ReviewDevelopmental Biology

Hippo Gains Weight: Added Insights and Complexity to Pathway Control

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Science Signaling  08 Oct 2013:
Vol. 6, Issue 296, pp. re7
DOI: 10.1126/scisignal.2004208

Abstract

The Hippo pathway is a kinase cascade, formed by Hippo, Salvador, Warts, and Mats, that regulates the subcellular distribution and transcriptional activity of Yorkie. Yorkie is a transcriptional coactivator that promotes the expression of genes that inhibit apoptosis and drive cell proliferation. We review recent studies indicating that activity of the Hippo pathway is controlled by cell-cell junctions, cell adhesion molecules, scaffolding proteins, and cytoskeletal proteins, as well as by regulators of apical-basal polarity and extracellular tension.

Introduction

How tissues regulate their growth and size during development is a fascinating question. The Hippo signaling pathway has emerged as a key regulator of cell proliferation and apoptosis in Drosophila melanogaster and mammals. We have begun to understand that the Hippo pathway integrates a large number of inter- and extracellular cues that are transformed into a well-balanced growth response of the cell. The Hippo pathway is critical in regulation of tissue growth, cell fate decisions, pluripotency, and suppression of cancer initiation and metastasis.

In Drosophila, the Hippo pathway consists of a core kinase cascade formed by the Ste20 kinase Hippo (Hpo) and the NDR family kinase Warts (Wts), as well as their adaptor proteins Salvador (Sav) and Mob as tumor suppressor (Mats), respectively (Fig. 1) [for reviews of the core pathway, see (16)]. Upstream regulators activate Hpo, and active Hpo subsequently phosphorylates Wts. Activated Wts phosphorylates the transcriptional coactivator Yorkie (Yki) at Ser168, which creates a binding site for 14-3-3 proteins and results in inhibition and cytosolic retention of Yki. Unphosphorylated Yki is active, enters the nucleus, and interacts with transcription factors, such as Scalloped (Sd), Teashirt (Tsh), Mothers against dpp (Mad), or Homothorax (Hth), to stimulate the transcription of growth-promoting and antiapoptotic genes, such as cyclinE, diap1, and the microRNA-encoding gene bantam (Fig. 1). Yki also promotes expression of genes encoding positive regulators of the Hippo pathway, such as ex, mer, and kibra, thereby providing negative feedback. Multiple cofactors, such as MASK, GAGA factor, and Tgi, modulate the transcriptional output of Yki (710). In this Review, we focus on control of the Hippo pathway in Drosophila by factors at the apical membrane, cell-cell junctions, and the cytoskeleton, which represent areas in which new insights into the regulation of Hippo signaling have been discovered in the last few years.

Fig. 1 The core of the Drosophila Hippo pathway.

Hpo corresponds to mammalian MST, Sav to Sav (also known as WW45), Wts to LATS1 and LATS2, Mats to Mob, Yki to YAP and TAZ, and MASK to MASK1 and MASK2 (also known as ANKHD1 and ANKRD17). On the left side, the Hippo kinase cascade is active, and Yki is excluded from the nucleus. On the right, the Hippo kinase cascade is inhibited, and Yki accumulates in the nucleus where it can interact with various transcription factors and regulate multiple target genes. Invertebrate epithelial cells have AJs and septate junctions, whereas vertebrate epithelial cells have AJs and tight junctions (TJs). In cells, inputs from the apical, basolateral, junctional regions, and the cytoskeleton may occur simultaneously or in various combinations, depending on the context. In Figs. 2 and 3, details of each regions inputs are detailed. c-Jun N-terminal kinase (JNK), Src, and many inputs only thus far identified in mammalian systems are omitted in the details of figures for clarity.

The Apical Membrane as a Platform for Hippo Signaling

Growing evidence suggests that the apical membrane of epithelial cells and apically localized junctions function as key regulators of Hippo signaling. In polarized epithelial cells of both vertebrates and invertebrates, the apical and basolateral cell surfaces are separated by adherens junctions (AJs) [reviewed in (11)]. Many members of the Hippo pathway are localized at the apical membrane or to the region just apical to AJs, the subapical membrane (Fig. 2A). Proteins that form AJs, as well as members of the apicobasal polarity complexes, have been implicated in upstream regulation of Hippo signaling in both mammalian and Drosophila systems.

Fig. 2 Upstream regulation of the Hippo pathway in fly epithelial cells from proteins localized in the apical and basolateral areas.

(A) Regulation by apically localized proteins and complexes. (B) Regulation by basolaterally localized proteins. Filled lines indicate direct biochemical interactions; dotted lines indicate regulation that has not, thus far, been demonstrated to be direct. Black lines connect signals that stimulate Hpo activity; green lines connect signals that stimulate Yki transcriptional activity.

At the subapical membrane of Drosophila epithelial cells, a complex formed by the FERM domain–containing proteins Expanded (Ex) and Merlin (Mer) and the WW domain–containing protein Kibra stimulates Hippo signaling (1218). Ex, Mer, and Kibra interact with each other, but are independently localized to the membrane. The proteins of this complex physically interact with the Hippo core pathway: Ex binds Hpo and Yki; Mer binds Sav; and Kibra binds Hpo, Sav, and Wts (1218). Although the molecular mechanisms of Hippo pathway activation are not well understood, interaction data suggest that the Ex-Mer-Kibra complex serves as a recruiting scaffold for active Hippo core pathway members at the subapical membrane. Bringing members of the core complex into close proximity might facilitate phosphorylation events within the kinase cascade. By binding Yki directly, independently of the phosphorylation state of Yki and canonical Hippo signaling, Ex retains Yki at the subapical membrane, preventing its translocation to the nucleus (19, 20). This might have a dual function, both to sequester unphosphorylated Yki and to recruit Yki to the Ex-Mer-Kibra complex and serve as a surface facilitating Wts phosphorylation of Yki.

The apical transmembrane protein Crumbs (Crb), a homophilic cell-cell adhesion molecule (2124), has a critical role in establishing and maintaining cell polarity (11) and in activating the Hippo pathway (2528). Experiments investigating the links between Ex and Crb suggest that localization of Ex is crucial for proper function. Deletion of Crb results in increased amounts of Ex, localization of Ex away from apical junctions, and inhibition of Hippo signaling, which leads to Yki activity and tissue overgrowth (2528). The degree of Ex mislocalization in cells lacking Crb varies in different reports and ranges from an almost complete loss from the apical surface (27) to a milder basal shift (25). In addition, overexpression of a truncated form of Crb lacking most of the extracellular domain depletes apical Ex (26, 28). However, expression of a different truncated Crb also leads to endocytosis of both the ectopic and the endogenous Crb protein (23, 29). Therefore, dissecting the effects of ectopically expressed truncated Crb is challenging.

A short FERM domain–binding motif in the intracellular domain of Crb directly interacts with Ex, and this interaction is proposed to recruit Ex to the membrane (25). This Ex-binding function of Crb is separable from its regulation of apical-basal polarity. Cell culture experiments showed that Ex is phosphorylated upon membrane localization by an unknown kinase (25). The exact function of phosphorylation of Ex remains to be determined, but correlates with Ex degradation (25). In mosaic wing imaginal discs with wild-type cells and crb-deficient cells, Ex is lost from the apical membranes of wild-type cells that are in contact with crb mutant cells, as well as from the apical membranes of the mutant cells themselves (27, 30). This suggests that Crb can regulate Ex in adjacent cells, possibly through Crb trans-homodimers across cells. This might represent a way of sensing social cues and transforming them into a growth response in the cell.

Although Crb has a clear effect on Hippo signaling through Ex, the overgrowth of crb mutant tissue can be mild (25, 27). Therefore, other factors are likely to be involved in Hippo and Ex regulation. Another candidate for Ex regulation is the large cadherin Fat (Ft). Genetic studies indicate that ft acts upstream to stimulate the Hippo pathway, and mutation of ft results in discs that are up to eight times larger than wild type (31). Loss of ft leads to increased expression of the Hippo pathway target genes diap1, cyclinE, and four-jointed, and tissue overgrowth is suppressed by loss of one allele of yki (12, 3234). In addition to regulating the Hippo pathway, Ft also regulates a form of tissue organization called planar cell polarity (PCP). Structure-function analysis has indicated that distinct regions of the cytoplasmic domain of Ft are involved in controlling PCP and growth (3539). Across cells, Ft binds the cadherin Dachsous (Ds), which is thought to function as a ligand for Ft (40). Ft-Ds binding is important for proper localization of each protein at the subapical membrane (4042), and the relative abundance of Ds in neighboring cells controls Ft’s activity in the Hippo pathway. Ds, which is expressed as a gradient in imaginal discs, transmits information to Ft about the steepness of its gradient and thereby the size of the tissue. Creating an artificial boundary of high versus low ds expression (for example, in clones) results in extra tissue growth in these regions (43, 44). The Golgi-localized kinase Four-jointed (Fj) regulates Ft-Ds binding affinity through phosphorylation of their extracellular domains (45, 46). Expression of ds and fj is, in turn, controlled by morphogens, such as Wingless, linking early patterning to growth regulation in development (44, 47). However, Ft’s function does not solely depend on Ds, because ft mutants have a stronger overgrowth phenotype than ds mutants, and ft mutant phenotypes can be largely rescued by expression of a form of Ft that cannot bind Ds on adjacent cells (35). Mutants of both ft and ds show additive growth defects, suggesting that they also have independent effects on Hippo signaling. This might be due to the ability of the Ds intracellular domain to regulate growth (35, 43). Ds can directly affect Hippo signaling by binding to the WD40 protein Riquiqui (Riq). Riq binds the kinase Minibrain (Mnb; known as Dyrk1a in mammals), which phosphorylates and inhibits Wts. Surprisingly, this interaction stimulates growth (48). Genetic studies suggest that Ft and Ds can form dimers in cis (interacting together on the same cell) (49), which may provide a mechanism for integrating these distinct pathways.

How does Ft regulate the Hippo pathway? Similar to crb mutant clones, ft mutant clones in the Drosophila eye and wing tissue show a reduction of Ex at the apical surface (3234). This might reflect an Ft-Ex signaling axis in which subapical Ex localization activates the Hippo core pathway by functioning as a recruiter for pathway members. However, Ft and Ex can also signal in parallel pathways, because in some tissues ft, ex double mutants show a stronger overgrowth phenotype than ft or ex single mutants (32, 50). Loss of ft also reduces the abundance of Wts. The effects of ft deficiency on the abundance of Wts are dependent on Dachs (D), an unconventional myosin that can interact directly with Wts in S2 cells and promotes Wts turnover (12, 51). D is a negative regulator of the Hippo pathway; thus, d mutant clones show the opposite phenotype of ft mutant clones (12, 51). Genetic experiments suggest that Ft inhibits D and that the palmitoyltransferase Approximated can relieve Ft’s inhibition of D and promote the apical localization of D (52). Whether inhibition of D can fully explain the stimulation of Hippo signaling by Ft and how D is molecularly linked to Ft remains to be addressed. D also binds the cytoplasmic domain of Ds, suggesting that the D impact on the Hippo pathway may be through Ds (53).

Ft signaling is also regulated by the casein kinase Discs overgrown (Dco), which phosphorylates and activates the intracellular domain of Ft. Ds binding to Ft promotes this phosphorylation (54, 55). Mutation of dco results in reduced Ft phosphorylation and increased tissue growth (54, 55).

It is striking that the best-described cell surface regulators of the Hippo pathway in Drosophila, Crb and Ft, are large cell-cell adhesion receptors, with unusually large well-conserved extracellular domains. We propose that their large size leads to exclusion from the adherens junctional area, setting up a subapical Hippo signaling domain. This size-dependent subdivision of signaling is reminiscent of the establishment of signaling domains in the “immunological synapse” (56).

Hippo Regulation from Basolateral Cell-Cell Contacts

Besides Crb, other members of polarity complexes have been implicated in Hippo signaling (Fig. 2B). Scribble (Scrib), Discs large (Dlg), and Lethal giant larvae (Lgl) are polarity proteins that are localized to the basolateral membrane and antagonize the apical Par complex, composed of atypical protein kinase C (aPKC), the polarity protein Par6, and the multiple PDZ domain protein Bazooka (also known as Par3). Cells mutant for scrib, dlg, or lgl show increased proliferation and increased CyclinE abundance. lgl mutant clones overproliferate without loss of apical-basal polarity (57), suggesting that the polarity complex proteins have roles in proliferation that are separable from their function in polarity. Genetic experiments indicate that the effect on cell proliferation of scrib, dlg, or lgl mutants involves Yki activation (28, 5760), but the molecular mechanism remains obscure. Drosophila Rassf (dRassf, Ras association domain family) functions as an inhibitor of Hpo by suppressing its interactions with Sav (61). The localization of Hpo and dRassf is basolaterally shifted or broadened in lgl mutant clones in the eye disc, which might be the cause of Yki activation (28). No direct biochemical links have been made between Yki and Scrib, Dlg, or Lgl in Drosophila epithelial cells. However, in mammalian cancer cells, Scribble binds to the Yki homolog TAZ and promotes its phosphorylation and degradation (62), suggesting a mechanism by which signals from the basolateral membrane lead to Hippo pathway activation and growth suppression. Whether a similar mechanism functions in Drosophila is unknown.

Another Par protein, the serine-threonine kinase Par-1, promotes proliferation by inhibiting the Hippo pathway. Genetic experiments indicate that Par-1 acts downstream of ft and ex and upstream of hpo. Par-1 phosphorylates and represses Hpo, inhibiting its kinase activity (63).

AJs as Signaling Centers

Drosophila epithelial cells are divided into apical and basolateral domains by AJs (Fig. 3A). AJs connect cells through classical cadherins and are associated with actin accumulation. Above the AJ lies the subapical membrane, which is marked by the accumulation of Ft, Ds, aPKC, and Crb. Along the basolateral surface lie septate junctions, which regulate the paracellular passage of small molecules between the basal and the apical sides, analogous to mammalian TJs [for comprehensive reviews on junctions in both systems, see (64, 65)].

Fig. 3 Upstream regulation of the Hippo pathway in fly epithelial cells by junctional-associated proteins and proteins associated with the cytoskeleton.

(A) Regulation by proteins associated with AJs. (B) Regulation by mechanical tension and the cytoskeleton. Filled lines indicate direct biochemical interactions; dotted lines indicate regulation that has not, thus far, been demonstrated to be direct. Black lines connect signals that stimulate Hpo activity; green lines connect signals that stimulate Yki transcriptional activity.

The transmembrane cell adhesion molecule Echinoid (Ed) is a component of AJs that stimulates Hippo pathway signaling (66). Ed RNAi (RNA interference) promotes Yki target gene expression and results in tissue overgrowth. In S2 cells, Ed binds Sav and promotes its stability. FRET (fluorescence resonance energy transfer) studies indicate that these proteins directly interact and that both Sav and Ed can also form homodimers. Ed and Sav colocalize at AJs in wing discs, and the abundance of Sav is reduced at AJs when Ed is depleted, suggesting that Ed recruits and stabilizes Sav at the AJ (66). Coimmunoprecipitation experiments from cell culture also uncovered Ex, Mer, Kibra, and Yki as Ed interactors, but the biological relevance of these interactions is unclear because the apical localization of Ex and Mer was unchanged upon Ed knockdown (66). Ed likely presents a different signaling branch than Ft, because D knockdown does not suppress Yki target gene expression induced by Ed RNAi, and knockdown of Ds and Ed has additive effects on a Yki reporter in the wing disc (66). In cell culture studies, Ed dimerization and membrane localization of Ed were critical for its stimulation of Hippo signaling (66). Ed mediates cell-cell adhesion by forming homophilic interactions with molecules from neighboring cells (67), and Sav and Ed preferentially colocalize at cell contact sites, suggesting that cell-cell contact can facilitate the Ed-Sav interaction (66). These findings imply that Ed may sense cellular social cues about cell density and cell contact and transmit them to the Hippo pathway.

Another protein associated with AJs is the Drosophila ortholog of mammalian Ajuba LIM domain proteins, dJub. dJub mutants mimic Yki depletion, with reduced proliferation and increased apoptosis, and the expression of Yki target genes is reduced in dJub mutant clones (68). Thus, dJub is a negative regulator of the Hippo pathway. Epistasis experiments suggest dJub signals downstream of hpo but upstream of wts (68), and dJub knockdown suppresses the overgrowth phenotypes caused by reduction of Ft and Ex by RNAi depletion (69). The epidermal growth factor receptor (EGFR)–Ras–Raf–mitogen-activated protein kinase (MAPK) pathway affects Hippo signaling by promoting dJub activity, and constitutively active EGFR signaling results in Yki-dependent tissue overgrowth (70). In cell culture overexpression experiments, dJub binds to Wts and Sav (68), and binding increases with activated MAPK signaling (70). dJub also weakly binds to Dachs (69). dJub is phosphorylated in an extracellular signal–regulated kinase (ERK)–dependent manner, and the abundance of overexpressed dJub is greater in S2 cells with ectopic MAPK activation, suggesting that ERK-mediated phosphorylation may stabilize dJub (70). We note that it will be important to determine whether endogenous dJub is regulated by EGFR signaling during fly development.

Similar to the fly system, mammalian homologs Ajuba, LIMD1, and WTIP bind the kinases LATS1 and LATS2 (collectively referred to as LATS1/2), the mammalian orthologs of Wts (68). EGFR-Ras-MAPK can signal through WTIP to promote YAP activity (70), suggesting a role for this pathway in inhibiting mammalian Hippo signaling. Mammalian Ajuba LIM protein localization depends on cell culture density: They are cytosolic in subconfluent settings and become junctional when confluency is achieved (71). Thus, Ajuba proteins might transmit cell density signals to the Hippo pathway and control contact inhibition. In addition, Ajuba proteins are phosphorylated by JNK, which promotes interactions with Wts or LATS proteins, providing integration with apoptotic and injury signaling (72).

Zyxin (Zyx), another LIM domain–containing protein, also inhibits the Hippo pathway. RNAi experiments have indicated that Zyx-depleted tissue shows reduced proliferation and Yki target gene expression and that Zyx is epistatic to ft, dco, and probably d, but not to ex (69). Zyx can bind D when both are expressed in S2 cells (69), and, like human Zyxin with LATS1/2 (73), the C-terminal LIM domains bind Wts, but only in the absence of the N-terminal portion (69). This hints that in a basal state, this interaction surface of Zyx is not accessible but opens upon stimulus. In wing discs, Zyx colocalizes with Dachs and E-cadherin. Despite their similar phenotypes when depleted, Zyx and dJub seem to play at least partially distinct roles, because Zyx overexpression fails to rescue dJub knockdown phenotypes and Zyx, dJub double knockdown has additive effects on growth reduction in the wing (69). In mammals, Zyxin associates with the cytoskeleton and can shuttle to the nucleus, raising the possibility that these features play a role in Hippo pathway regulation (74).

In addition, there are other apically localized factors, such as the FERM-containing tyrosine phosphatase Pez, which works with Kibra to restrict Yki activity in the developing intestine (75). The vertebrate ortholog of Pez, PTPN14, inhibits YAP activity and is localized to AJs (7678).

Cytoskeleton and Mechanical Forces in Hippo Signaling

How can cell-cell interaction within a tissue serve as a meaningful signal for growth during normal development? As soon as epithelial sheets are formed, cells are in constant contact with each other (except in disease states). This paradox has led to the investigation of cytoskeletal and mechanical forces as regulators of growth, following the rationale that as cells become more tightly packed, tensions arise in the sheet that could convey “crowding” information to cells. There is growing evidence both in Drosophila and in mammals that the cytoskeleton integrates mechanical cues like cell tension to the Hippo pathway to regulate a growth response (Fig. 3B).

In Drosophila, several lines of evidence suggest that filamentous actin (F-actin) inhibits the Hippo pathway. Mutation of genes encoding negative regulators of F-actin, such as capping protein a and b (cpa, cpb), increases Yki activity in cultured Drosophila S2 cells, whereas depletion of proteins that stimulate the formation of F-actin or destabilizing F-actin with cytochalasin D inhibits Yki activity (79). Loss of Cpa or Cpb or increased abundance of active Diaphanous (Dia), which is a stimulator of F-actin formation, results in overgrowth and Yki target gene expression (79, 80). Epistasis experiments suggest that F-actin signals upstream of wts but in parallel to hpo and ex. Similar results are observed in human HeLa cells, indicating a conserved role of F-actin in Hippo signaling (79). No major changes in cell architecture or other signaling pathways were observed upon Cpa loss or Dia activation, suggesting the effect is due to increased amounts of F-actin (79). F-actin also affects the Hippo pathway in a nonproliferating system, such as the adult Drosophila retina, an organ that requires Ft signaling through the Hippo pathway for maintenance of neuronal homeostasis (81). Knockdown of Cpa in photoreceptors results in neuronal degradation, which can be rescued by overexpression of hpo or ex or knockdown of Yki (82).

In turn, Hippo pathway components inhibit F-actin formation, because clones mutant for ex, hpo, sav, wts, or mats in wing discs show accumulated F-actin at the apical membrane, similar to cells with reduced Cpa abundance (80). However, in larval wing discs, regulation of F-actin by Hippo core pathway members does not seem to involve Yki, because overexpression of yki does not alter F-actin formation, and knockdown of Yki in ex clones does not rescue F-actin accumulation (80). In contrast, overexpression of Yki in the pupal wing leads to increased F-actin formation (83). Therefore, the Hippo pathway kinases may regulate actin organization in both Yki-dependent and Yki-independent pathways, and this is likely dependent on tissue and developmental stage.

Additional Regulators of the Hippo Pathway

The nonreceptor tyrosine kinase Src has been implicated in regulation of AJs, TJs, and the cytoskeleton [reviewed in (84)]. Overexpression of src in clones in the Drosophila eye and wing disc markedly induces proliferation of the surrounding wild-type cells (85). This nonautonomous proliferation depends on Yki and JNK signaling in the clone and leads to activation of Yki in the surrounding wild-type cells. It is not clear what is responsible for activating Yki in the surrounding cells, but induction of F-actin within the src mutant clone was essential for the Src-mediated cell nonautonomous activation of Yki (85). This is an exciting insight into how tumor cells, which tend to have disrupted cell junctions, may promote proliferation of normal cells during cancer development.

The Ste20 kinase Tao-1 is another newly described upstream regulator of the Hippo pathway (86, 87). In Drosophila, Tao-1–depleted tissue overgrows and has increased Yki target gene expression. In vitro, Tao-1 phosphorylates Hpo at Thr195 (86), a critical phosphorylation site necessary for Hpo autophosphorylation and Hpo catalytic activity (88). Epistasis experiments place tao-1 upstream of hpo, and in S2 cells, Tao-1 is required for Mer- and Ex-mediated phosphorylation and activation of Wts (86, 87). Tao-1 has also been implicated, in both Drosophila and mammalian systems, in microtubule regulation (89, 90), providing potential links to this part of the cytoskeleton.

Recent Insights from Mammalian Systems

Although we have here focused on recent insights from Drosophila, there is a large body of exciting data emerging from studies in mammalian systems. Mammalian tissue culture studies were the first to show that the Hippo pathway has a role in cell contact inhibition and as a sensor of social cues (91). As in Drosophila, cell junctions connect cells and present an ideal target for linking the detection of cell density to control of proliferation (Fig. 3A). There are two major forms of cell-cell junctions in mammals, AJs and TJs. The TJ, which is marked by zonula occludens 1 (ZO1) (92), lies apical to AJ and regulates the paracellular passage of small molecules. Mammalian data indicate that components of both AJs and TJs impact the activity of the two mammalian Yki homologs YAP and TAZ [reviewed in (6, 93, 94) and collectively referred to as YAP/TAZ].

Biochemical studies with mammalian cells have identified many components of AJs and TJs as regulators of YAP/TAZ activity. Molecules that are enriched at sites of cell contact or junctional complexes include Crb, PALS1, PATJ, ZO2, Angiomotin, NF2, Scribble, cadherins, and α-catenin (62, 95106). Although the core pathway [Hpo (represented by MST1 and MST2 in mammals), Sav (Sav), Wts (LATS1/2), Mats (Mob), and Yki (YAP/TAZ)] is well conserved from flies to man, conservation of the upstream regulators is not as clear. For example, Crb regulates the Hippo pathway in both flies and mammals; however, it works through Ex in flies (2528), and in mammals, it is thought to function through junctional proteins, such as PALS1 and PATJ (106). Whereas Ft regulates the Hippo pathway in flies, in mammals, the closest homolog Fat4 does not appear to regulate Hippo activity (37, 39, 107). However, the less closely related cadherin Fat1 has been implicated in control of YAP in zebrafish (108), as has the chicken Ft homolog Fat-j (109). Also, although there are strong data that D has an important role in the Drosophila Ft pathway, there is no D homolog in mammals.

An important emerging theme is that growth signaling at junctions sometimes includes the core Hippo pathway, but in other cases does not involve the core kinases. For example, YAP is found in a complex with α-catenin at junctions, and α-catenin is necessary for nuclear exclusion of YAP at high cell density. However, disruption of α-catenin does not alter YAP phosphorylation, and regulation of YAP activity by α-catenin seems independent of MST and LATS (9597). It is not yet understood how the many junctional proteins work together to communicate cell density to YAP/TAZ and Yki.

Mammalian cell culture experiments indicate that the Yki homologs YAP and TAZ are regulated by the stiffness of the extracellular matrix (ECM) or the size of available matrix islands. Cells on stiff ECM and large surfaces that allow cell spreading have active nuclear YAP/TAZ, whereas soft ECM and restricted adhesive surfaces lead to inactivation of YAP/TAZ and increased cytoplasmic retention (110, 111). The cellular response to these different types of mechanical inputs depends on F-actin and the myosin regulators Rho-dependent kinase (ROCK) and myosin light chain kinase (MLCK), because knockdown of ROCK or MLCK or inhibition of F-actin formation results in YAP/TAZ inhibition. However, YAP/TAZ regulation by mechanical forces seems to be independent of canonical Hippo signaling or phosphorylation mediated by LATS (110). The small GTPase (guanosine triphosphatase) Cdc42, which controls actin dynamics, stimulates YAP nuclear localization and activity in the developing mouse kidney (112). Under the control of Cdc42, YAP stimulates the expression of various genes involved in cell morphology and signaling, driving subsequent steps in the morphogenesis of developing nephrons (112). Thus, YAP appears to respond to cytoskeleton-mediated signals that occur as a tissue develops, thereby inducing subsequent gene expression programs.

YAP/TAZ activity is also regulated by G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs), which signal through F-actin and Rho GTPases to LATS (113). GPCRs respond to various extracellular ligands and can either positively (for example, by epinephrine or glucagon) or negatively (by lysophosphatidic acid and sphingosine 1-phosphate) regulate YAP/TAZ activity (113). This new dimension of signaling implies hormonal control of growth and that LATS activation status depends on ligand availability and the receptor profile of a cell. Thus, hormonal stimulation provides additional inputs to the regulation by cell contacts, cell tension, and cell polarity to control proliferation in vivo. In addition, loss of attachment to the ECM activates the Hippo pathway and results in anoikis, a type of apoptosis. YAP inhibition is key for initiating anoikis, and cancer cells that are resistant to anoikis can undergo this form of cell death upon YAP/TAZ knockdown in cell culture (114). Resistance to anoikis is a common feature of cancer cells and promotes metastasis.

An explosion of data in the past few years has identified multiple inputs from the cell surface and the cytoskeleton, greatly increasing our understanding of the intricate web of inputs that control Yki- and YAP/TAZ-dependent control of growth. In Drosophila, it is not well understood how the large adhesion molecules Ft and Crb function to regulate Hippo activity, although the FERM protein Ex may provide essential links. Tao-1–mediated kinase regulation of Hippo activity is conserved, but how Tao-1 in turn is regulated is unclear. It is also not yet understood if GPCRs regulate Yki activity in Drosophila, or if that control is a mammalian adaptation of the pathway. The actin cytoskeleton plays a central role in growth control, but we do not know how the state of actin polymerization and tension are communicated to regulate YAP/TAZ or Yki. Unraveling the intricate web of cytoskeletal and cell surface signals in growth will be an exciting and important challenge for future research.

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