Structural Insights into the Regulation of Hippo Signaling - ACS

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Structural Insights into the Regulation of Hippo Signaling Leah Cairns,† Thao Tran,† and Jennifer M. Kavran*,†,‡ †

Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and ‡Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States ABSTRACT: During development, the Hippo pathway regulates the balance between cell proliferation and apoptosis to control organ size. Appropriate Hippo signaling is associated with stem cell maintenance, while inappropriate signaling can result in tumorigenesis and cancer. Cellular and genetic investigations have identified core components and determined that complex formation and protein phosphorylation are crucial regulatory events. The recent spate of high-resolution structures of Hippo pathway components have begun to reveal the molecular mechanisms controlling these events, including the molecular determinates of complex formation between YAP and TEAD, the role of phosphorylation in controlling complex formation by Mob, and the conformational changes accompanying Mst1/2 kinase domain activation. We will review these advances and revisit previous structures to provide a comprehensive overview of the structural changes associated with the regulation of this pathway as well as discuss areas that could benefit from further mechanistic studies.

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Salvador, respectively.4,5,22,27−29 Together, these six components form the core of the pathway (Figure 1C) and represent the canonical view of Hippo signaling, which culminates in the phosphorylation dependent inhibition of YAP. A more comprehensive view of Hippo signaling can include additional proteins that can substitute for core components and alternate modes of phosphorylation independent of inhibition of YAP. The search for the external signal that activates Hippo signaling and the components which link that signal to core components has yielded numerous candidates. Multiple signals can independently activate the pathway including hormonal cues, cell number (through apical and tight junctions), Gprotein coupled receptors, Ras-associated family (RassF) proteins, and the extracellular matrix. Three proteins, Merlin, Kibra, and Expanded, have emerged as a potential node of upstream signaling and may be a conduit for external stimuli.15,30−33 RassF represents another upstream node, but unexpectedly, the output of this node increases apoptosis in human cells while it lowers it in flies.16,17 The search for additional components or signals is complicated by crosstalk between this pathway and others such as Wnt, Notch, and Hedgehog, and by the myriad of potential pathway components identified in large-scale interactome studies. While early work focused on the identification of key components and their function, the field is now poised to understand how these components interact on a molecular and structural level. This review focuses on recent structural studies of Hippo pathway components, which have begun to elucidate

he Hippo pathway regulates cell number by controlling the balance between proliferation and apoptosis and thus organ size. Genetic screens in Drosophila seeking overgrowth phenotypes identified several proteins that would go on to form the core of the pathway, and the phenotype of one set of flies inspired the name “Hippo.”1−9 In animal models, disruption of Hippo signaling leads to overgrowth and tumorigenesis.10−14 Aberrant pathway activity correlates with a variety of human cancers confirming Hippo signaling as a tumor suppressor pathway and a potential node for therapeutic intervention. Emerging evidence points to an additional role for Hippo signaling in stem cell maintenance, and attempts at manipulation of the pathway for tissue regeneration are underway. Both the structure and function of several core components of the Hippo pathway are conserved between flies and humans, but this conservation does not extend to the entire complement of upstream components.15−17 Homologues of core components have been identified in both humans and flies, and gene disruption in flies can be rescued with mammalian genes.10,18,19 The names of the proteins, however, are not conserved (Figure 1A,B). In this review, we will use the mammalian names in general but the species appropriate names when referring to specific results. On a molecular level, Hippo signaling regulates the localization of Yes-associated Protein (YAP), a transcriptional coactivator. YAP can enter the nucleus and form a functional complex with TEA-domain (TEAD) proteins.20−22 When the pathway is active, YAP is phosphorylated by the kinase Lats1/2, which promotes binding to 14−3−3 and is prevented from translocating into the nucleus.13,19−26 Activation of Lats1/2 requires phosphorylation by the upstream kinase Mst1/2.5,22 The activity of each kinase is regulated by either Mob or © 2017 American Chemical Society

Received: November 29, 2016 Accepted: February 2, 2017 Published: February 2, 2017 601

DOI: 10.1021/acschembio.6b01058 ACS Chem. Biol. 2017, 12, 601−610

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Figure 1. General Hippo pathway overview. Domain assignment of core Hippo pathway components from either Homo sapiens (a) or Drosophila melanogaster (b). Domains discussed in this review are shown in white, while others are shaded gray. Proline rich motifs (PRMs) are indicated by black triangles. The location of relevant post-translational modifications are indicated by black circles for serine or threonine phosphorylation or asterisks for sites of palmitoylation. (c) A schematic of the Hippo pathway organization in cells. Critical phosphorylations are indicated by a “P” in a circle, with the color of the circle corresponding to the kinase that catalyzed the addition.

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the mechanism of key regulatory events, and revisits previously determined structures of Hippo components in light of new data. This review is organized based on the protein domains found in the core components (Figure 1A,B) and will discuss evidence for potential allostery between domains.

FERM DOMAINS

FERM (Band Four-Point-One, Ezrin, Radixin, and Moesin) domain containing proteins share a common architecture and are often responsible for linking the cytoskeleton to the 602


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Figure 2. Frequently occurring domains in the Hippo pathway. (a) Schematic of canonical FERM domain activation. The FERM domain is represented by a light gray triangle, the linker region by a black line, and the CTD by a dark gray rectangle. A “P” in a circle indicates the site of phosphorylation. (b) Cartoon representation of the superposition of the Lats2 segment (light blue) onto the complex between Merlin FERM domain (light gray) and its CTD (dark gray).44 The three subdomains of FERM (A, B, and C), each with homology to the folds ubiquitin, acetylcoA binding protein, and phosphotyrosine binding (PTB)/pleckstrin homology (PH), respectively, are labeled.36 The newly explained set of mutations associated with neurofibromatosis type 2 are denoted with a dotted black circle. Residues in the blue box motif are colored blue. (c) Cartoon diagram of the second WW domain of YAP (red) bound to PRM peptide from Smad7 (cyan).55 Shown in sticks are the conserved tryptophans of the WW domain and the prolines of Smad7. (d) Cartoon diagram of the β-clam shell shaped homodimer formed by the second WW domain of Salvador with one monomer in hot pink and the other in light pink.54 Shown in sticks are the conserved tryptophans and the tyrosine substitutions. (e) Cartoon representation of a SARAH domain heterodimer between Mst1 (pale green) and RassF5 (yellow).73

Merlin FERM domain have been annotated in the Catalog of Somatic Mutations in Cancer (COSMIC) database since the initial FERM domain structures were published. Several of these substitutions disrupt Lats1/2 binding,41 and our analysis suggests three additional substitutions (M183T, R187K, and W191R) are located near the binding site for Lats1/2 and CTD and may also disrupt complex formation (Figure 2B). In flies, deletion of the blue box motif results in overproliferation.44 Absence of these residues may destabilize the nearby Lats1/2 binding site.

membrane. Merlin and Expanded, two FERM domaincontaining proteins, act together with Kibra upstream of the core pathway components in flies. These three form a membrane apical complex responsible for the recruitment and activation of Warts, Hippo, and Salvador.30−32,34,35 While no mammalian homologue of Expanded has been identified, the mammalian complex between Kibra and Merlin functions equivalently to the ternary complex in Drosophila.30,32,34 How exactly these binding events and the subsequent changes in localization stimulate pathway activity remains to be resolved. The FERM domain of Expanded can also exclude Yorkie from the nucleus by direct binding. This was the first example of a phosphorylation independent mode of Yorkie inhibition.15 The molecular details of Merlin and Expanded activation are not as well established as those for other FERM domain proteins. Typically, phosphorylation of the linker region destabilizes the interaction between the FERM and the Cterminal domain (CTD) resulting in a conformational change that, much like a switch, shifts the protein from a closed to an open state and reveals a binding site for other components10,36−39 (Figure 2A). Phosphorylation of the linker region of Merlin, however, does not fully disrupt the interface between the FERM and CTD, supporting the notion that Merlin activation may be more rheostat in nature than switch.40,41 The Merlin FERM domain has the clover-leaf structure characteristic of FERM domains42,43 (Figure 2B). Comparison of the structures of the Merlin FERM domain alone, bound to its CTD, or to a short segment of Lats1/2 reveals no structural changes upon complex formation (Figure 2B) but does demonstrate that the binding sites for the CTD and Lats1/2 overlap and are mutually exclusive41,44 (Figure 2B). Human Merlin attracted attention before the identification of the Hippo pathway because mutations to the human gene (Nf 2) lead to neurofibromatosis type 2.43,45−47 The majority of the missense mutations associated with this disease map to the core of the protein and presumably disrupt protein folding.42,43 The recently determined structure of Merlin bound to Lats1/2 provides additional constraints to interpret two additional sets of mutations. Fifty-seven additional missense mutations in the

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WW DOMAINS The first WW domain ever identified was from YAP.48−50 WW domains are small domains (roughly 30 to 40 residues) named for two conserved tryptophan residues that bind short prolinerich motifs (PRMs). As a result of their modular and autonomous nature, these domains often function as scaffolds and are compared to LEGOs.51 Three Hippo pathway members contain tandem WW domains (YAP, Salvador, and Kibra),4,48,52 and many pathway components have multiple PRMs including Mst1/2, Lats1/2, and Expanded. Consequentially, numerous WW domain-mediated complexes are possible, and it is impossible from sequence alone to determine which WW domain binds which PRM. Significant strides have been made in mapping these interactions with cellular approaches, but in only a few instances has a specific WW domain been linked to a specific PRM. Most structures of WW domains have been determined by NMR. These three stranded antiparallel β-sheets form a shallow groove that acts as the binding site for PRMs53 (Figure 2C). The second WW domain of Salvador is atypical because a tyrosine is substituted for the second conserved tryptophan. It was surprising when the crystal structure revealed this WW domain to be a homodimer, and solution measurements confirmed this dimerization54 (Figure 2D). The typical ligand binding site is buried at the dimer interface, implying that this subset of WW domains cannot bind ligands. This structure can explain previous data in which Salvador dimerization in cell lysates was mapped, unexpectedly, to its WW domains. 603


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ACS Chemical Biology Salvador also coimmunoprecipitates with Kibra, and this interaction has been attributed to heterodimerization between reciprocal WW domains, but experimental evidence is needed to prove this hypothesis.32 There is no structural information for a WW domain bound to a PRM from the Hippo pathway. However, biochemical analysis of the binding behavior of the WW domains of YAP to PRM peptides from SMAD, another binding partner, provide useful insights.55,56 An analysis of the binding affinities between each of the two WW domains of YAP and different PRMcontaining peptides revealed each WW domain has a unique substrate specificity. As a result, the tandem WW domain arrangement generates a bipartite binding surface with greater specificity than either single domain.55 This analysis also revealed WW domains can be promiscuous. A WW domain can bind a variety of PRMs with varying affinities. In the absence of its preferred partner, the WW domain bound another PRM with weaker affinity.55 These data should act as a cautionary tale for mapping interactions between PRMs and WW domains by substitution or deletion of a single PRM in the context of a protein that contains multiple PRMs. However, in cells, specific binding between WW domain containing proteins of the Hippo pathway and their PRM containing binding partners is ensured by both the tandem arrangement of WW domains and the stronger affinity for preferred PRMs.

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MOB The molecular mechanism of Mob is the most well understood in the Hippo pathway. Mob proteins are globular and predominantly α-helical with a flat acidic face and a flexible N-terminal region57−61 (Figure 3). The phosphorylation state of Mob regulates interactions with Mst1/2 and Lats1/2. Unphosphorylated Mob binds Mst1/2 and is then phosphorylated on two N-terminal residues.62,63 In flies, phosphorylated Mats (the Mob homologue) coimmunoprecipitates with Warts (the Lats1/2 homologue) more efficiently than unphosphorylated Mats.10,62 These data suggest a sequence of events in which unphosphorylated Mob binds Mst1/2, phosphorylation of Mob triggers complex disassociation, and phosphorylated Mob then binds Lats1/2. Recent structures of unphosphorylated and phosphorylated Mob bound to either a Mst2 linker-derived peptide or a fragment of Lats1, respectively, provide a structural explanation for these biochemical results.59,60 Two sites on Mob regulate complex formationone binds phosphorylated residues and another binds hydrophobic residues. When Mob1 is unphosphorylated, the phosphorylated Mst2 linker can bind each of these sites59 (Figure 3A). Phosphorylation of two residues on the N-terminal region, Thr12 and Thr35, induces conformational changes that occlude these sites. Phosphorylated Thr12 occupies the phospho-binding site, and residues adjacent to Thr35 occupy the hydrophobic site60 (Figure 3B). The rearrangement of residues adjacent to Thr35 also relieves a steric clash, facilitating binding to Lats160 (Figure 3B). The Nterminal region of Mob, thus, is a regulatory switch that controls complex formation in response to phosphorylation. Initially, however, the N-terminus was thought to be dispensable since it was sensitive to limited proteolysis and was thus removed from most Mob proteins used for structure determination.57,58,61,64,65 The first structure to include this region instead revealed an active role. In S. cerevisiae Mob1, the homologous region mediated homodimerization66 (Figure 3C). It is enticing to speculate that phosphorylation of these residues

Figure 3. Phosphorylation regulates Mob binding. (a) Cartoon representation of the unphosphorylated Mob1 (orange) core bound to a phosphorylated fragment of Mst2 linker (green).29 The hydrophobic and phospho-binding sites are indicated by gray boxes. (b) Cartoon representation of phosphorylated Mob1 (orange) bound to the NTR of Lats1 (blue).29 (c) Orthogonal views of S. cerevisiae Mob1 homodimer with each monomer represented in cartoon in different shades of orange.66 (d) Orthogonal views of a phosphorylated Mst2 linker, shown in green surface, bound to two complexes of Mob1− Mst2, shown in cartoon, with each complex colored different shades of orange and green.29 Zinc ions are shown as gray spheres and phosphorylated residues as sticks.

may regulate the oligomeric state of Mob. However, dimerization of yeast Mob1 could only be detected in solution at high concentrations (∼1 mM) and was undetectable in cell lysates.66 Analytical ultracentrifugation studies of mouse Mob1b reveal the protein is monomeric at lower concentrations (19 μM).60 Together these data suggest that if Mob does dimerize, it does so weakly. Mounting structural evidence suggests that two helices of Mob, H2 and H4, may be a more ubiquitous binding interface than previously appreciated. H2 and H4 contribute to the homodimerization interface of yeast Mob1 and the binding surface for Lats1/2.59,60,66 These helices also mediate a third complex found in the crystal lattice of Mob1 bound to a phosphorylated Mst2 peptide59 (Figure 3D). Crystals were grown with a 2-fold molar excess of peptide, and the resulting asymmetric unit contained six copies of the Mob1−peptide complex as well as three additional peptides. Each extra peptide 604


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Figure 4. Inactive and active kinase domain conformations. (a) Cartoon representation of the inactive, unphosphorylated Mst2 kinase domain29 (left) and an active Mst1 kinase domain bearing two phosphorylations (shown in sticks) on the activation loop (AL)82 (right). (b) Cartoon representation of Cbk1 (light blue) with a phosphomimetic substitution, T743E (orange), on the hydrophobic motif (dark blue surface) bound to Mob2 (pale orange). The disordered αC helix and AL are indicated by a dotted black line. The bound ATP analog is shown in white spheres.61

levels of Mst1 autophosphorylation, presumably by blocking homo-oligomerization.28 Cotransfection of Salvador leads to higher levels of substrate phosphorylation by Hippo (the Mst1/ 2 homologue) in flies, but how this happens is unclear.18 Interactions between SARAH domains may respond to structural cues from other domains of the protein. Immunoprecipitation experiments revealed that RassF binds unphosphorylated Hippo while Salvador binds phosphorylated Hippo.16 These mutually exclusive interactions could arise either from cross-talk between domains or phosphorylation dependent conformational changes in Hippo. The structure of a RassF5 SARAH domain bound to a near full-length unphosphorylated Mst2 variant did not reveal any interdomain crosstalk between the SARAH and kinase domains.29 The absence of an equivalent phosphorylated structure precludes analysis of the role of any phosphorylation induced conformational changes that may occur. SARAH domain interactions have been modeled with different oligomeric states. A trimer between Salvador, Hippo, and RassF68 was proposed based on sequence analysis, but attempts to isolate such a complex either from cell lysates or with purified proteins failed.16,74 A tetramer model, which could include a dimer of dimers, is supported by several solutionbased measurements. The residues that mediate complex formation between isolated SARAH domains were mapped by NMR.74 The residues that mediated homodimerization of Mst1 were the same as those that mediated heterodimerization with RassF5. Different residues, however, mediated binding to Salvador without disrupting the original homodimerization interface, suggesting that the complex between Salvador and Mst1 SARAH domains is a tetramer. Tetramers of isolated RassF5 SARAH domains were detected by gluteraldhyde crosslinking.74 The preponderance of evidence, however, supports a dimeric model. Each structure of a SARAH domain complex determined in solution is dimeric, and the thermal unfolding of both Mst1 and RassF SARAH domains fits a dimeric model.29,72−79 To fully resolve this issue, the oligomeric state for each possible SARAH domain complex needs to be determined systematically. The rate limiting factor in such experiments appears to be the difficulty in purifying Salvador SARAH domains.80

binds a groove formed by the H2 helices of two Mob1−peptide complexes. This peptide mediated dimer is different than the dimer observed in the yeast structure, and there is no evidence suggesting it is biologically relevant. The fact, however, that this region repeatedly mediates protein−protein interactions suggests that H2 and H4 may comprise an underappreciated, promiscuous docking site. It was predicted that the interaction between Mob1 and Lats1/2 would involve the flat acidic face of Mob1 and the Nterminal region (NTR) of Lats1/2,57,58,67 and the recently determined structures confirm those predictions. The NTR of Lats1/2 forms two long α-helices connected by a turn when bound to Mob (Figure 3D). In two structures of an NTR fragment of Lats1 bound to Mob 1, the NTRs are equivalent and superpose with an RMSD of 0.64 Å over 63 Cα atoms. In a third structure of a near full length Lats1/2 homologue bound to Mob2, the NTR is modeled as α-helix followed by a coil. The coil is in the equivalent position as the second α-helix in the fragment structures.61 Determination of the homologue structure involved tracing a de novo model in an electrondensity map at modest resolution, and the differences in the secondary structure assignment of its NTR most likely reflect this challenge rather than differences in the actual structure of the protein.

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SARAH DOMAINS SARAH domains are regions of heptad repeats found in the Ctermini of their namesake proteins Salvador, RassF, and Hippo68 and form higher-order coiled coils. They mediate complex formation between Mst1/2 and either RassF family members or Salvador but never between RassF and Salvador.4,5,7,16−18,69−71 Structures of isolated SARAH domain homodimers of Mst1, Mst2, and Nore1 (a murine RassF family member) and heterodimers of Mst1−RassF5 and Mst2− RassF5 have been determined.72−77 Each is an antiparallel coiled coil with a hydrophobic dimer interface (Figure 2E). Each monomer forms a short 310 helix followed by a long αhelix (Figure 2E). The angle between the two helices varies across the structures, suggesting a loose connection between the SARAH domain and the rest of the protein. RassF and Salvador have opposing effects on Mst1/2 kinase activity. The rate of autophosphorylation of full-length Mst1 is higher than that of a variant lacking the SARAH domain, presumably by increasing the local concentration of the kinase.29 Conversely, coexpression with RassF1 leads to lower

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KINASE DOMAINS Mst1/2. All kinase domains share a common bilobe architecture containing a β-strand rich N-lobe and an α-helical

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hydrophobic motif (T743E) are largely the same61 (Figure 4B). In both, the hydrophobic motif packs against Mob2 and the Nlobe of the kinase domain at a site distinct from that used in Protein Kinase B. The lack of structural changes raises the possibility either that activation of NDR-kinases is not sensitive to the phosphorylation state of the hydrophobic motif, in contrast to biochemical data, or that the phosphomimetic substitution does not accurately mimic phosphorylation, in contrast to cellular data.61 Additionally, the αC helix is disordered in both structures, implying that neither binding of Mob nor phosphomimetic substitution of the hydrophobic motif are sufficient to stabilize the active conformation of the kinase domain.61 The deviations from the typical ACG activation mechanism beg the question of how phosphorylation of the hydrophobic motif leads to activation of NDR-family kinases including Lats1/2.

C-lobe and undergo similar conformational changes upon activation.81 Structures of phosphorylated and unphosphorylated kinase domains of Mst1/2 display the prototypical structural rearrangements associated with activation. Upon phosphorylation, the activation loop adopts an extended conformation, and the αC helix repositions, leading to formation of the active site cleft between the two lobes29,82 (Figure 4A). While multiple residues can be phosphorylated in the activation loop of Mst1/2, only phosphorylation of a single residue, Thr180 for Mst1 or Thr183 for Mst2, confers catalytic activity.28,69,83 The upstream trigger for autophosphorylation is unknown but most likely promotes SARAH domain mediated homodimerization of Mst1/2.28,29 The recently determined structure of an Mst2 variant bound to the SARAH domain of RassF5 showed no interactions between the SARAH and kinase domains, providing no evidence for interdomain cross-talk or allostery.29 In the Mst2 variant crystallized, the wild-type linker was substituted with a shorter, synthetic linker to facilitate crystallization because NMR analysis revealed the wild-type linker to be disordered.29,74 Therefore, potential interactions between the native linker and kinase domains of Mst1/2 could not be evaluated. The wild-type linker, however, increases the thermal stability of the Mst1 SARAH domain and mediates binding to Mob, suggesting a more active role than previously anticipated, perhaps as a conduit for cross-talk between the kinase and SARAH domains.59,79 Isolated kinase domains also dimerize, albeit weakly (at tens of micromolar), in solution.27,29 Single residue substitutions of surface exposed residues on the C-lobe failed to inhibit kinasedomain dimerization.29 In general, biologically relevant interfaces are more likely to mediate crystal lattice contacts than nonbiologically relevant interfaces,84 and a lattice contact conserved among different crystal forms may represent a biologically relevant interface. Analysis of the lattice contacts in all available Mst1/2 kinase domain structures fails to identify such a conserved lattice contact. While these data do not exclude the possibility that such a dimer exists, they do suggest that if it does it is either weak or transitory. Lats1/2. Lats1/2 is part of the NDR kinase family, which is a subset of the ACG family. NDR kinases are regulated by two phosphorylation events.85 The hydrophobic motif on the C-tail is first phosphorylated by an upstream Sterile 20-like kinase, such as Mst1/2,17,71,86 and this triggers autophosphorylation of the activation loop.10 Binding of Mob stimulates both autophosphorylation and substrate phosphorylation by Lats1/ 2.10,62,63,87 The mechanism behind this activity increase is unknown but may arise from either allostery or from the recruitment of Lats1/2 to the membrane.67,88 There is no structural information on the kinase domain of Lats1/2. Instead, homologous structures may provide a framework for thinking about Lats1/2 activation. Structures of the inactive and active forms of Protein Kinase B, a prototypical ACG family kinase, revealed the canonical active kinase conformation is stabilized by phosphorylation of both the activation loop and hydrophobic motif. The phosphorylated hydrophobic motif packs against the N-lobe and stabilizes the active conformation of the αC helix.89,90 It was surprising, therefore, that the recently determined structures of the first NDR-kinase, Cbk1, did not reveal a similar role for the hydrophobic motif. The structures of a complex between Mob2 and either unphosphorylated Cbk1 or a Cbk1 variant with a phosphomimetic substitution on the

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YAP/TEAD Disruption of the complex between YAP and TEAD represents a potential mode of therapeutic intervention. Structures of the YAP binding domain of TEAD proteins in the presence and absence of YAP reveal an immunoglobulin G-like (IgG) fold that undergoes only minor rearrangements upon binding YAP.91−95 In contrast, the TEAD binding domain of YAP is flexible in the absence of TEAD and becomes ordered upon complex formation.91,92,94 The complex between YAP and TEAD is mediated by three interfaces91,92 (Figure 5). Interface

Figure 5. Complex formation between YAP and TEAD. Cartoon representation of the heterodimeric complex between the TEAD binding domain of YAP (red) and the YAP-binding domain of TEAD2 (purple).91 The complex is mediated by three interfaces (gray boxes). The structure of palmitoylated TEAD295 was superposed on the YAP−TEAD complex, and the resulting position of the palmitoylation is shown in sticks (yellow). Adapted with permission from ref 96, copyright 2012, Elsevier.

3 is predominantly hydrophobic and most likely contributes the majority of the binding energy since single residue substitutions both impair binding to TEAD and reduce transcription levels in cell based assays. Substitutions of residues at the other interfaces do not elicit such dramatic changes.91,92 The relative strength of the interfaces may vary between species. In insect cells, a single residue substitution in Interface 1 of Yorkie (the YAP homologue) is sufficient to disrupt interactions with Scalloped (the TEAD homologue),21 but in mammals multiple substitutions of Interface 1 are required to achieve a similar result.92 TEAD forms heterodimeric complexes with other coactivator proteins, and a structure of one such complex revealed that only Interfaces 1 and 2 mediate binding, suggesting that disrupting Interface 3 may be a way to specifically target TEAD−YAP complexes.96 Peptides designed to disrupt binding between YAP and TEAD based on the recent structures have had initial success.93 606


ACS Chemical Biology The structures of TEAD proteins have revealed palmitoylation as an unanticipated factor regulating TEAD function. The electron density map of the structure of TEAD3 revealed unexpected electron density connected to the side chain of a universally conserved cysteine95 (Figure 5), and reanalysis of previous electron density maps revealed the same additional density. Mass spectrometry analysis determined this addition to be a palmitate, and lower levels of other lipids were also detected.95 This post-translational modification is the result of autopalmitoylation97 and increases the thermal stability of the protein.95 The biological significance of this discovery is not yet fully understood. Unpalmitoylated TEAD proteins, bearing a substitution of the conserved cysteine, immunoprecipitate YAP less efficiently and have lower transcriptional activity in cell based assays.97 The binding pocket for palmitoylation had been previously identified as a druggable site and bound flufenamates with modest results.98 The identification of this post-translational modification provides new constraints to guide small molecule targeting of TEAD activity.

CONCLUSIONS Since the discovery of the Hippo pathway, the field has worked at an amazing speed to identify additional components, cellular functions, and genetic linkages. Having established this groundwork, the field is poised to understand how these components function on a molecular level to control the equilibrium between the inactive and active states of the pathway. The findings discussed here demonstrate the importance of complex formation and phosphorylation in regulating pathway activity. Recent structural advances have revealed novel post-translational modifications for TEAD, deciphered the function of Mob phosphorylation, and provided a framework to interpret the disease associated mutations in Merlin. The molecular mechanisms regulating both kinases are less well understood. For Mst1/2, we look forward to learning what triggers autophosphorylation, the potential role of allosteric regulation, and how Salvador stimulates kinase activity. For Lats1/2, further work is needed to understand the molecular mechanisms of key activation events including the allosteric roles of both Mob and the hydrophobic motif. Understanding the mechanisms that regulate pathway activity will help guide targeted therapeutic interventions and direct cellular and biochemical investigations aimed at deciphering this pathway.

NOMENCLATURE

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REFERENCES

(1) Justice, R. W., Zilian, O., Woods, D. F., Noll, M., and Bryant, P. J. (1995) The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation. Genes Dev. 9, 534−546. (2) Xu, T., Wang, W., Zhang, S., Stewart, R. A., and Yu, W. (1995) Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase. Development 121, 1053−1063. (3) Kango-Singh, M. (2002) Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila. Development 129, 5719−5730. (4) Tapon, N., Harvey, K. F., Bell, D. W., Wahrer, D. C. R., Schiripo, T. A., Haber, D. A., and Hariharan, I. K. (2002) Salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines. Cell 110, 467−478. (5) Harvey, K. F., Pfleger, C. M., and Hariharan, I. K. (2003) The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell 114, 457−467. (6) Jia, J., Zhang, W., Wang, B., Trinko, R., and Jiang, J. (2003) The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. Genes Dev. 17, 2514−2519. (7) Pantalacci, S., Tapon, N., and Léopold, P. (2003) The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila. Nat. Cell Biol. 5, 921−927. (8) Udan, R. S., Kango-Singh, M., Nolo, R., Tao, C., and Halder, G. (2003) Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway. Nat. Cell Biol. 5, 914−920. (9) Bryant, P. J., Watson, K. L., Justice, R. W., and Woods, D. F. (1993) Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila. Dev. Suppl., 239− 249.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jennifer M. Kavran: 0000-0001-9117-5209 Notes

The authors declare no competing financial interest.

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Allostery, process by which binding of a molecule to one site of a macromolecule alters the function of a second, distal site on the same macromolecule; kinase, protein enzyme that catalyzes the transfer of the gamma phosphate of an ATP to a residue on a protein substratethe mammalian Hippo pathway kinases include Mst1/2 and Lats1/2, and both catalyze the phosphorylation of serine and threonine residues; molecular mechanism, how a macromolecule performs its function on a structural, biophysical, and biochemical levelthis process includes complex formation, conformational changes, and enzyme activity; signaling pathway, process by which a cell interprets an environmental cue to elicit a specific cellular response through a coordinated series of molecular events which can include complex formation, cellular localization, and changes in enzyme activity; FERM domain, cloverleaf-shaped fold with three subdomains that localizes to the membranein the Hippo pathway, FERM domains are found in Expanded and Merlin; WW domain, domain, named for two conserved tryptophan residues, that binds proline-rich motifs and mediates protein−protein interactionsthis domain is found in Hippo pathway components YAP, Salvador, and Kibra; MOB, family of tumor-suppressor proteins that shares a globular, α-helical fold and can function as allosteric activators for NDR-kinases, such as Lats1/2; SARAH domain, domain that mediates oligomerization through coiled-coil formation SARAH domains are found exclusively in Salvador, RassF, and Hippo homologues; YAP/TEAD, transcriptional coactivator complex that promotes transcription of pro-apoptotic and antiproliferative genesin Hippo signaling, phosphorylation of YAP prevents it from entering the nucleus and forming a complex with TEAD

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ACKNOWLEDGMENTS

We would like to thank S. Bailey and F. Fowl for critical reading of this manuscript and apologize to those whose work we could not cite due to space constraints. This work was funded in part by a grant (T32GM007445) from the National Institutes of Health. 607


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