BIOLOGICAL OVERVIEW

Merlin (from moesin-ezrin-radixin-like) is a novel member of the ERM family of proteins (named after the founding members of the family: ezrin, radixin and moesin). Interest in the ERM family has been stimulated by the discovery that human neurofibromatosis 2 (NF2) is caused by mutations in merlin protein. The hallmark of NF2 is the presence of bilateral acoustic schwannomas (acoustic neuromas) affecting the eighth cranial nerve, in addition to other neurally associated tumors, such as meningiomas and ependymomas. Identified Drosophila proteins that have sequence resemblance to ERM family members include Moesin-like, Coracle, Expanded, and Inscuteable (McCartney, 1996).

The cellular functions of the ERM family in vertebrates are thought to be performed in two specific cellular domains: the microvilli, where ezrin is a component, and the adherens junction, from which radixin was first isolated. McCartney and Fehon (1996) have characterized two Drosophila ERM-like proteins, Merlin and Moesin-like. Although Merlin and Moesin-like proteins are frequently coexpressed in developing tissues, they display distinct subcellular localizations. In polarized epithelia, such as the embryonic hindgut, salivary gland, or the imaginal disc, both Moesin-like and Merlin are found in the highest concentration in the most apical part of the cell. Colocalization experiments in wing imaginal discs demonstrate that at least part of the detected Merlin and Moesin-like protein is associated with the adherens junction as is the beta-Catenin homolog, Armadillo. Moesin-like is localized to the apical cap of imaginal disc cells, a region known to contain abundant microvilli. Both Moesin-like and filamentous actin are localized in microvilli present during cellularization in the preblastoderm embryo. Merlin does not localize with actin or Moesin-like in apical buds. While Moesin-like is observed in continuous association with the plasma membrane, as is typical for an ERM family protein, Merlin is found in punctuate structures at the membrane and in the cytoplasm. Investigation of Merlin distribution in cultured cells demonstrates that it is associated with endocytic compartments. Merlin may not interact with cytoskeletal actin in the same way that has been proposed for other members of the ERM family. It is thought that Merlin is associated with membrane internalized from the cell surface. As a result of these studies, it is proposed that whereas Moesin-like may fulfill all the functions of vertebrate ERM proteins, Merlin protein has unique functions in the cell which differ from those of other ERM family members, perhaps being involved in endocytosis. It is concluded that Merlin is a functionally distinct ERM family member (McCartney, 1996).

Genetically marked clones, mutant for Merlin, were generated during the larval period. Comparison of wild-type (control) and mutant clones in the eye suggested that the mutant clones are consistently larger than their wild-type sisters. The number of ommatidia within each mutant clone was compared to the number of ommatidia in its sister wild-type clone and a ratio of mutant clone size to wild-type sister clone size was generated. Merlin mutant clones range from 2.1 to 2.7 times the size of their wild-type sisters, depending on the allele of Merlin examined. This observation suggests that the Merlin mutant cells either proliferate more rapidly than their wild-type neighbors or that they continue proliferating later in development. Another possible explanation is that the mutant cells have a defect in cell death leading to apparent overproliferation. Acridine orange staining of Merlin mutant imaginal discs did not indicate any changes in the level of cell death, however. Because loss of Merlin function in clones results in overproliferation without any gross morphological defects, it is concluded that Merlin functions in a process that specifically affects the regulation of proliferation (LaJeunesse, 1998).

Besides the overproliferation observed in Mer eye clones, a second Mer phenotype has been observed. A single viable allele, Mer³ is due to a missense mutation of Met177 to Isoleucine. Flies homozygous for Mer³ survive as viable, sterile adults and display a broadened wing phenotype along with low and variably penetrant expression of weakly roughened eyes and the development of abnormal head cuticle structures. The broadening of the wing is characteristic of mutations that increase cell proliferation in the wing. Further analysis was carried out to determine the structural domains within Merlin required for proper function, both in terms of the subcellular distribution of Merlin and the overproliferation phenotype (LaJeunesse, 1998).

Mutational analysis of Merlin protein has been carried out to try to understand how Merlin functions to restrain cell proliferation. The molecular organization of Merlin is similar to that of ERM family members and consists of a tripartite domain structure: an NH₂-terminal protein 4.1 domain, a putative coiled-coil domain, and a COOH-terminal region. Because of the apparent modular nature of Merlin, several Merlin truncations were generated that contain portions of the NH₂- or COOH-terminal halves of the protein and a truncation containing only the central coiled-coiled region. Wild-type Merlin is initially targeted to the membrane, and within 3 h, much of the protein localizes to punctate cytoplasmic structures. A similar pattern (membrane-associated and cytoplasmic staining) is also observed in endogenously expressed Merlin in S2 cells and within the imaginal disc epithelium (McCartney, 1996). Removal of the conserved COOH-terminal 35 amino acids results in a protein, mycMer^1-600, that is localized almost exclusively at the plasma membrane and does not appear to internalize. This observation suggests that the COOH-terminal 35 amino acid residues play a role in Merlin internalization. As will be described below, internalization is likely to be important in Mer function. In the imaginal epithelia of transgenic larvae, this truncated Merlin protein is localized to the plasma membrane (similar to the localization of wild-type Merlin) but did not display the punctate cytoplasmic localization characteristic of the wild-type protein. It is concluded that components of both the NH₂- and COOH-terminal halves of Merlin are required for its correct targeting to the plasma membrane (LaJeunesse, 1998).

The conserved NH₂-terminal region (CNTR) was examined in greater detail. The CNTR is nearly 60% identical between Merlin and the ERM proteins; however, a closer examination of this region reveals seven amino acids (¹⁷⁰YQMTPEM¹⁷⁷) that are identical in human and Drosophila Merlin but are divergent from the ERM proteins. The possible functional significance of this region is further supported by the presence of the Mer³ missense mutation (the viable allele described above) at amino acid 177. This region has been named "the Blue Box" (BB). To investigate the functional significance of the BB, two Merlin proteins were generated, one lacking the BB region (mycMer^deltaBB) and one in which the BB is replaced by a polyalanine stretch (mycMer^BBA). Removal or replacement of the entire BB results in proteins that are properly targeted to the plasma membrane but are not internalized. It is concluded that the BB region is required for internalization of Merlin, but is not required for association with the plasma membrane. Genetic rescue of Mer-lethality by truncated proteins indicate that all essential Merlin functions reside in the NH₂-terminal 350 amino acids of the protein (LaJeunesse, 1998).

Nonmutant class siblings that carried ubiquitously expressing Mer transgenes were examined for dominant phenotypes. None of the NH₂- or COOH-terminal deletions display any dominant phenotype in a Mer⁺ background. In contrast, dominant phenotypes, suggesting hyperproliferation, results from expression of the Merlin BB mutant proteins. The dominant phenotype includes a broadening of the wing blade, variably penetrant ectopic wing vein material (primarily around the second wing vein), anterior and posterior cross vein defects, and ectopic bristles or sense organs. The most striking quality of this dominant phenotype is the enlargement of the wing blade, resulting in a curvature of the wing surface. High levels of expression of the Merlin BB mutant proteins in the wing blade under two different Gal4 drivers (engrailed^ciBeGal4, apterous^md5 Gal4) result in an outheld wing phenotype associated with alterations in the wing hinge morphology (LaJeunesse, 1998).

To examine the wing blade phenotype in more detail, the wing surface areas were compared for the expression of Merlin BB proteins or wild-type Merlin under the engrailed GAL4 driver. As expected of an overproliferation phenotype, a significant increase in the total surface area of wings expressing BB mutant proteins was observed. The area of overgrowth is localized to the posterior half of the wing where the engrailed Gal4 specifically drives expression, although a decrease in surface area is observed between wing veins 2 and 3, a region anterior to the expression of the transgene. To determine whether the observed increase in area is due to increased cell number or increased cell size, the densities of wing hairs were measured at several positions within the wing blade. (Each wing blade cell secretes a single hair). The overall wing hair density in wings expressing BB Merlin proteins is indistinguishable from that in wings expressing a wild-type protein, indicating that the broadening of the wing results from an increase in cell number rather than in cell size. As shown in the somatic mosaic analysis, loss of Mer function results in overproliferation of mutant cells. Similarly, ectopic expression of BB mutant proteins resulted in increased proliferation. These results suggest that the BB mutant proteins act in a dominant-negative manner and therefore interfere with the activity of wild-type protein (LaJeunesse, 1998).

MycMer^1-600, a truncated Merlin protein missing the C-terminal 35 amino acids acts as an activated protein that displays greater rescuing activity and suppresses the phenotypes produced by a dominant-negative form of Merlin. Pulsed expression of Mer^1-600 in S2 cultured cells under an inducible promoter indicates that the levels of expression and the stability of this protein are not significantly different from wild-type Merlin. The simplest explanation for this phenomenon is that the COOH-terminal region contains a domain important for reducing the activity of Merlin. Recent studies demonstrate that ERM protein activity is regulated by a Rho-based signaling pathway (Takaishi, 1995, Hirao, 1996 and Mackay, 1997). Rho-Kinase has been shown to phosphorylate a conserved threonine within the COOH-terminal 35 amino acids of all ERM proteins and thereby regulate the conformational change that occurs during a putative transition from an inactive to an active state (Matsui, 1998). Although the COOH-terminal 35 amino acids of Merlin are divergent from those of the ERM proteins, the threonine residue phosphorylated by Rho-Kinase is conserved, suggesting that Merlin could use a similar mechanism for switching between an inactive and an active state. Creation of an activated Merlin protein by the removal of the COOH-terminal regulatory region is consistent with this notion, but further studies are required to confirm this regulatory role (LaJeunesse, 1998).

Two Merlin mutations described here, the dominant-negative Mer^DeltaBB and the activated Mer^1-600, are found primarily at the plasma membrane. This apparently contradictory result, that two mutant forms of Merlin with opposite functions both localize to the plasma membrane, indicates that localization to the plasma membrane is not sufficient for Merlin function, that is to say, internalization is likely to be important in Mer function. The requirement for internalization distinguishes Mer from other ERM family members, Moesin-like for example, which are exclusively located at junctions. It is proposed that Merlin normally undergoes an activation process that occurs at the cytoplasmic face of the plasma membrane. In this model, not only is Mer^DeltaBB refractory to activation, but it also inhibits activation of endogenous wild-type Merlin, thus causing wild-type Merlin to accumulate at the plasma membrane in a nonfunctional state and producing a dominant phenotype. In contrast, Mer^1-600 exists in a constitutively activated state, thereby evading the block presented by Mer^DeltaBB and suppressing the dominant BB mutant phenotype. The two functional states of wild-type Merlin may represent two distinct conformations of the protein, as has been shown for ERM proteins. Alternatively, Merlin may associate with two distinct binding partners at the membrane. In either case, one state may serve to associate Merlin with the membrane, and the second may be required for Merlin activation. (LaJeunesse, 1998).

Spatial Organization of Hippo Signaling at the Plasma Membrane Mediated by the Tumor Suppressor Merlin/NF2

Although Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. In contrast to the commonly depicted linear model of Merlin functioning through Hpo/Mst, this study shows that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. This study further shows that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. These findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction (Yin, 2013).

Since its initial discovery as a human disease gene underlying NF2, the tumor suppressor Merlin has been the subject of intense investigation. Besides the Hippo pathway, Merlin has been linked to a variety of mechanisms such as transmembrane receptor endocytosis/localization (EGFR and CD44) and signaling by Ras, Rac/PAK, and PI3K pathways. Paradoxically, as a membrane-associated tumor suppressor, Merlin was also reported to suppress tumorigenesis in mammalian cells by translocating to the nucleus to inhibit a specific E3 ubiquitin ligase. Among these proposed targets, the linkage between Merlin and Hippo signaling has attracted much attention given the similarity of the respective mutant phenotypes in Drosophila and the dosage-sensitive genetic suppression of Merlin mutant phenotypes by heterozygosity of the Hippo effector YAP in multiple mouse tissues (Yin, 2013).

Despite the genetic evidence implicating Merlin in Hippo signaling, the molecular basis of this functional link was unknown. The current study addresses this outstanding issue in two important ways. First, molecular evidence is provided showing that Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. These studies therefore disprove the prevailing assumption that Merlin functions biochemically upstream of Hpo activation. Along this line, it is noted that current models of Hippo signaling are actually a composite of true molecular relationships (such as Hpo acting upstream of Wts or Wts acting upstream Yki) and genetic epistasis relationships (such as Mer acting upstream of Hpo). In light of the current study, it is cautioned that biochemical and epistasis relationships should be clearly distinguished in signaling diagrams because mixing and matching them can be misleading. Second, direct physical interactions between Merlin and Wts/Lats were elucidated, and it was shown that such interactions promote Hippo signaling by recruiting Wts/Lats to the plasma membrane. The discovery of physical interactions between Merlin and a key component of the Hippo pathway therefore provides molecular support for a Merlin-Hippo connection that has so far been based largely on genetics and indirect evidence. Interestingly, interactions between Merlin and Wts are regulated by the actin cytoskeleton, underscoring Merlin as a potential mediator of actin-regulated Hippo signaling (Yin, 2013).

Besides identifying a conserved molecular function for Merlin, these studies also revealed quantitative differences between Drosophila and mammalian Merlin. WT Mer normally does not associate with Wts in Drosophila S2R+ cells, yet WT NF2 suffices to bind Lats1/2 in human cells. Such differences correlate with an intrinsically more open conformation of NF2 compared to Mer. These findings agree with previous reports that the intramolecular interaction in NF2 is relatively weak and dynamic. It is noted that the intrinsically more active/open state of NF2 is consistent with the role of S518 phosphorylation in antagonizing NF2 activity and the absence of this negative regulatory site in Drosophila Mer. Obviously, such negative regulation would be of more functional relevance in the context of an intrinsically more active Merlin protein as in mammals (Yin, 2013).

The plasma membrane is the entry point of diverse environmental stimuli and is intimately involved in spatial organization of signaling proteins. Although many reported upstream regulators of the Hippo pathway in Drosophila are transmembrane proteins (e.g., Fat and Crumbs) or are localized in apical membrane domains (e.g., Mer, Ex, and Kibra), how these membrane-associated inputs spatially organize the Hippo kinase cassette was poorly understood. This question is further complicated by the possible evolutionary divergence of upstream inputs into the pathway between Drosophila and mammals. Notably, among these upstream inputs, Merlin is the only protein whose contribution to Hippo signaling has been genetically validated in both flies and mammals (Yin, 2013).

This study demonstrates that an important and evolutionarily conserved molecular function of Merlin is to promote the membrane association of Wts/Lats. Sav is also implicated as a membrane-associated scaffold that promotes the membrane association of Hpo/Mst, the upstream kinase of Wts/Lats. Thus, two predominantly membrane-associated proteins, Merlin and Sav, are involved in targeting the two essential kinases of the Hippo kinase cassette to the plasma membrane. It is tempting to speculate that at least some of the other upstream regulators of Hippo signaling may function in a similar manner by promoting the membrane association of the Hippo kinase cassette. It is noted that a functional role for Sav in membrane association of Hpo does not preclude the other previously described roles for the Sav scaffold in Hippo signaling, such as tethering Hpo and Wts. It is possible that Sav potentiates Hippo signaling both by tethering multiple signaling components and by localizing signaling activity to specific subcellular compartments, as shown in other well-studied scaffold signaling proteins such as Ste5 and KSR. Nevertheless, this study has uncovered a role for Sav in spatial organization of the Hippo pathway (Yin, 2013).

Wts/Lats is known to be subjected to two modes of regulation, including phosphorylation and protein stability. This study extends previous studies by showing that the membrane association of Wts represents an additional mode of regulation. In addition, this study suggests that Wts may be activated by alternate upstream kinase(s) besides Hpo. Identifying the kinases that mediate Hpo-independent activation and understanding the regulation of such kinases should greatly expand knowledge about the physiological regulation of Hippo signaling. With its activity subjected to multiple modes of regulation, it is becoming increasingly clear that Wts/Lats represents as a critical node in the Hippo signaling network. These different modes of regulation are not exclusive of each other and are indeed functionally intertwined, as membrane association of Wts/Lats also enhances its phosphorylation. Understanding how the multiple regulatory inputs into Wts/Lats are coordinated will shed light on the physiological regulation of Hippo signaling in normal development and offer new strategies for therapeutic intervention in pathological conditions such as NF2 (Yin, 2013).

PROTEIN STRUCTURE

Amino Acids - 635

Structural Domains

Merlin is 55% identical to human merlin, while only 45% identical to human ezrin and mouse radixin, and 39% identical to human moesin. The similarity between Drosophila and human Merlin extends over the entire lingth of the two sequences, and is greatest in the N-terminus, where all ERM proteins share high identity. In this region, however, areas can be identified where the Merlin sequences, while similar to each other, diverge from the human and Drosophila Moesin sequences. The similarity between Drosophila Merlin and human merlin is particularly notable at the C-terminus, where the merlin proteins diverge from the other ERM family members. Sequences from the second Drosophila ERM family member, Moesin-like, are found to share 58% overall identity with both human moesin and human ezrin, 57% identity with mouse radixin, and only 41% identity with human merlin. Designation of this gene as Moesin-like, rather than as ezrin or radixin, is based primarily on its lack of the polyproline tract characteristic of the latter two proteins. furthermore, Moesin-like shares a greater degree of identity in the C-terminal divergent region with human moesin (26%) than with human ezrin (25%) or mouse radixin (22%) (McCartney, 1996).

date revised: 15 September 2022

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