FEBS Letters 588 (2014) 2743–2752

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Review Molecular insights into NF2/ tumor suppressor function ⇑ ⇑ Jonathan Cooper , Filippo G. Giancotti

Cell Biology Program, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY, United States article info abstract

Article history: The FERM domain Merlin, encoded by the NF2 tumor suppressor , regulates cell prolif- Received 26 February 2014 eration in response to adhesive signaling. The growth inhibitory function of Merlin is induced by Revised 1 April 2014 intercellular adhesion and inactivated by joint integrin/ tyrosine kinase signaling. Merlin Accepted 2 April 2014 contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling Available online 12 April 2014 at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes Edited by Shairaz Baksh, Giovanni Blandino uncontrolled mitogenic signaling and tumorigenesis. Merlin’s predominant tumor suppressive and Wilhelm Just functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4DCAF1 E3 Keywords: ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function Merlin has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti- Neurofibromatosis Type 2 mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4DCAF1 and activation of Hippo signaling. The biochemical events underlying Merlin’s normal Contact inhibition function and tumor suppressive activity will be discussed in this Review, with emphasis on recent DDB1 and Cul4-Associated Factor 1 discoveries that have greatly influenced our understanding of Merlin biology. CRL4 E3 ubiquitin ligase Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction the Schwann cells comprising the myelin sheath surrounding sen- sory and motor neurons, arachnoid cap cells within the arachnoid The ability of normal cells to survive and proliferate is dictated villi in the meninges, and ependymal cells lining the CSF-filled ven- by environmental cues, including intercellular and matrix tricles of the brain and central spinal canal, respectively. In addi- adhesions as well as the presence of growth factors. Normal cells tion to the autosomal dominant NF2 disorder, non-germline undergo growth arrest when detached from the extracellular ma- Merlin deficiency is a driving force in sporadic occurrences of trix or when they come into contact with adjacent cells and form nearly all vestibular schwannomas, a majority of meningiomas, intercellular junctions [1]. Neoplastic cells evade contact inhibition and a notable fraction of ependymomas. Moreover, Merlin was of proliferation, which leads to the disruption of tissue organiza- found to be inactivated in a large proportion of malignant mesot- tion – a distinguishing event in cancer [2]. The NF2 (Neurofibroma- heliomas [8,9] and to a lesser extent in other solid tumors [10–13]. tosis Type 2) tumor suppressor gene encodes the FERM (4.1 Due in large part to Merlin’s high to the protein/Ezrin/Radixin/Moesin) domain protein Merlin, which is ERM (Ezrin/Radixin/Moesin) family of cytoskeletal linker , coordinately regulated by intercellular adhesion and attachment it was widely assumed that Merlin suppresses mitogenic signaling to the extracellular matrix [3–7]. Cadherin engagement inactivates at the cell cortex to mediate contact inhibition and tumor suppres- PAK, causing an accumulation of the active, dephosphorylated sion [14]. Active Merlin suppresses Rac-PAK signaling [7,15–17], form of Merlin. Notably, since integrin attachment to the extracel- restrains activation of mTORC1 independently of Akt [18,19], lular matrix activates PAK, Merlin can therefore be regulated inhibits PI3K-Akt and FAK-Src signaling [20,21], and negatively independently of contact-mediated signaling events [7]. regulates the EGFR-Ras-ERK pathway [22,23]. However, the mech- Neurofibromatosis type 2 patients carry a single mutated NF2 anisms by which Merlin inhibits these pathways remain unclear, allele and develop a highly specific subset of central and periphe- and furthermore, the relative contribution of these pro-mitogenic ral nervous system tumors. NF2-associated tumors include signals to various Merlin-deficient malignancies is unknown. Inter- schwannomas, meningiomas, and ependymomas, which arise from estingly, Merlin activates the Hippo tumor suppressor pathway to suppress the transcriptional coactivators YAP/TAZ in mammals or

⇑ Fax: +1 212 639 3917 (F.G. Giancotti). the Drosophila ortholog Yorkie, revealing a conserved role for E-mail addresses: [email protected] (J. Cooper), [email protected] Merlin in regulation of organ size, stem cell behavior, and cell pro- (F.G. Giancotti). liferation [24–26]. Notably, few germline or somatic mutations of http://dx.doi.org/10.1016/j.febslet.2014.04.001 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2744 J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752

Hippo pathway components have been discovered in human tu- to suppress oncogenic gene expression [29]. Moreover, Merlin’s mors – the exception being Merlin, which remains as one of the regulation of YAP/TAZ is a burgeoning and highly provocative field only bona fide tumor suppressors in the Hippo pathway. due to the multifarious roles of Hippo signaling in organ growth, Merlin’s upstream regulation has been extensively stem cell maintenance, and cancer. Recent studies have also shed characterized, and recently reported models of regulation and light on Merlin’s conformational changes that regulate its intramo- post-translational modification will be highlighted in this Review. lecular associations and downstream signaling, providing funda- Post-translational modification of Merlin is vitally important to its mental insight into Merlin’s regulation and biochemical function. conversion between dormant and growth-inhibitory states, where In this Review, we will explore Merlin’s rich biochemical back- the dephosphorylated and more open state is now considered to be ground and examine new insights that are shaping our understand- active in contact inhibition and tumor suppression [27]. However, ing of Merlin’s role in normal biology and how its loss leads to the without crystallographic analyses of full-length Merlin in both its deregulation of a multitude of signaling pathways leading to active and inactive conformations, structural inferences drawn tumorigenesis. from biochemical experiments must be approached with caution. Apropos of the myriad upstream signals and modifications affect- 2. The NF2 gene ing Merlin, the downstream biochemical functions of Merlin have been the subject of intensive research for two decades, yet a con- NF2 maps to the long arm of 22 and encodes two sensus mechanism for Merlin’s function in normal tissues has not Merlin isoforms. The longer, dominant Merlin isoform 1 (Merlin-1 been reached. It is becoming apparent that Merlin functions or Merlin), has an extended carboxy-terminal tail that is encoded primarily in two branches – contact inhibition of proliferation by exon 17 (Fig. 1A). Merlin isoform 2 (Merlin-2), on the other and tumor suppression. Although these branches are naturally hand, contains the alternatively spliced exon 16 which ends in a intertwined, the distinct locations of Merlin function and respec- stop codon, encoding 11 unique residues following amino acid tive interactors in those subcellular compartments lend credence 579 as compared to Merlin-1 [10]. Notably, Merlin-2 lacks to a concept of a bimodal function. carboxy-terminal residues required for intramolecular binding be- Recent studies among the ever-increasing breadth of NF2 litera- tween the amino-terminal FERM domain and the carboxy-terminal ture have revealed important high-affinity interactors governing tail, possibly leading to a constitutively open conformation [30,31]. Merlin’s biochemical function. Notably, Merlin regulates tight Recent studies have found that Merlin-2 inhibits cell proliferation junction-associated Angiomotin to inhibit Rac signaling [28], and and attenuates downstream mitogenic signaling to the same DCAF1 nuclear-localized Merlin inhibits the CRL4 E3 ubiquitin ligase extent as Merlin-1 [27,32].

A FERM Coiled-Coil C-term 1 17 98 112 212 220 311 506 595 Subdomain A Subdomain B Subdomain C Exon 1 2 3 4567 8 9 1011 12 13141517 Mutation Frequency 1 59

B Contact-inhibited cells Proliferating cells

GPCR

Nucleus Active Mer Nucleus

AJs cAMP

PAK PKA PAK

P P Rac Inactive Mer Inactive Mer MYPT1 Active Mer

Integrins RTKs

Matrix

Fig. 1. (A) Merlin structure and overview of pathogenic mutations. Merlin is a 595-residue protein divided into three structurally distinct regions – an amino-terminal FERM domain, an a-helical coiled-coil domain, and a carboxy-terminal hydrophilic tail. Merlin’s FERM domain is further subdivided into three globular subdomains. Nf2 encodes 16 exons, terminating with exon 17 in the canonical Merlin isoform. The positions of patient-derived missense mutations or single residue deletions are indicated below the exon schematic, with mutation frequency indicated by a heat map. Either these mutations underlie NF2 tracked within a family, were found in two or more unrelated patients, or experimental evidence was obtained to confirm their pathogenicity. Mutational data were obtained from [48] and [29]. (B) Adhesion-mediated regulation of the Merlin activation cycle. (Left) In contact-inhibited cells, dephosphorylated Merlin accumulates as a result of intercellular adhesions which lead to PAK inhibition. Merlin may also be activated via MYPT1-mediated dephosphorylation. (Right) Conversely, in proliferating cells, integrin-mediated anchorage to the cell matrix and stimulation of receptor tyrosine kinases (RTKs) activate Rac, in turn activating PAK and leading to phosphorylation of Merlin at Serine 518. In response to high cyclic AMP levels, PKA also phosphorylates Merlin at serine 518. Serine 518 phosphorylation increases the interdomain binding between Merlin’s carboxy-terminus and FERM domain, maintaining Merlin in a more closed, inactive form. J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752 2745

3. Merlin protein structure and intramolecular association not suppress cell growth, whereas Merlin-2 and the S518A mutant – which are both defective in interdomain binding and therefore The biochemical functions of Merlin are imparted by three more open – suppress growth to the same extent as wild-type Mer- mechanisms – post-translational modification, localization, and lin. Cumulatively, these results reveal that S518 phosphorylation interaction with downstream effectors. Merlin is composed of a or mutations that cause Merlin to become more closed result in de- globular amino-terminal FERM domain and a carboxy-terminal creased growth inhibition, whereas more open forms of Merlin – hydrophilic tail joined by a flexible coiled-coil segment, displaying those that are increasingly defective in interdomain binding – are structural resemblance to the FERM domain-containing ERM pro- more active in growth inhibition. This finding contributes greatly teins (Fig. 1A) [33,34]. This resemblance is most apparent in the to a consensus of Merlin structure and function as it relates to nor- first 300 residues of Merlin’s FERM domain, which share 65% se- mal biology and tumor suppression, resolving a decade-old schism quence identity with canonical ERMs. However, Merlin has several between biochemical observations and in vivo genetic studies. notable differences in its primary sequence that distinguish it in Biochemical studies have provided essential insight into Mer- form and function from its ERM counterparts. Merlin’s amino-ter- lin’s conformational changes and resulting activation, which are minus contains a 7-residue conserved Blue box motif and 17 un- now largely corroborated by genetic data. Most patient-derived ique amino-terminal residues [35,36]. Importantly, Merlin lacks a truncations and a vast majority of non-truncating mutations occur canonical actin-binding motif that is otherwise conserved among within the FERM domain (Fig. 1A), lending credence to its quintes- ERMs and paramount to their function at the cortical cytoskeleton sential role in anti-mitogenic signaling and tumor suppression [14]. [48]. Initially discovered in Drosophila, the ‘‘Blue box motif’’ is a A central mechanism in Merlin and ERM function is the forma- conserved region within the FERM subdomain F2 corresponding tion of homo- and heterotypic interactions that regulate their to residues 177–183 in human Merlin. Substitution of these resi- activity. The majority of ERMs within cells are held in a dormant dues with alanines imparts loss of contact inhibition and promotes state imparted by an intramolecular association between the FERM tumorigenesis in NIH-3T3 cells [49]. Mutations in the Blue box act domain and the C-terminal tail [37–39]. Disruption of this associ- in a dominant negative manner in both Drosophila and mammals, ation and subsequent activation is achieved by phosphorylation displaying the importance of the FERM domain in Merlin’s tumor of an ERM C-terminal threonine residue by Rho kinase. Active suppressive function. As compared to the FERM domain, mutations ERMs bind to cytoplasmic domains of cell-adhesion receptors such are scarce in Merlin’s carboxy-terminal tail and are dramatically as CD44 and ICAM via their FERM domains while their C-termini reduced in the central a-helical domain, again implying that Mer- interact with and regulate the cortical actin cytoskeleton [40,41]. lin’s downstream tumor suppressive functions are imposed by the Furthermore, once opened, active ERM proteins may form interac- FERM domain (Fig. 1A) [48]. Notably, replacement of Merlin’s F2 tions amongst themselves and with Merlin [37]. Merlin’s intramo- subdomain with that of the ERM protein Ezrin abolishes its growth lecular associations and subsequent activation states have been a suppressive activity, while individual or combined replacement of subject of intensive study for nearly 20 years [27,30,31,38,42– subdomains F1 and F3 do not affect proliferation [32]. These results 44], yet a consensus model to explain the relationship between imply that subdomain F2 of Merlin’s FERM domain is largely its phosphorylation and interdomain binding in the context of responsible for Merlin’s growth inhibitory effects. Truncating seemingly contrary genetic evidence has only recently been mutations correlate with increased NF2 severity and predominate uncovered. among NF2 patients, while missense mutations – although more It is well established that Merlin switches from an active state rare and generally less severe – allow for a more systematic inter- to an inactive state by phosphorylation at serine 518 (Fig. 1B) pretation of Merlin’s biochemical role in tumor suppression. Mer- [45]. Phospho-mimetic (S518D) or phospho-deficient (S518A) per- lin’s FERM domain interacts directly with the CRL4DCAF1 E3 mutations strongly correlate with Merlin’s growth-permissive or ubiquitin ligase, inhibiting its ubiquitination activity and down- growth-inhibitory functions, respectively [27,29,46]. Essentially, stream oncogenic signaling. Multiple patient-derived missense serine 518 phosphorylation acts as a binary switch in which mutants, including those mapping to subdomain F2, are defective dephosphorylated Merlin functions in adhesion signaling while in CRL4DCAF1 interaction, despite the fact that some of these muta- phosphorylated Merlin remains dormant, although studies within tions were predicted to cause little structural perturbation [29,34]. the last five years have shown this to be an oversimplified interpre- Recent biochemical insights combined with more than a decade tation. Several lines of evidence suggest that Merlin exists in multi- of patient-derived genetic data provide crucial insight into Merlin’s ple states that vary between ‘‘fully open’’ and ‘‘fully closed’’ forms normal biochemical function and role in tumor suppression. Mer- which are regulated primarily by S518 phosphorylation, and more- lin’s a-helical domain and carboxy-terminus function in maintain- over, FRET studies reveal that Merlin’s FERM domain and carboxy- ing Merlin in an inactive conformation during normal biological terminus are constitutively held in close proximity, undergoing function, and may further be necessary for functions underneath only subtle changes upon post-translational modification [27,47]. the cell cortex in the organization of cell junctions and in contact A classic study found that Merlin-2 – which lacks the carboxy-ter- inhibition. However, while interdomain binding is an important minal residues necessary for intramolecular interaction – is unable part of Merlin’s regulation and normal cellular functions, the over- to function in contact inhibition or tumor suppression [30]. This all structure and specific protein-interacting domains within Mer- observation led to a hypothesis that was held for over a decade lin’s amino-terminal FERM domain are essential for contact positing that dephosphorylated and active Merlin functions in a inhibition and tumor suppression. closed conformation. Recently, two independent laboratories found that Merlin-2 4. Post-translational modification of Merlin suppresses growth in mammalian cell lines, suggesting that inter- domain binding is dispensable for Merlin’s adhesion signaling Merlin is regulated by multiple post-translational modifications [27,32]. Through comprehensive biochemical investigation, downstream of an ever-expanding array of intracellular and extra- Anthony Bretscher’s group reported that phosphorylated Merlin cellular cues. Receptor tyrosine kinases and integrins relay pro- displays higher interdomain binding and that Merlin therefore mitogenic signals by activating CDC42 and Rac, which in turn acti- inhibits cell growth in its open state [27]. These results are under- vate p21-activated kinase (PAK) (Fig. 1B). Activated PAK directly scored by the observation that a stably closed Merlin mutant does phosphorylates Merlin at serine 518, leading to an increase in 2746 J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752 carboxy-terminal association with the FERM domain and A Merlin Regulation of the subsequent inactivation, presumably through masking of protein- Hippo Pathway interacting domains on the FERM domain which are necessary Sav1 Mer CRB for downstream signaling or occlusion of a nuclear localization sig- Mst1/2 Lats1/2 TJs 2 Pals1 nal [29,33]. Since Merlin has a long half-life within cells, it is a Mst1/2 Patj compelling hypothesis that phosphatases exist to dephosphorylate Lats1/2 AMOT Merlin at serine 518 and dynamically control levels of the dephos- AJs 3 YAP/TAZ Mer phorylated active form. MYPT1-PP1d (MYPT1) is a phosphatase 1 β-cat that was shown to dephosphorylate Merlin [22] (Fig. 1B), and α-cat recent evidence revealed that it may be inhibited by integrin- Mer Mer linked kinase (ILK) [50]. It remains to be determined whether 4 AMOT P CRL4DCAF1 MYPT1-mediated Merlin activation via dephosphorylation is a YAP/TAZ YAP/TAZ Ub major mechanism in NF2-mediated tumor suppression or whether UbUb P Lats1/2 TEAD nascent and dephosphorylated Merlin largely functions in this capacity. P YAP/TAZ The phosphorylation status of serine 518 drives Merlin’s activa- tion cycle, although other modifications modulate Merlin’s confor- Suppression of CRL4DCAF1-mediated mation and downstream biochemical functions. Phospho-peptide B Gene Expression mapping and band-shift experiments revealed that Merlin is phos- phorylated at multiple sites and that serine 518 phosphorylation P may be necessary for subsequent phosphorylation at other residues Active Mer Mer [51,52]. In addition to PAK-mediated phosphorylation, PKA was Inactive shown to independently phosphorylate Merlin at serine 518 and AJs Mer serine 10, the latter modification affecting the actin cytoskeleton PAK DCAF1 [53,54]. Merlin’s phosphoregulation by PKA may be an important CRL4

mechanism in cells which are sensitive to the cyclic AMP-PKA sig- Ub UbUb P Transcription factors naling axis, including the pathogenically relevant Schwann cells Lats1/2 Epigenetic regulators P YAP/TAZ [55]. AKT phosphorylates Merlin at two additional sites – threonine P 230 and serine 315 – and this phosphorylation causes decreased YAP/TAZ interdomain binding, intermolecular binding to phosphoinositides Oncogenic and PIKE-L (a GTPase that enhances PI3K activity), and ubiquitina- gene expression tion [56]. The observation that AKT-mediated phosphorylation blocks Merlin’s interaction with PIKE-L supports an inhibitory role for this interaction [20,56]. This finding also raises the possibility Matrix that constitutive activation of the PI3K-Akt pathway resulting from PTEN loss or activating PI3K mutations would lead to Merlin inacti- Fig. 2. (A) Merlin activates the Hippo pathway to suppress YAP/TAZ through vation, imparting a PI3K feed-forward mechanism. However, spec- regulation of core kinase components at the plasma membrane, through inhibition of the CRL4DCAF1 E3 ubiquitin ligase in the nucleus, and by regulating cell junction- ulation that Merlin is inactivated by decreased interdomain associated proteins that modulate Hippo signaling. (1) At adherens junctions, E- binding conflicts with the recent observations that the open form cadheren promotes Hippo signaling while a-catenin sequesters 14-3-3 protein- of Merlin is active [27,32]. Moreover, the importance of the ubiqui- bound YAP. Merlin interacts with a-catenin at maturing adherens junctions in skin tin–proteasome system in Merlin’s degradation is debatable, as epithelium, plausibly influencing Hippo signaling at this subcellular compartment. cycloheximide chase experiments revealed that Merlin has a half- (2) At the plasma membrane, Merlin recruits the Lats kinases and coordinates their activation by Mst1, driving phosphorylation and inhibition of YAP/TAZ. (3) The life exceeding 24 h [29], although this does not rule out other ubiq- Crumbs homolog (CRB) complex recruits Angiomotin (AMOT), which directly binds uitination-mediated effects on Merlin function. Akt- and PAK-med- Hippo pathway components. AMOT serves as a scaffold for Mst1/2 and Lats1/2 iated phosphorylation of Merlin was recently shown to promote its activation and also directly binds and inhibits YAP. Conversely, the p180 isoform of sumoylation – an ubiquitin-like modification – which was found to AMOT promotes YAP function in the nucleus. Merlin interacts with AMOT at tight junctions to suppress Rac activity, however, it remains to be determined how affect Merlin’s subcellular localization, interdomain binding, Merlin influences Angiomotin’s inhibition of YAP at tight junctions or YAP- and growth-inhibitory activity [57]. However, the requirement activating functions in the nucleus. (4) Dephosphorylated Merlin translocates to for sumoylation in Merlin’s normal function and in tumor suppres- the nucleus and inhibits CRL4DCAF1, preventing this E3 ligase from ubiquitinating sion remains incompletely understood since serine 518 phosphory- and inhibiting Lats1/2. These core Hippo pathway components, activated by an lation, which renders Merlin inactive, appears to promote its upstream kinase, phosphorylate and inactivate YAP/TAZ. (B) Merlin’s regulation of CRL4DCAF1 impinges on multiple downstream epigenetic mechanisms and blocks sumoylation. oncogenic gene expression. In contact inhibited cells, active Merlin translocates to the nucleus where it binds to and inhibits the CRL4DCAF1 E3 ubiquitin ligase. Active DCAF1 5. Merlin localization and function or dysregulated CRL4 induces an oncogenic gene expression program by promoting downstream Hippo pathway targets via inhibition of the Lats1/2 kinases and through modulation of epigenetic modifiers and transcription factors. 5.1. Functions at the plasma membrane, cell cortex, and cytoskeleton

As cells become confluent, Merlin interacts with membrane- Merlin is recruited to the plasma membrane [36,61]. These observa- associated proteins where it regulates the formation of membrane tions were corroborated by Merlin’s role in the establishment of domains [58]. Merlin’s organization of intercellular contacts is cen- adherens junctions in confluent cells and localization in lamellipo- tral to its normal function in contact inhibition, which is disrupted dia and membrane ruffles [5,7,62–64]. Merlin interacts with a num- in Merlin-deficient tissues [59]. Indeed, Merlin was initially inter- ber of proteins that are localized at or underneath the plasma preted as functioning at the cell cortex due to its homology to membrane, including other ERMs, NHERF, the intracellular domain ERM proteins and its localization by immunostaining [60]. This of CD44, a-catenin at maturing adherens junctions, and Angiomotin notion was supported in Drosophila by the observation that nascent at tight junctions [6,28,59,60,65]. J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752 2747

In keratinocytes and skin epithelium, Merlin is recruited to a- [78], which could function in promoting the transport of anti-mito- catenin and promotes its binding to Par3, aiding in the maturation genic biomolecules or regulating the availability of growth factors of adherens junctions (Fig. 2A) [59]. This is consistent with the dis- at the plasma membrane. Merlin’s effects on the actin cytoskeleton ruption of adherens junctions in Merlin-deficient primary fibro- do not appear to be pathogenically relevant, and it remains to be blasts and keratinocytes [5]. Interestingly, a-catenin can bind to demonstrated whether regulation of microtubule dynamics is nec- 14-3-3 protein, which sequesters phosphorylated YAP in the cyto- essary in Merlin-mediated contact inhibition and tumor suppres- plasm and suppresses YAP-mediated transcription [66,67]. More- sion in vivo. Therefore, current models imply that Merlin largely over, it was recently discovered that a-catenin interacts with the enforces contact inhibition and the organization of membrane do- APC tumor suppressor to promote the ubiquitination and degrada- mains by acting at the plasma membrane or cortex, while Merlin’s tion of b-catenin as well as repression of Wnt target in the strongest tumor suppressive functions are enforced in another cel- nucleus [68]. Since Merlin’s interaction with a-catenin and its role lular compartment. in junction formation was exclusively studied in skin and fibro- blasts, it is difficult to put this mechanism in the context of NF2 5.2. Nuclear localization and inhibition of CRL4DCAF1 pathogenesis and other Merlin-deficient malignancies. It will be of great interest to see whether Merlin’s interaction with a-catenin Dephosphorylated Merlin translocates to the nucleus where it leads to the suppression of YAP- and b-catenin-mediated onco- binds to and inhibits the CRL4DCAF1 E3 ubiquitin ligase (Fig. 2B) genic gene expression in pathogenically-relevant tissues. [29], which was the first identification of a nuclear binding partner Merlin interacts with the scaffold and signaling protein Angi- for Merlin. Observation that the dephosphorylated form of Merlin omotin at mature tight junctions, regulating contact inhibition preferentially translocates to the nucleus while the phosphorylated and potentially tumor suppression (Fig. 2A) [28]. Angiomotin ex- form is largely excluded is consistent with the long-standing ists in two isoforms – both of which interact with Merlin – and observation that Merlin suppresses tumorigenesis in its dephos- associate with tight junctions through interactions with Patj, Pals1, phorylated form [27,29,32,49]. Moreover, several reports have and Mupp1 [69,70]. At assembled tight junctions, Merlin binds to characterized Merlin’s localization in the nucleus, and Merlin mi- Angiomotin and displaces Rich – a GTPase Activating Protein for grates towards the nucleus by microtubule-based transport in Dro- Rac – thus suppressing Rac-PAK signaling [28]. Therefore, Merlin’s sophila [57,75,79,80]. Although Merlin lacks a canonical nuclear inhibition of Rac at tight junctions promotes contact inhibition and localization sequence, it contains a motif on its carboxy-terminus acts as a barrier to mitogenic signaling [7]. One major caveat to this that promotes nuclear export by the CRM1-exportin pathway finding is that Angiomotin interacts not only with Merlin’s a-heli- [80], and truncations that remove these residues show increased cal and carboxy-terminal domains, but also with phospho-defi- nuclear localization [29,32]. Interestingly, the isolated FERM do- cient and phospho-mimetic Merlin mutants [28]. These results main of Merlin is necessary and sufficient for nuclear localization, strongly imply that Merlin binds to Angiomotin regardless of its once again highlighting the importance of Merlin’s FERM domain activation status, and therefore, independently of growth suppres- in its normal function (Table 1) [29]. Moreover, the dephosphoryl- sive stimuli. Further work is needed to fully understand the role of ated form of Merlin preferentially localizes to the nucleus in con- Angiomotin in Merlin’s function in normal cells and in Merlin-defi- fluent and growth factor-deprived cells, when Merlin’s anti- cient tumorigenesis. mitogenic effects would be most active [29]. As the phosphory- A fraction of Merlin has been found to localize to cholesterol- lated, more closed form of Merlin is excluded from the nucleus, dependent membrane domains – which were interpreted as lipid interdomain binding imposed by S518 phosphorylation may oc- rafts – as a result of phosphoinositide binding [71,72]. In several clude the FERM domain residues necessary for nuclear transloca- studies, active Merlin was shown to bind phosphoinositides, tion [27,29]. Since a large proportion of Merlin shuttles to the although there is contradicting evidence as to whether this occurs nucleus in contact-inhibited cells, Merlin’s nuclear function – in Merlin’s active or inactive forms [72,73], raising uncertainty particularly inhibition of the oncogenic CRL4DCAF1 ubiquitin ligase over the relevance of this interaction to adhesion signaling. How- – may be vital for growth suppression [29]. Indeed, multiple lines ever, a Merlin mutant lacking residues necessary for phosphoinosi- of evidence suggest that Merlin suppresses tumorigenesis largely tide binding – K79, K80, K269, E270, K278, and K279 – was through inhibition of CRL4DCAF1. Genetic epistasis experiments in significantly defective in suppressing growth of Merlin-deficient several cell lines – including pathogenically relevant human cells [72]. This observation was supported by experiments con- schwannoma and mesothelioma cells – reveal that Merlin firming that the Merlin mutant retained normal folding. However, mediates its tumor suppressive and anti-mitogenic effects through the ability of the phosphoinositide-binding mutant to interact with other proteins that were previously shown to mediate Merlin’s Table 1 DCAF1 function was not tested. Notably, mutation of Merlin E270 abol- Patient-derived NF2 mutations abrogate Merlin’s regulation of CRL4 . The indicated patient-derived missense and truncation mutants are defective in nuclear ishes interaction with CRL4DCAF1, which could explain the translocation, direct binding to CRL4DCAF1, suppression of CRL4DCAF1-mediated growth-suppression defects of the phosphoinositide-binding ubiquitination, or a combination of these mechanisms. As a result, the NF2-derived mutant [29]. Merlin’s phosphoinositide binding and potential missense mutants are unable to interact with CRL4DCAF1 in vivo, permitting the localization to lipid rafts requires further scrutiny to determine dysregulated ligase to promote tumorigenesis. NT, not tested. Data were obtained its relevance both in normal biological function and in Merlin- from [29]. deficient pathogenesis. Patient-derived Biochemical defect (relative to wild-type) In addition to Merlin’s localization at the cortex and contact- Merlin mutation dependent membrane domains, Merlin functions in the cytoplasm Nuclear Reduced In vitro Reduced DCAF1 exclusion DCAF1 binding ligase inhibition to control cytoskeletal dynamics and vesicular transport. Merlin associates with actin and tubulin in the cytoplasm [32,74,75], L46R + + NT F62S À +NT although Merlin’s association with the actin cytoskeleton is dis- L64P + + NT pensable for tumorigenesis [32,76]. Merlin migrates along micro- L141P + + NT tubules in a kinesin-1- and dynein-dependent manner [75], and A211D + À NT Merlin was recently found to slow the turnover of microtubules, E270G À +NT thus stabilizing them [77]. In addition, Merlin has been found to 466X NT NT + 341X ÀÀ + promote microtubule-mediated anterograde transport of vesicles 2748 J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752 inhibition of CRL4DCAF1. Notably, multiple patient-derived Merlin intrinsic kinase activity towards histone H2A to downregulate missense mutants are unable to interact with DCAF1 as a result anti-mitogenic genes [98]. Combined, these studies reveal that of nuclear localization defects, disruption of Merlin-DCAF1 bind- DCAF1 can interact with multiple target proteins using several dis- ing, or a combination of these aberrations (Table 1) [29]. Under- tinct regions spanning its primary sequence. However, without fur- standing more about CRL4DCAF1 biology and its interaction with ther structural characterization of full-length DCAF1, it cannot be Merlin and other binding partners is an important step in concep- ruled out that these domains are juxtaposed, and that one or more tualizing how Merlin’s inhibition of this E3 ligase is an important protein-interacting regions act in a single multifunctional recruit- stepping stone in the control of multiple pro-oncogenic signaling ment domain. pathways. DCAF1 was established as the substrate recognition component DCAF1 (DDB1 and Cul4-Associated Factor 1) was originally of the CRL4DCAF1 E3 ligase (Fig. 3B) nearly ten years ago [83], and identified as VprBp (Vpr-binding protein) – a cytoplasmic protein several CRL4DCAF1 targets have been discovered. Vpr- and Vpx- that binds to the HIV-1 viral accessory protein Vpr [81,82], before mediated targeting of SAMHD1 and the uracil DNA glycosylase it was found to function as the substrate recognition subunit of the UNG2 direct these proteins to proteasomal degradation [91,99]. CRL4DCAF1 E3 ubiquitin ligase [83]. Viral Vpr and the related Vpx Moreover, it was found that CRL4DCAF1 is recruited by Vpr to acti- proteins promote HIV and SIV infection by functioning as adaptors vate the SLX4 complex, a structure-specific endonuclease whose that bind to DCAF1 and usurp its recruitment of substrates to de- ectopic activation results in G2/M arrest and evasion of innate grade proteins that normally suppress viral infectivity [84,85].In immunity in HIV-infected cells [100]. Native DCAF1-binding pro- fact, viral subversion of E3 ubiquitin ligases occurs in a number teins include Merlin, the nuclear orphan receptor Rora, histones of human diseases [83,86–89]. SAMHD1 is a viral restriction factor H2A and H3, HDAC, the polarity protein Lgl, the lymphoid cell-spe- which functions in myeloid-lineage cells and resting CD4+ T-cells cific V(D)J recombination protein RAG1, and the replication factor by reducing the concentration of cellular dNTPs and thus inhibiting Mcm10 [29,97,101–104]. Several of these proteins – particularly the viral reverse transcriptase. In HIV-2 and SIV, Vpx binds to the histones and HDAC – can broadly affect epigenetics, suggesting DCAF1 and acts as an adaptor to co-opt the ubiquitin-conjugating that Merlin’s inhibition of CRL4DCAF1 can greatly alter gene expres- machinery to target and degrade SAMHD1 [90–95]. A recent land- sion. It was recently observed that CRL4DCAF1 interacts with and mark study found that Vpx binds the WD40 domain of DCAF1 to promotes activation of TET proteins, a family of enzymes that con- produce a new surface that recruits SAMHD1, providing the first vert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) [105]. structural elucidation of lentiviral hijacking of the cell’s ubiqui- These conversions cause epigenetic modifications that regulate tin–proteasome system [96]. These studies provide a starting point DNA demethylation and therefore gene transcription, which were to understand how CRL4DCAF1 binds its targets, and furthermore, shown to have dramatic effects during embryonic development how Merlin may serve as an inhibitory pseudo-substrate (Fig. 3A). and tumorigenesis. Notably, loss of TET2 is a common occurrence Structural analysis of the DCAF1–Vpx–SAMHD1 complex re- in myeloid leukemia and 5hmC reduction is a distinguishing fea- vealed that Vpx creates a new binding surface by enclosing a por- ture of melanoma and is observed in a range of other solid tumors tion of the DCAF1 WD40 domain. The WD40 domain of DCAF1, [106]. However, the mechanism by which CRL4DCAF1 regulates TET near the carboxy-terminus, is a seven-bladed b-propeller that proteins – whether by ubiquitination or another modification – is forms an overall fan shape [96]. Residues within the WD40 domain currently unknown. Indeed, the mechanism by which CRL4DCAF1 are necessary for interacting with DDB1 and therefore docking to modulates several bona fide interactors remains elusive, and it is the CRL4 complex [83]. As Vpx-associated DCAF1 is able to recruit unclear whether ubiquitination of these targets is necessary or suf- SAMHD1 and mediate its ubiquitination [96], it is plausible that ficient for their regulation. Regardless, these findings suggest that DCAF1 uses portions of its WD40 domain to bind native targets. CRLDCAF1 can multifariously modulate epigenetics (Fig. 2B), and A recent study found that DCAF1 can recruit mono-methylated its dysregulation following Merlin deficiency shifts cells towards proteins using the hydrophobic pocket within its chromo domain, an oncogenic gene expression program. Indeed, Merlin expression which resides in the amino-terminal half of the protein [97].In and DCAF1 knockdown in Merlin-deficient cells reveal that Merlin addition to the role of CRL4DCAF1 in ubiquitination, DCAF1 has a suppresses tumorigenesis largely through DCAF1 inhibition, pro- putative kinase domain near the amino-terminus which imparts viding further support that Merlin regulates oncogenic gene expression through its inhibition of the CRL4DCAF1 E3 ubiquitin ligase [29]. A Merlin-bound CRL4DCAF1 B Active CRL4DCAF1 Merlin’s FERM domain binds the carboxy-terminal acidic tail of

N Target N DCAF1, which is outside of the WD40 domain [29]. Since the Merlin Target Ub DCAF1 acidic tail is adjacent to its WD40 domain in its primary se- Ub Ub quence, it is possible that Merlin’s binding to the tail brings it in E2 E2 DCAF1 C DCAF1 C close proximity to the binding surface of the WD40 propeller

Rbx1 Rbx1 (Fig. 3A). However, many possibilities exist to explain how Merlin can reduce CRL4DCAF1 activity [29]; this is further complicated by DDB1 DDB1 Cul4 Cul4 the multiple protein-interacting domains within DCAF1. A ground- work explanation is that Merlin occludes one or more of the DCAF1 targeting domains without significant structural perturbation. In addition to – or instead of – direct competition of target recruit- Tumor Suppressive Growth-promoting/oncogenic ment, Merlin’s binding may cause dramatic structural alterations, thus attenuating DCAF1 targeting abilities. Further biochemical Fig. 3. (A) Active Merlin translocates to the nucleus and binds to the carboxy- terminus of DCAF1, acting as a pseudo-substrate to block CRL4DCAF1 from recruiting studies and structural analyses will be paramount in gaining DCAF1 and ubiquitinating target proteins. Merlin’s inhibition of CRL4DCAF1 suppresses mechanistic insight into Merlin’s inhibition of CRL4 . oncogenic gene expression. (B) CRL4DCAF1 is an E3 ubiquitin ligase consisting of the substrate-recruiting DCAF1 protein which is linked to the amino-terminal domain 5.3. Convergence on YAP and TAZ of a cullin (Cul4A/B) backbone through DDB1, which is itself a large multi-domain protein. Bound to the cullin carboxy-terminal domain, Rbx1 uses its RING domain to recruit an ubiquitin-charged E2, which directs conjugation of ubiquitin to a The size of multicellular organisms and their composite tissues DCAF1-bound target protein. is tightly regulated by restricting cell size and proliferation J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752 2749

[107,108]. The Hippo pathway has emerged as a potent regulator of or cells that have had their tight junctions disrupted. Combined, organ size throughout the animal kingdom. Notably, genetic exper- these results suggest Angiomotin has tumor suppressive and iments in Drosophila and mice revealed that the Hippo pathway growth-promoting functions, which may depend on the tissue, cel- controls cell proliferation and promotes apoptosis, and disruption lular, or biochemical environment. Identification of the contexts in of the pathway at multiple levels leads to tissue overgrowth and which Angiomotin functions to inhibit or promote Hippo signaling tumorigenesis [109,110]. This is particularly evident in mouse ge- – including the biochemical function of Merlin in these processes – netic models, where dysregulation of Hippo pathway proteins in will provide important mechanistic insight into both Merlin func- the liver elicits dramatic hepatomegaly and development of a tion and Hippo signaling. range of tumors [24,111–114]. The Hippo pathway is canonically Drosophila genetics experiments provided strong evidence that driven by a core kinase cascade consisting of the Ste20-like kinases Merlin functions with Ex to activate Hippo signaling [25]. How- Mst1 and Mst2 (Mst1/2), which in conjunction with Sav1, phos- ever, two recent landmark studies revealed separate, non-linear phorylate and activate the NDR family kinases Lats1 and Lats2 mechanisms by which Merlin promotes Hippo signaling. DJ Pan’s (Lats1/2) along with cofactors Mob1A and Mob1B. Active Lats ki- group found that Merlin could directly bind to Wts/Lats in both nases phosphorylate and inactivate the YAP and TAZ transcrip- Drosophila and mammals, recruiting these proteins to the plasma tional coactivators (Fig. 2A). In the absence of Hippo signaling, membrane to be phosphorylated by active Mst1/2 (Fig. 2A) [129]. active YAP and TAZ translocate to the nucleus to promote tran- This study further used genetic experiments in mammalian cells scription of genes that drive proliferation, evasion of apoptosis, to show that Merlin promotes downstream Hippo signaling inde- and stemness [26,115]. The atypical cadherin Fat, along with the pendently of Mst1/2, revealing Merlin’s direct biochemical role in apical polarity protein Crumbs, function in Drosophila to activate regulating the Hippo pathway. More recently, however, our group the Hippo pathway. These proteins activate the FERM domain pro- found that dysregulated CRL4DCAF1 promotes YAP function and tein Expanded, which complexes with Merlin and Kibra to activate TEAD-dependent transcription by ubiquitinating and inhibiting the core kinase cascade [116–119]. Although the core kinase Lats1/2 in the nucleus (Fig. 2A) [130]. Genetic experiments and cascade is evolutionarily conserved between Mammals and analyses of Merlin mutations revealed that CRL4DCAF1-mediated Drosophila, the upstream signaling promoting activation of the inhibition of Lats1/2 sustains the tumorigenicity of Merlin-defi- Hippo pathway has diverged. cient cells. Compellingly, analysis of clinical samples strongly sug- It has recently become evident that some of the key proteins gested that this YAP- and TAZ-promoting circuit of the Hippo regulating the core Hippo cascade in Drosophila evolved after the pathway functions in Merlin-deficient malignancies. Furthermore, divergence of arthropods. It is unclear whether Drosophila Ex- our experiments revealed that tumor-derived Merlin mutants are panded (Ex) and Fat have orthologs in vertebrates, and the mam- invariably defective in binding to CRL4DCAF1 yet retain Lats1 bind- malian proteins which bare closest resemblance appear to ing, providing genetic evidence that Merlin’s interaction with Lats1 function primarily through Hippo-independent mechanisms at the plasma membrane is not sufficient to suppress tumorigene- [120–123]. The adherens junction protein Echinoid and the uncon- sis. Merlin’s recruitment of Lats1 at the plasma membrane is an ventional myosin Dachs – a downstream target of Fat – regulate important and evolutionarily conserved non-canonical Hippo the Drosophila Hippo pathway but are absent in vertebrates. Angi- pathway mechanism. However, it appears that Merlin’s suppres- omotin was retained during the evolution of chordates as a regula- sion of the CRL4DCAF1 E3 ubiquitin ligase is a paramount tumor sup- tor of Hippo signaling but was replaced with Ex at the base of the pressive clamp embedded in the human Hippo pathway. In vivo arthropod lineage [123]. The evolution of a novel domain in Ex to insight into the contribution of both of these mechanisms in mam- bind Yki – the Drosophila ortholog of YAP – reestablished mals will be an essential step in validating their significance in the Crumbs-mediated regulation of Hippo signaling which was uncou- context of normal function and tumorigenesis. These findings fur- pled when the PDZ-binding motif of Yki was lost during arthropod ther emphasize the lack of conservation between the linear model evolution. In addition to these evolutionary shifts in regulation of for Hippo activation in Drosophila and the multifarious regulation Hippo signaling, Merlin’s input into the pathway appears to be of YAP and TAZ in mammals. Combined, insights over the past five more complicated in mammals. years have shown that Merlin regulates the Hippo pathway at mul- It is becomingly increasingly clear in mammals that multiple tiple levels, yet these signals invariably converge to regulate YAP signals converge to affect the Hippo core kinase cascade in addition and TAZ activation [115]. As a next step, it will be important to to canonical Hippo signaling [115]. Upstream of the core cassette, determine the spatio-temporal hierarchy of Merlin’s effects on Merlin can interact with Kibra to activate the Hippo kinase Hippo signaling and the importance of these pathways in patho- cascade, although the biochemical mechanism is unclear genically relevant tissues, which may unmask the most significant [24,117]. Merlin is recruited to tight junctions by Angiomotin, druggable targets for Merlin-deficient malignancies. which serves as a scaffold for PDZ- and WW-domain-containing proteins, including Hippo pathway components (Fig. 2A) 6. Conclusions [124–127]. Interestingly, tight junction-associated Angiomotin di- rectly binds phosphorylated YAP and TAZ and retains them at the The biochemical mechanisms underlying Merlin-mediated cortex [124,127] and also serves as a scaffold for activation of the growth suppression are unraveling at an accelerating rate, with Hippo core kinases Mst2 and Lats2, promoting canonical YAP/TAZ several critical observations occurring within the past five years. inhibition [125]. Since Merlin binds to Angiomotin and displaces It is becoming increasingly clear that Merlin suppresses cell prolif- other complexed proteins, it is plausible that Merlin’s binding eration through two modes that are defined by functions at the could modulate Angiomotin recruitment of Hippo components. It cortex or in the nucleus. By linking Par3 to a-catenin at adherens remains to be tested whether Merlin promotes or inhibits Angi- junctions, Merlin promotes cell adhesions and thereby tissue orga- omotin’s direct effects on Hippo signaling. Recently it was found nization in skin epithelium [59]. At tight junctions, Merlin’s that the p130 isoform of Angiomotin promotes YAP function by binding to Angiomotin releases Rich, which inactivates Rac and disrupting its association with Lats1 in the cytoplasm, and further- thereby attenuates mitogenic signaling [7,28]. As Angiomotin di- more, by binding to YAP in the nucleus to drive expression of a sub- rectly suppresses Hippo signaling in the cytoplasm [124,125,127] set of YAP target genes to drive tumorigenesis (Fig. 2A) [128].It and promotes YAP activity in the nucleus [128], further insight is will be of great interest to determine whether Angiomotin is needed to understand the contextual relevance of these shuttled to the nucleus to promote YAP function in sparse cells observations as well as how Merlin may be involved. Moreover, 2750 J. Cooper, F.G. Giancotti / FEBS Letters 588 (2014) 2743–2752 as a-catenin inhibits YAP and Wnt-b-catenin signaling, Merlin’s [11] Dalgliesh, G.L. et al. (2010) Systematic sequencing of renal carcinoma reveals potential regulation of these pathways through interactions with inactivation of histone modifying genes. Nature 463, 360–363. [12] Lau, Y.K., Murray, L.B., Houshmandi, S.S., Xu, Y., Gutmann, D.H. and Yu, Q. a-catenin warrants investigation. 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