The NF2 interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), associates with merlin in the "open" conformation and suppresses cell growth and motility

The neurofibromatosis 2 tumor suppressor protein, merlin or schwannomin, functions as a negative growth regulator; however, its mechanism of action is not known. In an effort to determine how merlin regulates cell growth, we analyzed a recently identified novel merlin interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). We demonstrate that regulated overexpression of HRS in rat schwannoma cells results in similar effects as overexpression of merlin, including growth inhibition, decreased motility and abnormalities in cell spreading. Previously, we showed that merlin forms an intramolecular association between the N- and C-termini and exists in "open" and "closed" conformations. Merlin interacts with HRS in the unfolded, or open, conformation. This HRS binding domain maps to merlin residues 453-557. Overexpression of C-terminal merlin has no effect on HRS function, arguing that merlin binding to HRS does not negatively regulate HRS growth suppressor activity. These results suggest the possibility that merlin and HRS may regulate cell growth in schwannoma cells through interacting pathways.     

The neurofibromatosis 2 tumor suppressor protein interacts with hepatocyte growth factor-regulated tyrosine kinase substrate

The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.     

Cover Image

Background: The tumor suppressor protein merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of merlin is associated with Neurofibromatosis Type 2 (NF2) tumors. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. We investigate how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. Results: We identify two conserved arginine residues (R325 and R335) in Merlin which, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over‐expression of Merlin^R325A and/or Merlin^R335L in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co‐over‐expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. Conclusions: Sip1 facilitates the activation of Merlin as a tumor suppressor protein. Thus, our work provides insight into how Merlin functions as a tumor suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis. Developmental Dynamics 243:1554–1570, 2014. © 2014 Wiley Periodicals, Inc.     

Regulation of cell proliferation and adhesion by means of a novel region of drosophila merlin interacting with Sip1

Background: The tumor suppressor protein merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of merlin is associated with Neurofibromatosis Type 2 (NF2) tumors. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. We investigate how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. Results: We identify two conserved arginine residues (R325 and R335) in Merlin which, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over‐expression of Merlin^R325A and/or Merlin^R335L in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co‐over‐expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. Conclusions: Sip1 facilitates the activation of Merlin as a tumor suppressor protein. Thus, our work provides insight into how Merlin functions as a tumor suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis. Developmental Dynamics 243:1554–1570, 2014. © 2014 Wiley Periodicals, Inc.     

Defects in neurofibromatosis 2 protein function can arise at multiple levels

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.      

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading

The neurofibromatosis 2 ( NF2 ) gene product, merlin, is a tumor suppressor protein mutated in schwanno-mas and several other tumors. Merlin, which shares significant homology with the actin-associated proteins ezrin, radixin and moesin (ERM proteins), inhibits cell growth when overexpressed in cell lines. The similarities between merlin and ERM proteins suggest that merlin's growth-regulatory capabilities may be due to alterations in cytoskeletal function. We examined this possibility in rat schwannoma cell lines overexpressing wild-type merlin isoforms and mutant merlin proteins. We found that overexpression of wild-type merlin resulted in transient alterations in F-actin organization, cell spreading and cell attachment. Merlin overexpression also impaired cell motility as measured in an in vitro motility assay. These effects were only observed in cells overexpressing a merlin isoform capable of inhibiting cell growth and not with mutant merlin molecules (NF2 patient mutations) or a merlin splice variant (isoform II) lacking growth-inhibitory activity. These data indicate that merlin may function to maintain normal cytoskeletal organization, and suggest that merlin's influence on cell growth depends on specific cytoskeletal rearrangements.      

Functional analysis of neurofibromatosis 2 (NF2) missense mutations.

Neurofibromatosis 2 (NF2) is a tumor predisposition syndrome in which affected individuals develop nervous system tumors at an increased frequency. The most common tumor in individuals with NF2 is the schwannoma, which is composed of neoplastic Schwann cells lacking NF2 gene expression. Moreover, inactivation of the NF2 gene is observed in nearly all sporadic schwannomas, suggesting that the NF2 gene is a critical growth regulator for Schwann cells. In an effort to gain insights into the function of the NF2 gene product, merlin or schwannomin, we performed a detailed functional analysis of eight naturally occurring non-conservative missense mutations in the NF2 gene. Using a regulatable expression system in rat schwannoma cells, we analyzed proliferation, actin cytoskeleton-mediated events and merlin folding. In this report, we demonstrate that mutations clustered in the predicted alpha-helical region did not impair the function of merlin whereas those in either the N- or C-terminus of the protein rendered merlin inactive as a negative growth regulator. These results suggest that the key functional domains of merlin lie within the highly conserved FERM domain and the unique C-terminus of the protein.     

Structural basis of DDB1‐and‐Cullin 4‐associated Factor 1 (DCAF1) recognition by merlin/NF2 and its implication in tumorigenesis by CD44‐mediated inhibition of merlin suppression of DCAF1 function

Merlin, a tumor suppressor encoded by the neurofibromatosis type 2 gene, has been shown to suppress tumorigenesis by inhibiting the Cullin 4‐RING E3 ubiquitin ligase CRL4^DCAF1 in the nucleus. This inhibition is mediated by direct binding of merlin to DDB1‐and‐Cullin 4‐associated Factor 1 (DCAF1), yet the binding mode of merlin to DCAF1 is not well defined. Here, we report structural and biophysical studies of the merlin binding to DCAF1 and its interference with CD44 binding. The crystal structure of the merlin FERM domain bound to the DCAF1 C‐terminal acidic tail reveals that the hydrophobic IILXLN motif located at the C‐terminal end of DCAF1 binds subdomain C of the FERM domain by forming a β‐strand. The binding site and mode resemble that of merlin binding to the CD44 cytoplasmic tail. Competition binding assay showed that CD44 and DCAF1 compete for binding to the merlin FERM domain in solution. The CD44 cytoplasmic tail is known to be cleaved for nuclear translocation by regulated intra‐membrane proteolysis (RIP). Our structure implies that, in the nucleus, the CD44 cytoplasmic tail cleaved by RIP could release DCAF1 from merlin by competing for binding to the merlin FERM domain, which results in the inhibition of merlin‐mediated suppression of tumorigenesis.     

NF2 deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions

Mutation of the Neurofibromatosis 2 (NF2) tumor suppressor gene leads to cancer development in humans and mice. Recent studies suggest that Nf2 loss also contributes to tumor metastasis. The Nf2-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane:cytoskeleton-associated proteins. However, the cellular mechanism whereby merlin controls cell proliferation from this location is not known. Here we show that the major cellular consequence of Nf2 deficiency in primary cells is an inability to undergo contact-dependent growth arrest and to form stable cadherin-containing cell:cell junctions. Merlin colocalizes and interacts with adherens junction (AJ) components in confluent wild-type cells, suggesting that the lack of AJs and contact-dependent growth arrest in Nf2(-/-) cells directly results from the absence of merlin at sites of cell:cell contact. Our studies indicate that merlin functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell:cell contact.     

Characterization of chicken Nf2/merlin indicates regulatory roles in cell proliferation and migration.

The neurofibromatosis 2 (NF2) tumor suppressor protein merlin, or schwannomin, functions as a negative growth regulator such that inactivating mutations in Nf2 predispose humans to tumors. In addition, merlin has a critical role during embryonic development. Nf2-deficient mice die early during embryogenesis, with defects in gastrulation and extraembryonic tissues. To investigate the function of Nf2/merlin during embryonic development, we first identified the homologous Nf2 gene in chicken (cNf2) and examined the distribution of chicken merlin (c-merlin) during myogenesis. cNf2 encoded a full-length mRNA of 1,770 nucleotides and a protein of 589 residues. C-merlin shared high sequence homology and common protein motifs with vertebrate and Drosophila merlins. In addition, cNF2 functions as a negative growth regulator similar to human and Drosophila merlin in vitro. In vivo, c-merlin was expressed diffusely in the forming dermomyotome but down-regulated in migratory muscle precursors in the forelimb. As muscle formed in the limb, c-merlin expression was up-regulated. As an initial examination of c-merlin function during myogenesis, c-merlin was ectopically expressed in muscle precursors and the effects on muscle development were examined. We show that ectopic merlin expression reduces the proliferation of muscle precursors as well as their ability to migrate effectively in limb mesoderm. Collectively, these results demonstrate that c-merlin is developmentally regulated in migrating and differentiating myogenic cells, where it functions as a negative regulator of both muscle growth and motility.     

The tumor suppressor merlin interacts with microtubules and modulates Schwann cell microtubule cytoskeleton

The lack of neurofibromatosis 2 tumor suppressor protein merlin leads to the formation of nervous system tumors, specifically schwannomas and meningiomas. Merlin is considered to act as a tumor suppressor at the cell membrane, where it links transmembrane receptors to the actin cytoskeleton. Several tumor suppressors interact with another component of the cytoskeleton, the microtubules, in a regulated manner and control their dynamics. In this work, we identify merlin as a novel microtubule-organizing protein. We identify two tubulin-binding sites in merlin, one residing at the N-terminal FERM-domain and another at the C-terminal domain. Merlin's intramolecular association and phosphorylation of serine 518 regulate the interaction between merlin and tubulin. Analysis of cultured glioma cells indicates colocalization between merlin and microtubules especially during cell division. In primary mouse Schwann cells only minor colocalization at the cell periphery of interphase cells is seen. However, these cells drastically change their microtubule organization upon loss of merlin indicating a functional association of the proteins. Both in vitro assays and in vivo studies in Schwann cells indicate that merlin promotes tubulin polymerization. The results show that merlin plays a key role in the regulation of the Schwann cell microtubule cytoskeleton and suggest a mechanism by which loss of merlin leads to cytoskeletal defects observed in human schwannomas.     

NF2: the wizardry of merlin

Neurofibromatosis type II (NF2) is an autosomal dominant cancer syndrome characterized by the formation of tumors of the nervous system, particularly schwannomas and meningiomas. The NF2 gene is also implicated in the development of sporadic schwannomas and meningiomas, as well as tumor types seemingly unrelated to the NF2 disorder, such as malignant mesotheliomas. Inactivation of NF2 occurs by a "two-hit" mechanism, as proposed by Al Knudson, and the NF2 gene behaves as a classical tumor suppressor gene. The NF2 gene product, merlin, exhibits homology with the ezrin-radixin-moesin family of membrane-cytoskeleton-linking proteins. During the past several years, there has been intensive investigation aimed at elucidating the mechanisms underlying merlin's functions. In this review, we summarize the involvement of NF2 inactivation in tumorigenesis. We also discuss observations implicating merlin in cell motility and cell proliferation, with a focus on recent findings linking merlin to Rac signaling.     

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.     

Transduction of wild-type merlin into human schwannoma cells decreases schwannoma cell growth and induces apoptosis.

Mutations in both alleles of the tumour suppressor gene coding for merlin/schwannomin, an ERM family protein, cause the hereditary disease neurofibromatosis type 2 (NF2). NF2 is characterized by the development of multiple nervous system tumours especially vestibular schwannomas. Efficient oncoretrovirus-mediated gene transfer of different merlin constructs was used to stably re-express wild-type merlin in primary cells derived from human schwannomas. Using two-parameter FACS analysis we show that expression of wild-type merlin in NF2 cells led to significant reduction of proliferation and G0/G1 arrest in transduced schwannoma cells. In addition, we show increased apoptosis of schwannoma cells transduced with wild-type merlin. Our findings in primary schwannoma cells from NF2 patients strongly support the hypothesis of merlin acting as a tumour suppressor and may help in understanding development of human schwannomas in NF2.     

The neurofibromatoses: when less is more

The study of cancer predisposition syndromes presents unique opportunities to gain insights into the genetic events associated with tumor pathogenesis. Individuals with two inherited cancer syndromes, neurofibromatosis 1 (NF1) and neurofibromatosis 2 (NF2), develop both benign and malignant tumors. The corresponding genes mutated in these two disorders encode tumor suppressor proteins, termed neurofibromin (NF1) and merlin (NF2), which function in novel ways to regulate cell growth and differentiation. Neurofibromin inhibits cell proliferation, at least in part, by modulating mitogenic pathway signaling through inactivation of p21-ras. In contrast, merlin may act as a membrane-associated molecular switch that regulates cell-cell and cell-matrix signals transduced by cell surface receptors. Significant progress in our understanding of the genetics and biology of NF1 and NF2 has elucidated the roles of these genes in tumor initiation and progression.     

Neurofibromatosis 2 (NF2) tumor suppressor schwannomin and its interacting protein HRS regulate STAT signaling

Mutations in the neurofibromatosis 2 (NF2) gene with the resultant loss of expression of the NF2 tumor suppressor schwannomin are one of the most common causes of benign human brain tumors, including schwannomas and meningiomas. Previously we demonstrated that the hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) strongly interacts with schwannomin. HRS is a powerful regulator of receptor tyrosine kinase trafficking to the degradation pathway and HRS also binds STAM. Both of these actions for HRS potentially inhibit STAT activation. Therefore, we hypothesized that schwannomin inhibits STAT activation through interaction with HRS. We now show that both schwannomin and HRS inhibit Stat3 activation and that schwannomin suppresses Stat3 activation mediated by IGF-I treatment in the human schwannoma cell line STS26T. We also find that schwannomin inhibits Stat3 and Stat5 phosphorylation in the rat schwannoma cell line RT4. Schwannomin with the pathogenic missense mutation Q538P fails to bind HRS and does not inhibit Stat5 phosphorylation. These data are consistent with the hypothesis that schwannomin requires HRS interaction to be fully functionally active and to inhibit STAT activation.     

Reduced apoptosis rates in human schwannomas

Schwannomas, tumors originating from Schwann cells, represent a frequent neurological tumor and can occur both in a genetic disorder called neurofibromatosis type 2 (NF2) and sporadically. In both cases the genetic background is identical as all schwannomas are caused by biallelic mutations in the tumor suppressor gene NF2 coding for merlin. Mutations in this gene have also been found to be responsible for 50% to 60% of spontaneous and 100% of the NF2 associated meningiomas. The NF2 gene product, merlin, links transmembrane proteins to the cytoskeleton and is involved in intracellular signaling processes. It has previously been shown that reexpression of wild-type merlin in primary human schwannoma cells leads to an increase in the number of apoptotic cells. Here, we report in vivo and in vitro evidence that the basal apoptosis rate of primary human schwannoma cells is reduced in comparison to that of normal Schwann cells, supporting the idea that in this benign tumor type, apoptosis has a role in tumorigenesis.     

Involvement of p21 (waf1) in merlin deficient sporadic vestibular schwannomas

Previous studies have demonstrated that merlin acts as a tumor suppressor by blocking Ras-mediated signaling. However, the mechanism by which merlin controls cell proliferation has remained obscure. Here we show that merlin deficient tumors exhibited loss of p21, concomitant with elevated CDKs/cyclin D1 levels in sporadic vestibular schwannomas (VS) from clinic patients. Likewise, silencing of merlin gene expression in the cell lines resulted in down-regulation of p21. Furthermore, we find that merlin-enhanced p21 protein stability, rather than increased RNA accumulation, was responsible for the elevated p21 levels. Interestingly, p21 was required to maintain merlin levels and the inhibitory effect of merlin on Ras signaling was partially overridden by knockdown of p21. Consistent with the observation that over-expression of merlin arrested cell growth at G1-phase, the current study indicates that merlin exerts its antiproliferative effect, at least in part, by maintaining p21 expression, and loss of p21 is a prominent feature of merlin deficient schwannomas.