskeleton can influence YAP/TAZ activity [32]. Other regulators of the Hippo pathway

© The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 29

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National Center for Protein Science Shanghai, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China, and 2 School of Life Sciences and Technology, Tongji University, Shanghai 200092, China

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Core Kinase Cascade MST1/2–SAV1/RASSFs complex MST1/2 kinases belong to STE20 germinal center kinase (GCK) II subfamily. They consist of an amino (N)-terminal kinase domain, an inhibitory region and a carboxy (C)-terminal SARAH (Sav/Rassf/ Hpo) domain [36,37]. SAV1 contains two WW domains and a SARAH domain in the C-terminal region [38]. RASSF family contains 10 members, namely RASSF1 to RASSF10 [39,40]. RASSF5 is also known as new Ras effector 1 (NORE1) and regulator for cell adhesion and polarization enriched in lymphoid tissues (RAPL). They share a conserved Ras association (RA) domain in their N- or C-terminal regions. A SARAH domain is present in the C-terminal regions of RASSF1–6, but not in those of RASSF7–10. The SARAH domain mediates the homo- and heterodimerization of MST1/2, SAV1, and RASSFs [19,36,37,41–43]. The kinase domains of MST1/2 adopt a canonical kinase fold, with N- and C-lobes (Fig. 1A) [44]. The solved structure of MST1 kinase domain exhibits an active conformation (Protein Data Bank [PDB] code: 3COM). Its activation loop is phosphorylated at residues Thr177 and Thr183 and adopts an extended conformation, with the phosphate group of phosphorylated Thr183 forming electrostatic interaction with Arg148 in the His-Arg-Asp (HRD) motif and Arg181. The residue Thr183 in the activation loop is a primary autophosphorylation site, and may be autophosphorylated in a similar manner observed for other GCK members, such as MST4 and oxidative stress-responsive 1 [45–47]. In contrast, the structure of MST2 kinase domain, in which residue Asp146 in the DFG (Asp-Phe-Gly) motif is mutated to asparagine, adopts an inactive conformation (PDB code: 4LG4) [44]. The activation loop of MST2 is unphosphorylated and folds into a helix. Both MST1 and MST2 kinase domains form homodimers in the crystals, which are mainly mediated by the αEF and αG helices in their C-lobes. In the inactive MST2 kinase domain, the activation loop is also involved in the dimerization. However, the relative orientations of two monomers are distinct in MST1 and MST2 homodimers, which may be caused by crystal packing. Although mutations in the dimeric interface of MST2 kinase domain do not affect its

dimerization in solution, MST2 kinase activity is indeed impaired in some cases, suggesting that the dimeric conformation is at least partially required for MST2 activation [44]. The inhibitory domain between the kinase domain and the SARAH domain of MST1/2 is an intrinsically unfolded region and structurally disordered [44,48,49]. This region contains caspase recognition sites [50]. Caspase cleavage removes the inhibitory domain of MST1/2, which leads to MST1/2 activation in apoptosis. Unexpectedly, the MST1 inhibitory domain increases the thermodynamic stability of its SARAH domain dimer and the homodimer affinity [49]. However, the inhibitory mechanism of the MST1/2 inhibitory domain is still poorly understood. The SARAH domain of MST1/2 mediates its homodimerization and heterodimeric interactions with other SARAH domain-containing proteins such as SAV1 and RASSFs [19,36,41–43]. The SARAH domain-mediated homodimerization of MST1/2 is required for their full activation [51]. The monomeric state of MST1/2 SARAH domain is thermodynamically unstable and its dimerization has been coupled with structural folding [49,52]. The structures of MST1/2 and RASSF5 SARAH homodimers or heterodimers have been determined (Fig. 1B) [44,48,52–54]. The SARAH domain of MST1/2 or RASSF5 alone forms a symmetric homodimer (PDB codes: 2JO8, 2YMY, 4HKD, 4L0N, 4NR2, and 4OH9). Each monomer of MST1/2 SARAH homodimer contains two helices, H1 and H2, while that of RASSF5 has only one helix, corresponding to the H2 helix of MST1/2 SARAH. In one type of heterodimeric interactions, the RASSF5 SARAH and the H2 helix of MST1/2 SARAH fold into antiparallel coiled coil. In general, most coiled coils are structurally organized as multiple ‘heptad repeat (a–g positions)’. However, the heptad register is interrupted by two stutters in the MST1/2 and RASSF5 SARAH domains, and thus the coiled coils have two transitions in periodicity from heptad (a–g) to tetrad (d–g), resulting in a low degree of supercoiling. Each H1 helix of MST1/2 SARAH contacts H2 helices from the two monomers. Hydrophobic interactions dominate the SARAH domain homo- or heterodimerization, while electrostatic interactions and hydrogen bonds further stabilize dimeric interfaces. Although the overall structures of these SARAH domains are similar, their local conformations vary, even in different structures of the same SARAH domain, implying a dynamic nature of the SARAH domain. Sequence conservation indicates that these structural features exist in all SARAH domains [52,54]. SAV1 binding enhances MST1/2 kinase activity, while the influence of RASSFs on MST1/2 activity is uncertain [41–43,51,55,56]. RASSF5 suppresses MST1 kinase activity in vitro, whereas it appears to promote MST1 activation in mouse pre-B cell line BAF cells [42,43,51]. The SARAH domain of MST1/2 forms heterodimer with that of RASSF5 in a manner similar to their homodimerization (Fig. 1B) (PDB codes: 4LGD and 4OH8) [44,53,57]. The interface of MST1/2–RASSF5 heterodimer also resembles those of their homodimers, but the heterodimers have more intermolecular hydrogen bonds and more extensive hydrophobic interactions than the homodimers, which makes them more stable than the homodimers, as supported by results from urea-induced denaturation experiment [53]. Therefore, SARAH domains function as a universal dimerization module to form homodimer or heterodimer with a preference for heterodimer. The SAV1 SARAH domain is expected to adopt a similar structural fold when forming homodimer or heterodimer with MST1/2. In this regard, the differential effects of SAV1 and RASSFs on MST1/2 activation may be caused by recruiting distinct partners and are dependent on different cellular contexts. Besides the SARAH domain, all RASSFs have a RA domain, which binds to Ras protein in a GTP-dependent manner [58,59]. In the

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include Ras association domain-containing proteins (RASSFs), G protein-coupled receptor (GPCR), thousand and one amino acid protein kinase 1 (TAO1), microtubule-associated protein/microtubule affinity-regulating kinase 1 (MARK1, also known as PAR1), saltinducible kinase 1 (SIK1), ankyrin repeat and KH domain-containing protein 1 (MASK), serine/threonine protein phosphatase 2A (PP2A). Some regulators of the Hippo pathway are also important components of other signaling pathways, indicating extensive crosstalk between Hippo and these pathways [33]. For example, several components of Wnt signaling, including Dishevelled (DVL) and β-catenin, are associated with YAP/TAZ to regulate their localization, degradation, and activity [34]. YAP/TAZ cooperate with SMAD family members to maintain stem cell pluripotency or induces specific stem cell differentiation [35]. During the past decade, growing biochemical and structural studies were carried out to better understand the nature of the Hippo signaling transduction. A prominent example is that several important modular protein domains, especially the WW domain and its Pro-Pro-Xaa-Tyr (PPxY) motif, are revealed to widely mediate protein–protein interactions in the Hippo pathway. Here, we summarize the structural studies of the Hippo core kinase complexes and TEAD transcriptional complexes and discuss the regulatory mechanism derived from their structural aspect.

Structural dissection of Hippo signaling

Structural dissection of Hippo signaling

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structure of RASSF5 RA domain in complex with Ras, Ras binds a GTP analog guanyl-5′-yl imidodiphosphate (GppNHp) and a magnesium ion (PDB code: 3DDC) [60]. The RASSF5 RA domain has a ubiquitin fold similar to Ras binding domains (RBDs) of other Ras effectors, but contains additional N-terminal region and an insertion between β1 and β2. The switch I region of Ras forms an intermolecular β-sheet with the strand β2 of RASSF5 RA domain. Unlike other Ras effectors, the N-terminus of RASSF5 RA domain contributes to extra contact with the switch II region of Ras via hydrophobic interactions. RASSF1 and RASSF5 have a C1 domain in the N-terminal part, which is a phorbol-ester/diacylglycerol (DAG)-type zinc finger. The C1 domain of RASSF5 consists of a distorted five-stranded, antiparallel β-sheet with a helix (PDB codes: 1RFH and 2FNF) [61]. Two zinc ions exist in the RASSF5 C1 domain, and each zinc ion is coordinated by three cysteines and one histidine to stabilize the C1 structure. The RASSF5 C1 domain can form an intramolecular interaction with the

RA domain, and Ras-GTP binding to the RA domain disrupts this interaction and displaces the C1 domain. The free C1 domain may bind phosphatidylinositol 3-phosphate to localize toward membrane. SAV1 has two WW domains, WW1 and WW2, which may bind to LATS1/2 [15]. The WW domain was named so as it contains two conserved tryptophan residues [62]. Like typical WW domain, the SAV1 WW1 domain is a monomer consisting of a triple-stranded antiparallel β-sheet (PDB code: 2YSB) (Fig. 2A). The WW1 domain of SAV1 may associate with PPxY motifs to mediate its interaction with other proteins (see the section ‘LATS–YAP/TAZ complex’), but the specific partner has yet to be identified. The second conserved tryptophan residue important for partner binding is substituted by a tyrosine in the SAV1 WW2 domain and this tyrosine residue is evolutionarily conserved [63]. Although the SAV1 WW2 domain has a WW-like fold, unlike the canonical monomeric WW domain, it forms a symmetric β-clam-like homodimer (PDB code: 2DWV) [63]. The molecular surface corresponding to that of the canonical WW domain used for

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Figure 1. Structures of MST1/2 kinases and their complexes with RASSF5 (A) Structures of MST1/2 kinase domains. MST1/2 kinase domains form homodimers. (B) Structures of MST1/2 and RASSF5 SARAH homodimers and heterodimers. Following structures in PDB were used: 3COM (MST1_KD), 4LG4 (MST2_KD), 4NR2 (MST1_SARAH), 4HKD (MST2_SARAH), 2YMY (RASSF5_SARAH), 4OH8 (MST1-RASSF5_SARAH) and 4LGD (MST2-RASSF5_SARAH). Structure images were generated by PyMOL (www.pymol.org). KD represents kinase domain.

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Structural dissection of Hippo signaling

Figure 3. Structures of MOB1 (A) Structures of human MOB1 and yeast Mob1p (PDB codes: 1PI1 and 2HJN). (B) Structure of yeast Mob2p in complex with Cbk1p (PDB code: 4LQP). (C) Structure of human MOB1 in complex with a phosphor-peptide (PDB code: 4JIZ).

binding partners mediates homodimerization in the WW2 domain, which results from the presence of hydrophobic residues, such as Val244, Ala261, and Phe249 and a residue deletion between

Gly250 and Thr251 in the WW2 domain. The function of the SAV1 WW domains, particularly the dimeric WW2 domain, remains largely unknown.

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Figure 2. Structures of LATS1 and YAP WW domains (A) Structures of LATS1 WW domains. The LATS1 WW1 domain is a monomer, while its WW2 domain is a dimer. (B) Structures of YAP WW domain in complex with its binding partners. Following structures in PDB were used: 2YSB (SAV1_WW1), 2DWV (SAV1_WW2), 1JMQ (YAP_WW1-WBP1), 2LAX (YAP_WW1-SMAD1), 2LAY (YAP_WW1-SMAD1), 2LTW (YAP_WW1-SMAD7), 2L4J (YAP_WW2), 2LAW (YAP_WW2-SMAD1), and 2LTV (YAP_WW2-SMAD7).

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Structural dissection of Hippo signaling

LATS1/2–MOB1 complex

pombe [70]. In MEN, Cdc15p kinase first phosphorylates the scaffold protein Nud1p and then the phosphorylated Nud1p binds Mob1p to recruit the Dbf2p–Mob1p complex to spindle pole body [71]. Cdc15p further phosphorylates and activates the Dbf2p–Mob1p complex. The structure of human MOB1 in complex with an optimized Nud1p-like phosphor-peptide TVARIYHpSVVRYAPS (pS, phosphor-serine) was recently solved (PDB code: 4JIZ) (Fig. 3C) [71]. This peptide forms an intermolecular β-sheet with the β2 strand of MOB1, where the strand S-1 exists in the Mob1p structure. The phenolic side chain of residue Tyr-2 from the peptide packs against the side chain of Lys84 in the H3/H4 loop of MOB1. Residues at the +1 to +3 positions, which prefer hydrophobic residues in peptide library screening, form hydrophobic interaction with MOB1. The phosphorylation-dependent MOB1 binding is explained by extensive electrostatic interactions between phosphoserine of the peptide and three basic residues Lys132, Arg133, and Arg136 from MOB1. Although the binding site of phosphor-peptide in MOB1 is conserved in yeast and human, the Nud1p homologue protein has not been identified in fly or mammal. Thus, whether conserved proteins or even similar phosphor-peptides exist in higher organism warrants further investigation.

Linker between Upstream and Downstream of Signaling LATS–YAP/TAZ complex YAP/TAZ have an N-terminal TEAD-binding domain (TBD), a transactivation domain, and a PDZ-binding motif at their C termini. Besides, the longest isoform of YAP contains two WW domains, WW1 and WW2, while TAZ has only one WW domain in human. YAP/TAZ interact with signaling proteins, such as LATS1/2, SMAD1/7, Runt-related transcription factor 2 (RUNX2), DVL, AMOT, and P73, via the WW domains to regulate multiple signaling pathways [4,72–74]. LATS1 and LATS2 have two and one PPxY motif, respectively. The second PPxY motif in LATS1 is conserved in LATS2; and this PPxY motif interacts with YAP [75]. Although the LATS–YAP/TAZ complex has no structural information, several structures of YAP WW1 or WW2 domain alone or in complex with other partners have been solved (Fig. 2B) [76–80]. Like SAV1 WW1 domain, each WW domain of YAP consists of a three-stranded antiparallel β-sheet. The linker between two WW domains of YAP adopts a helix–loop–helix fold [78]. Based on sequence similarity, the structure of TAZ WW domain should resemble that of YAP WW1 domain. The WW1 and WW2 domains of YAP have different preference for binding motifs, which is caused by variant residues in the loop 1 between the first two β strands [78,81]. The WW2 domain binds to PPxY motifs, while the WW1 domain can bind both PPxY motifs and phosphor-serine-proline peptides [76,78]. Residues around PPxY motifs also affect their selective binding with WW domain. In the YAP WW1 and WW domain-binding protein 1 (WBP-1) complex (PDB code: 1JMQ), two prolines and one tyrosine in the WBP-1 PPxY motif contact the hydrophobic cavity in YAP WW1 domain [76,77]. The N- and C-terminal parts of SMAD7 PPxY motif extend its interface with YAP via polar interactions (PDB code: 2LTW) [81]. Compared with the WW1 domain, the YAP WW2 domain has lower affinity for SMAD7 PPxY motif since its loop 1 repels the N- and C-terminal extensions of SMAD7 PPxY motif (PDB code: 2LTV). YAP associates with SMAD1 via its tandem WW domains [78]. The YAP WW1 and WW2 domains recognize the phospho-serine-proline motif and the PPxY motif of SMAD1, respectively (PDB codes: 2LAW, 2LAX, and 2LAY). Besides the hydrophobic

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In the Hippo pathway, MST1/2 kinases phosphorylate MOB1 and LATS1/2 to activate LATS1/2 kinases and enhance their affinities with MOB1 [18,21]. Phosphorylated MOB1 binding further promotes the LATS1/2 autophosphorylation and kinase activity, leading to full activation of LATS1/2. LATS1/2 are members of protein kinase A/G/C-like (AGC) kinases nuclear dbf2-related (NDR) family. LATS1/ 2 contain a UBA domain, an S100B and MOB association (SMA) domain, a kinase domain and an AGC kinase C-terminal domain. Although the structure of LATS1/2 has not been determined, the general mechanism of AGC kinase activation has been studied [64]. AGC kinase activation requires phosphorylation of two conserved motifs, the activation loop in the kinase domain and the hydrophobic motif in the AGC kinase C-terminal domain. The two phosphorylation sites of LATS1 are Ser909 and Thr1079, which are phosphorylated by MST1/2 [19]. The phosphorylated hydrophobic motif binds to the N-lobe of AGC kinases to stabilize the αC helix conformation. Phosphorylation of the activation loop alters conformation of the catalytic center. Phosphorylation of both the activation loop and the hydrophobic motif is required for positioning the αC helix in an active conformation and further activation of AGC kinase [64]. Unlike other AGC kinases, LATS1/2, as well as NDR1/2, have an SMA domain preceding the kinase domain and an autoinhibitory sequence (AIS) in the kinase domain. MOB1 binds to the SMA domain of LATS1/2 and may relieve autoinhibition of the AIS [65,66]. MOB1 is highly conserved in eukaryotes from yeast to mammal, which is composed of a left-handed four-helix bundle and several flexible loops (PDB codes: 1PI1, 1R3B, and 2HJN) (Fig. 3A) [67–69]. Four conserved residues, two cysteines (Cys79 and Cys84 in human MOB1A) and two histidines (His161 and His166 in human MOB1A) coordinate a zinc atom to stabilize the structure of MOB1. Saccharomyces cerevisiae MOB1 (Mob1p) forms a dimer in the crystal (PDB: code 2HJN), which was confirmed by static light-scattering analysis [69]. Three structural elements, short strand S-1, extended strand S0, and helix H0, in the N-terminal region of Mob1p were not observed in the solved structures of human and Xenopus laevis MOB1 due to different fragments used for structure determination. The strand S0 and helix H0, but not strand S-1, are conserved in MOB1. The strand S0 associates with the conserved surface composed of helices H2–H4 and the H4/H5 loop. The helix H0 mediates the homodimerization and is important for Mob1p function in budding yeast. Whether the MOB1 associates as a functional dimer in other species, especially in human, remains to be addressed. Nuclear magnetic resonance (NMR) titration showed that the X. laevis NDR1 peptide (amino acids 68–86, AHARKETEFLRLKRTRLGL), as part of the SMA domain, leads to specific chemical shift perturbations in H3 and H4 helices and H4/H5 loop of MOB1, suggesting this region is involved in the NDR–MOB1 interaction [68]. Several studies imply that the positively charged basic and hydrophobic residues in the SMA domain of NDR kinases contact a negatively charged surface on MOBs [65–68]. Recently, three structures of S. cerevisiae MOB2 (Mob2p) in complex with NDR family kinase CBK1 (Cbk1p) were deposited in PDB (PDB codes: 4LQP, 4LQQ and 4LQS) (Fig. 3B). The SMA domain preceding the Cbk1p kinase domain interacts with helices H1, H2, and H7 of Mob2p through extensive electrostatic and hydrophobic interactions. The Cbk1p–Mob2p interaction might be stabilized by phosphorylation of the C-terminal hydrophobic motif of Cbk1p. LATS1/2 and MOB1 may interact with each other in a similar fashion. The Hippo pathway is known as the mitotic exit network (MEN) in S. cerevisiae and the septation initiation network in Schizosaccharomyces

34 interaction between SMAD1 and YAP WW1 domain, the phosphate group of residue Ser206 phosphorylated by cyclin-dependent kinase 8/ 9 (CDK8/9) in SMAD1 forms a hydrogen bond with the YAP WW1 domain. Further phosphorylation of SMAD1 at Thr202 by glycogen synthase kinase-3 (GSK3) destabilizes the interaction of SMAD1 with YAP. SMAD1 binds to YAP WW2 domain mainly via core residues and C-terminal extension of its PPxY motif. Several YAP-binding proteins have two or more PPxY motifs, which may enhance their affinities with tandem WW domains of YAP [73,79].

YAP/TAZ–14-3-3 complex

TEAD Transcription Complex YAP/TAZ–TEAD complex All TEAD1–4 are composed of an N-terminal TEA/ATTS (AbaA, TEC1p, TEF-1 sequence) domain and a C-terminal YAP-binding domain (YBD) [83,84]. YAP/TAZ form a complex with TEAD YBD via their TBDs to transactivate downstream genes expression, such as CTGF and Cyr61 [4,85]. Structuralwise, TEAD YBD adopts an immunoglobulin-like β-sandwich fold with four short helices (PDB codes: 3JUA, 3KYS, and 3L15) (Fig. 4A) [86–88]. YAP TBD consists of one β strand, one α helix, and one twisted coil [87]. YAP TBD interacts with TEAD YBD via three interfaces [86,87]. The interface 1 constitutes the β strand of YAP TBD and the β7 strand of TEAD YBD, forming an antiparallel β-sheet. Α helix of YAP packs into a hydrophobic groove in TEAD YBD on interface 2. On the interface 3, the twisted coil of YAP TBD embeds into a hydrophobic pocket in TEAD YBD. Mutation studies revealed that the interface 3 is critical for YAP– TEAD complex formation.

VGLLs–TEAD complex Drosophila vestigial (Vg) directly interacts with Sd to activate wingspecific gene expression during Drosophila wing development [89– 92]. Mammalian vestigial-like protein 1 to 4 (VGLL1–4) can act as transcriptional coactivator or corepressor [93–101]. VGLL1 or VGLL2 activates TEAD, myocyte-specific enhancer factor 2 (MEF2) or myoblast determination protein 1 (MYOD1)-dependent transcription, while VGLL4 suppresses TEAD-mediated transcription. VGLL3 is amplified and over-expressed in soft tissue sarcomas, and in contrast, it may act as a tumor suppressor in epithelial ovarian cancer [102,103]. VGLL1–4 proteins bind YBDs of TEADs via their Tondu (TDU) domains [93,94,96,104]. VGLL1–3 proteins have one TDU domain, while VGLL4 has two TDU domains, namely TDU1 and TDU2, respectively. VGLL4 can bind two molecules of TEAD via its tandem TDU domains [96]. The TDUs of VGLL1 and VGLL4 interact with TEAD in a similar manner to YAP (PDB codes: 4EAZ

and 4LN0) (Fig. 4B–D) [96,105]. Unlike YAP, the interface corresponding to interface 3 of the YAP–TEAD complex is lacking in VGLL1–TEAD and VGLL4–TEAD complexes (Fig. 4E). YAP contains a conserved LxxLF (Leu-Xaa-Xaa-Leu-Phe) motif in interface 2, while VGLLs have the VxxHF (Val-Xaa-Xaa-His-Phe) motif. The VGLL1 TDU domain possesses interfaces 1 and 2 [105]. The VGLL4 TDU1 domain has only interface 2, while the TDU2 domain has interfaces 1 and 2 [96]. There is only one helix in the interface 2 of YAP TBD and VGLL1 TDU domain, as well as in the TDU1 domain of VGLL4, while an extra helix is present in interface 2 of VGLL4 TDU2 domain, strengthening its interaction with TEAD. Collectively, interface 2 of VGLL1 or VGLL4 TDU domains appears to play an essential role in their interactions with TEAD.

TEAD–DNA complex The TEA domain of TEAD is a DNA-binding domain that is highly conserved from yeast to mammal [83,84]. The TEA domain binds specific DNA elements such as M-CAT (muscle-CAT, 5′-CATTCCT-3′) in the promoter regions of TEAD target genes and 5′-TGGAATGT-3′ in the simian virus 40 (SV40) enhancer [106–108]. The TEAD1 TEA domain is a helix-turn-helix (HTH) fold and belongs to the homeodomain structural family (PDB code: 2HZD) [109]. The TEA domain is composed of three α helices. NMR titration assay identified the H3 helix and L2 loop immediately preceding H3 in TEAD1 TEA domain as DNA-binding interfaces. The structure of TEAD in complex with DNA has not been experimentally determined. According to DALI search, the structure of TEA domain is similar to those of paired box 6 (PAX6) paired domain (PDB code: 6PAX), telomeric repeatbinding factor 1 (TERF1) HTH myb-type domain (PDB code: 1W0 T) and homeobox-containing protein 1 (HMBOX1) homeobox domain (PDB code: 4J19), all of which bind DNA in each structure [110–113]. Thus, it is very likely that TEAD TEA domain may interact with DNA in a similar manner.

Perspective The Hippo signaling involves multiple protein–protein and protein– DNA interactions, which are mediated by several shared modular protein domains. Examples representing modularity of the Hippo pathway include the WW domain and its partner PPxY motif [72–74]. In the Hippo pathway, many proteins such as SAV1, YAP, TAZ, and KIBRA possess one or more WW domains, while one or more PPxY or PPxF (Pro-Pro-Xaa-Phe) motifs were found in other components, including MST1/2, LATS1/2, CRB1-3, AMOT, angiomotin-like protein 1/2 (AMOTL1/2), fat homolog 1-4 (FAT1-4), and dachsous homolog 1/2 (DCHS1/2). The PDZ domain is another modular domain participating in Hippo signaling [72]. PALS1, Pals1-associated tight junction protein, SCRIB, and DLG have PDZ domain, while YAP, TAZ, KIBRA, CRB1-3, AMOT, and AMOTL1/ 2 contain PDZ-binding motif. These proteins form a complicated network to regulate Hippo signaling. Components of the Hippo core kinase cascade are tumor suppressors, while YAP/TAZ are oncoproteins. Germline or somatic mutations of the Hippo pathway genes are rare in human cancers. However, expression levels and nuclear localization of YAP are elevated in various tumors, including liver, lung, breast, colon, ovarian, and gastric cancers [29,114]. YAP/TAZ also regulate stem and progenitor cell self-renewal and expansion and function in tissue repair and regeneration [4,29]. Therefore, targeting YAP/TAZ may provide novel strategies for anticancer therapy, whereas suppressing the Hippo

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LATS1/2 and Wts phosphorylate YAP/TAZ at Ser127/Ser89 and Yki at Ser168, respectively, to promote YAP/TAZ and Yki associations with 14-3-3 proteins, leading to their cytoplasmic retention [17,23–25]. The crystal structure of 14-3-3σ in complex with YAP peptide phosphorylated at Ser127 (124-RAHpSSPASLQ-133) has been reported (PDB code: 3MHR) [82]. The 14-3-3σ exists as a dimer with a typical W-shape. The YAP peptide binds to the amphipathic groove of 14-3-3σ created by helices 3, 5, 7, and 9. The phosphorylated Ser127 of the YAP peptide is coordinated by residues Lys49, Arg56, Arg129, and Tyr130 of 14-3-3σ. The orientation of the YAP peptide in the 14-3-3 cavity indicates a comparable mode II 14-3-3-binding motif, although the N-terminus of the peptide fits more closely to a mode I binding motif from a sequence point of view.

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core kinases may promote tissue regeneration. Structure determination of Hippo pathway components and related complexes can provide atomic insights into its regulatory mechanism. Based on detailed three-dimensional structures, one can design specific and targetable compounds including small molecules and peptides to inhibit kinases or YAP/TAZ activity for therapeutic purposes like tissue regeneration and cancer treatment. A representative example is targeting the YAP– TEAD complex by a small molecule verteporfin and a peptide SuperTDU to suppress YAP-driven liver overgrowth and gastric cancer in mice, respectively [96,115]. Although a number of new discoveries on the Hippo pathway have been reported, many crucial questions await answers before the regulatory mechanism of Hippo pathway can be fully elucidated. Recently, several groups identified Hippo interactome by affinity purification and mass spectrometry (AP-MS) techniques, which may facilitate further understanding of the Hippo pathway [116–119].

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Funding This work was supported by the grants from the 973 program of the Ministry of Science and Technology of China (No. 2012CB910204), the National Natural Science Foundation of China (Nos. 31270808, 31300734, 31470736, and 31470868), the Science and Technology Commission of Shanghai Municipality (Nos. 11JC14140000 and 13ZR1446400), and the ‘Cross and Cooperation in Science and Technology Innovation Team’ Project of the Chinese Academy of Sciences.

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Figure 4. Structures of TEAD–YAP/VGLLs complexes (A–D) Structures of TEAD in complex with YAP, VGLL1, and VGLL4. (E) Structural comparison of TEAD–YAP/ VGLLs complexes. Following structures in PDB were used: 3KYS (TEAD1–YAP), 4EAZ (TEAD4–VGLL1), and 4LN0 (TEAD4–VGLL4).

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