Subcellular localization of vertebrate merlin

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, the expression pattern and subcellular localization in human fibroblasts was studied by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. Higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa were also detected. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to the formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa are quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, is not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (1) perinuclear in a compartment resembling endoplasmic reticulum; (2) in ruffling membranes and at the leading edges; (3) in filopodia, and (4) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments is found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions, as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin reveal a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting that NF2 protein contributes to the regulation of cell growth by interaction with cytoskeleton-associated proteins (Schmucker, 1997).

Merlin is expressed in Schwann cells, where it is localized in vitro to the cell membrane by immunohistochemistry and subcellular fractionation. Exogenous expression of merlin fragments confirms this subcellular distribution and suggests that both the N-terminal and C-terminal portions of the molecule are required for this localization. In addition, merlin is expressed in rat sciatic nerve Schwann cells at paranodal membranes, where it colocalizes with RhoA. Lastly, expression of the NF2 gene increases during postnatal rat sciatic nerve development, consistent with its role as a negative growth regulator for Schwann cells. These results collectively suggest that merlin may function at the cell surface to modulate cell growth in Schwann cells and to link cell membrane proteins to the cytoskeleton (Scherer, 1996).

Polyclonal and monoclonal antibodies have been generated that detect merlin as an approximately 66 kD protein in many different cell types. Using indirect immunofluorescence, endogenous merlin has been visualized and it has been localized to the motile regions, such as leading or ruffling edges, in human fibroblast and meningioma cells. Merlin co-localizes with F-actin in these motile regions but is not associated with stress fibers. Merlin does not localize to the same structures as either ezrin or moesin in human meningioma cells, suggesting a function distinct from these ERMs. Thus, merlin is associated with motile regions of the cell and its participation in these structures may be intimately involved in control of proliferation in Schwann cells and meningeal cells (Gonzalez-Agosti, 1996).

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. 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 results directly from the absence of merlin at sites of cell:cell contact. These studies indicate that merlin functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell:cell contact (Lallemand, 2003).

Developmental expression and function of vertebrate merlin

The neurofibromatosis type II (NF2) tumor suppressor encodes a putative cytoskeletal associated protein, the loss of which leads to the development of Schwann cell tumors associated with NF2 in humans. The NF2 protein merlin belongs to the band 4.1 family of proteins that link membrane proteins to the cytoskeleton and is thought to be involved in dynamic cytoskeletal reorganization. Beyond its membership in this family, however, the function of merlin remains poorly understood. In order to analyze the function of merlin during embryogenesis and to develop a system to study merlin function in detail, the mouse Nf2 gene has been inactivated by homologous recombination in embryonic stem cells. Most embryos homozygous for a mutation at the Nf2 locus fail between embryonic days 6.5 and 7.0, exhibiting a collapsed extraembryonic region and the absence of organized extraembryonic ectoderm. The embryo proper continues to develop, but fails to initiate gastrulation. These observations are supported by the expression patterns of markers of the extraembryonic lineage and the lack of expression of mesodermal markers in the mutant embryos. Mosaic studies demonstrate that merlin function is not required cell autonomously in mesoderm, and support the proposition that merlin function is essential for the development of extraembryonic structures during early mouse development (McClatchey, 1997).

To determine the pattern of NF2 gene expression in mouse embryos, the mouse NF2 gene was sequenced and in situ hybridization and antischwannomin antibodies were used to determine the developmental expression of the NF2 gene. Schwannomin is detected in most differentiated tissues but is undetectable in undifferentiated tissues. In particular, schwannomin was not detectable in mitotic neuroepithelial cells, the perichondrium, the liver, the neocortex, and the ventricular zone of the developing cerebral cortex. In the heart, expression is observed at all developmental stages beginning on embryonic day 8. In the eye, which shows developmental abnormalities in NF2 patients, expression is detected in the cells of the lens and in the pigment epithelium but weakly detected in retinal neurons. The most striking example of tightly regulated NF2 expression is observed in cells migrating from the ventricular zone to the cortical plate on embryonic days 15 and 16. Only cells in the intermediate zone express schwannomin, indicating that schwannomin may play an important role in cellular migration (Huynh, 1996).

Erbin and the NF2 tumor suppressor Merlin cooperatively regulate cell-type-specific activation of PAK2 by TGF-beta

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. Therefore this study investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding (Wilkes, 2009).

The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals

The conserved Hippo signaling pathway regulates organ size in Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using conditional knockout mice, it was demonstrated that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. These studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2 (Zhang, 2010).

Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

The Hippo pathway effectors YAP and TAZ (see Drosophila Yorkie) act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. This study uncovered a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 (see Drosophila Pak) signaling and negating the functions of Merlin and LATS1/2 (see Drosophila Merlin and Warts). It was further shown that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. It is proposed that Agrin acts as a mechanotransduction signal in the ECM (Chakraborty, 2017).

The tumor suppressor Nf2 regulates corpus callosum development by inhibiting the transcriptional coactivator Yap

The corpus callosum connects cerebral hemispheres and is the largest axon tract in the mammalian brain. Callosal malformations are among the most common congenital brain anomalies and are associated with a wide range of neuropsychological deficits. Crossing of the midline by callosal axons relies on a proper midline environment that harbors guidepost cells emitting guidance cues to instruct callosal axon navigation. Little is known about what controls the formation of the midline environment. This study found that two components of the Hippo pathway, the tumor suppressor Nf2 (Merlin) and the transcriptional coactivator Yap (Yap1; see Drosophila Yorkie), regulate guidepost development and expression of the guidance cue Slit2 in mouse. During normal brain development, Nf2 suppresses Yap activity in neural progenitor cells to promote guidepost cell differentiation and prevent ectopic Slit2 expression. Loss of Nf2 causes malformation of midline guideposts and Slit2 upregulation, resulting in callosal agenesis. Slit2 heterozygosity and Yap deletion both restore callosal formation in Nf2 mutants. Furthermore, selectively elevating Yap activity in midline neural progenitors is sufficient to disrupt guidepost formation, upregulate Slit2 and prevent midline crossing. The Hippo pathway is known for its role in controlling organ growth and tumorigenesis. This study identifies a novel role of this pathway in axon guidance. Moreover, by linking axon pathfinding and neural progenitor behaviors, these results provide an example of the intricate coordination between growth and wiring during brain development (Lavado, 2014).

Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP

The Drosophila TEAD ortholog Scalloped is required for Yki-mediated overgrowth but is largely dispensable for normal tissue growth, suggesting that its mammalian counterpart may be exploited for selective inhibition of oncogenic growth driven by YAP hyperactivation. This hypothesis was tested genetically and pharmacologically. A dominant-negative TEAD molecule was shown not to perturb normal liver growth but potently suppresses hepatomegaly/tumorigenesis resulting from YAP overexpression or Neurofibromin 2 (NF2)/Merlin inactivation. Verteporfin was identified as a small molecule that inhibits TEAD-YAP association and YAP-induced liver overgrowth. These findings provide proof of principle that inhibiting TEAD-YAP interactions is a pharmacologically viable strategy against the YAP oncoprotein (Liu-Chittenden, 2012).

Merlin mutation is associated with tumors

Neurofibromatosis type 2 (NF2) is a monogenic dominantly inherited disease predisposing carriers to develop nervous system tumours. To identify the genetic defect, the region between two flanking polymorphic markers on chromosome 22 was cloned and several genes identified. One is the site of germ-line mutations in NF2 patients and of somatic mutations in NF2-related tumours. Its deduced product has homology with proteins at the plasma membrane and cytoskeleton interface, a previously unknown site of action of tumour suppressor genes in humans (Rouleau, 1993).

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by the occurrence of bilateral vestibular schwannomas and other central nervous system tumors including multiple meningiomas. Genetic linkage studies and investigations of both sporadic and familial tumors suggest that NF2 is caused by inactivation of a tumor suppressor gene in chromosome 22q12. A candidate gene for the NF2 tumor suppressor has been identified that has suffered nonoverlapping deletions in DNA from two independent NF2 families and alterations in meningiomas from two unrelated NF2 patients. The candidate gene encodes a 587 amino acid protein with striking similarity to several members of a family of proteins proposed to link cytoskeletal components with proteins in the cell membrane. The NF2 gene may therefore constitute a novel class of tumor suppressor gene (Trofatter, 1993).

Sporadic and inherited schwannomas were scanned for the nature, frequency, and distribution of mutations in the NF2 locus encoding the merlin tumor suppressor protein on 22q. Of 58 tumors, 47% displayed loss of heterozygosity for NF2, leaving a total of 89 NF2 alleles to be examined. Pathogenic alterations were identified in 62 of these alleles, including 36 frameshifts with premature termination, 14 nonsense mutations, and 12 changes presumed to affect splicing. Effects of ten of the latter were confirmed in the NF2 transcript and indicate that activation of cryptic splice sites in coding sequence is another frequent mechanism leading to truncation of merlin. The mutations are relatively evenly distributed across both the protein 4.1 superfamily (exons 1-9) and the alpha-helical (exons 10-15) domains of merlin, but they do not occur at all in exons 16 and 17, which encode the protein's alternative COOH-termini. The data support the "two-hit" tumor suppressor model for formation of schwannomas and indicate that loss of merlin function can be achieved by truncation at various locations in the protein. However, the absence of mutations in exons 16 and 17 suggests that an inactivating mutation affecting only one of merlin's alternative termini may not be sufficient to eliminate tumor suppressor function (Jacoby, 1996). \

Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2-encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene (Bianchi, 1998).

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins that link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations are nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, protein truncation test was performed and it was found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) were expressed in a fibroblast cell line. The wt-NF2 shows intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain shows granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells reveals that wt-NF2 protein induces a progressive elongation of cell processes. Furthermore, cells that express mu-NF2 have decreased adhesion, which results in detachment from the substratum. These findings suggest that the exon-missing mutations in the ERM-homology domain may affect cell membrane-cytoskeleton signaling and consequently disrupt cell-to-cell or cell-to-matrix interaction (Koga, 1999).

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Cleavage of merlin by the ubiquitous protease calpain has been demonstrated along with considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway (Kimura, 1999).

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. This mechanism also applies for the neurofibromatosis type-2 gene (NF2). NF2, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain show high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein are normal. These results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages (Giovannini, 1999).

Hemizygosity for the NF2 gene in humans causes a syndromic susceptibility to schwannoma development. However, Nf2 hemizygous mice do not develop schwannomas but mainly osteosarcomas. In the tumors of both species, the second Nf2 allele is inactivated. Conditional homozygous Nf2 knockout mice with Cre-mediated excision of Nf2 exon 2 in Schwann cells show characteristics of neurofibromatosis type 2. These included schwannomas, Schwann cell hyperplasia, cataract, and osseous metaplasia. Thus, the tumor suppressor function of Nf2, here revealed in murine Schwann cells, is concealed in hemizygous Nf2 mice because of insufficient rate of second allele inactivation in this cell compartment (Giovannini, 2000).

Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver

The molecular signals that control the maintenance and activation of liver stem/progenitor cells are poorly understood, and the role of liver progenitor cells in hepatic tumorigenesis is unclear. This study reports that liver-specific deletion of the neurofibromatosis type 2 (Nf2) tumor suppressor gene in the developing or adult mouse specifically yields a dramatic, progressive expansion of progenitor cells throughout the liver without affecting differentiated hepatocytes. All surviving mice eventually developed both cholangiocellular and hepatocellular carcinoma, suggesting that Nf2(-/-) progenitors can be a cell of origin for these tumors. Despite the suggested link between Nf2 and the Hpo/Wts/Yki signaling pathway in Drosophila, and recent studies linking the corresponding Mst/Lats/Yap pathway to mammalian liver tumorigenesis, these molecular studies suggest that Merlin is not a major regulator of YAP in liver progenitors, and that the overproliferation of Nf2(-/-) liver progenitors is instead driven by aberrant epidermal growth factor receptor (EGFR) activity. Indeed, pharmacologic inhibition of EGFR blocks the proliferation of Nf2(-/-) liver progenitors in vitro and in vivo, consistent with recent studies indicating that the Nf2-encoded protein Merlin can control the abundance and signaling of membrane receptors such as EGFR. Together, these findings uncover a critical role for Nf2/Merlin in controlling homeostasis of the liver stem cell niche (Benhamouche, 2010).

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Merlin: Biological Overview | Protein Interactions | Developmental Biology | Effects of Mutation | References

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