Evolution and origin of merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor suppressor gene, belongs to the ezrin-radixin-moesin (ERM) subgroup of the protein 4.1 superfamily, which links cell surface glycoproteins to the actin cytoskeleton. While merlin's functional activity has been examined in mammalian and Drosophila models, little is understood about its evolution, diversity, and overall distribution among different taxa. By combining bioinformatic and phylogenetic approaches, this study demonstrates that merlin homologs are present across a wide range of metazoan lineages. While the phylogenetic tree shows a monophyletic origin of the ERM family, the origin of the merlin proteins is robustly separated from that of the ERM proteins. The derivation of merlin is thought to be in early metazoa. The expansion of the ERM-like proteins is observed within the vertebrate clade, which occurred after its separation from Urochordata (Ciona intestinalis). Amino acid sequence alignment reveals the absence of an actin-binding site in the C-terminal region of all merlin proteins from various species but the presence of a conserved internal binding site in the N-terminal domain of the merlin and ERM proteins. In addition, a more conserved pattern of amino acid residues is found in the region containing the so-called 'Blue Box,' although some amino acid substitutions in this region exist in the merlin sequences of worms, fish, and Ciona. Examination of sequence variability at functionally significant sites, including the serine-518 residue, the phosphorylation of which modulates merlin's intra-molecular association and function as a tumor suppressor, identifies several potentially important sites that are conserved among all merlin proteins but divergent in the ERM proteins. Secondary structure prediction reveals the presence of a conserved alpha-helical domain in the central to C-terminal region of the merlin proteins of various species. The conserved residues and structures identified correspond to the important sites highlighted by the available crystal structures of the merlin and ERM proteins. Furthermore, analysis of the merlin gene structures from various organisms reveals the increase of gene length during evolution due to the expansion of introns; however, a reduction of intron number and length appears to occur in the merlin gene of the insect group. These results demonstrate a monophyletic origin of the merlin proteins with their root in the early metazoa. The overall similarity among the primary and secondary structures of all merlin proteins and the conservation of several functionally important residues suggest a universal role for merlin in a wide range of metazoa (Golovnina, 2005).

Alternative splicing and postranslational modification of vertebrate merlin

The recently isolated gene for neurofibromatosis type 2 (NF2) encodes a 595 amino acid protein, named merlin, which is related to the cytoskeleton-associated proteins moesin, ezrin and radixin. To identify evolutionarily conserved regions and to provide sequence information necessary for the establishment of a mouse model for NF2, the cDNA sequence has been determined for the mouse NF2 tumor suppressor gene, and it has been mapped in the mouse genome. Mouse merlin is a 596 amino acid protein, 98% identical to human merlin, but one amino acid longer due to the insertion of a proline residue near the C-terminus. Of the nine amino acid differences between mouse and humans, seven occur in the C-terminal 20% of the protein, far from the protein 4.1 domain that defines this family. Two of the NF2 cDNA clones reveal evidence of alternative splicing events that alter the predicted merlin product: one removes a 45 amino acid segment from the middle section of the protein and the other changes the C-terminus. The existence of several different forms of merlin, each potentially with a different primary role, will complicate the identification of the precise function that must be disrupted to cause the NF2-associated tumors. The mouse NF2 homolog maps to Chr 11, in a region homologous to human Chr 22, but devoid of any mouse mutations that could be models of the human disorder (Haase, 1994).

Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17', which lacks exon 16 and 'NF2-16', which contains exon 16 andencodes a merlin protein truncated at the C-terminus. In rat schwannoma cells, overexpression of NF2-17 inhibits cell growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibitionby merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. Thisassociation does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminalsequences. These data indicate that merlin has a unique mechanism for tumor suppression: inhibiting cell proliferation viaself-association (Sherman, 1997).

During the characterization of mouse NF2transcripts, four different transcripts were identifed by cDNA analysis and reverse-transcribed PCR. These transcripts containdifferent sequences in the 3' ends of the coding sequences. In human cell lines three isoforms encoding two distinctschwannomins (an alternative name for merlin) are detected. The mouse and human transcripts containing 61 and 60 bp inserts, respectively, have notbeen previously described. The isoforms encode schwannomins with significantly altered C-termini and are expressedat different relative levels in adult mouse tissues and during mouse embryogenesis. These results suggest thatschwannomin isoforms have distinct functional roles and predict the existence of human mutations involving theC-terminus of schwannomin (Huynh, 1994).

Examination of merlin in several cell lines reveals that the protein migrates as two distinct species near 70 kDa. Phosphatase treatment and orthophosphate labeling demonstrates that the species with decreased mobility is phosphorylated. Given Merlin's localization to cortical actin structures, the effect of cell-cell contact or other forms of growth arrest were examined on Merlin expression and post-translational modification. Under conditions of confluency or serum deprivation, the levels of phosphorylated and unphosphorylated Merlin species increases significantly. Cells arrested in G1 by other methods or other phases of the cell cycle do not show changes in Merlin levels. Furthermore, loss of adhesion results in a nearly complete dephosphorylation of Merlin, which is reversed when cells are re-plated, suggesting Merlin phosphorylation may be responsive to cell spreading or changes in cell shape. Thus, the tumor suppressor function of Merlin may involve the regulation of cellular responses to cues such as cell-cell contact, growth factor microenvironment, or changes in cell shape (Shaw, 1998a).

Domain structure of vertebrate merlin

An examination was carried out of the localization and effects of overexpressing epitope-tagged full-length isoforms of merlin as well as amino- and carboxyl-terminal truncations. The full-length and the amino-terminal domain of merlin localize to cortical actin, particularly areas of dynamic actin rearrangements such as membrane ruffles. Furthermore, overexpression of the carboxyl half of merlin induces cell death in NIH3T3 cells. The effect is splice-form specific and is not observed in the context of the full-length molecule. Thus, as has been described for the erzin, radixin, and moesin proteins, the activities of the carboxyl-terminal domain of merlin may be suppressed by the amino-terminal domain (Shaw, 1998b).

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Both transiently expressed wild-type isoforms are observed underneath the plasma membrane. At this location they are detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrate identical properties. Three different truncated schwannomins, prototypical for most naturally occurring NF2 mutations, are affected neither in their location nor in their cytochalasin D sensitivity. However, they are detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility is also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain have lost the submembraneous localization and tend to accumulate in perinuclear patches that are unaffected by cytochalasin D treatment. A similar behavior is observed when the N-terminal domain is entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane (Deguen, 1998).

The neurofibromatosis 2 (NF2) tumor suppressor gene product, merlin (schwannomin) forms an intramolecular association thatis required for negative growth regulation in vitro and in vivo. In an effort to develop a molecular model for merlin relevant to itstumor suppressor function, merlin intramolecular folding was further characterized. Merlin forms two intramolecular associations, one between the amino terminal (N-term) domain and the carboxyl terminal (C-term) domain andanother within the amino terminal domain (N-term/N-term) itself. The N-term/C-term domain interaction requires contactbetween residues 302-308 in the N-term and an intact exon 17 (residues 580-595) in the C-term domain. In addition, the N-term/N-term domain self-interaction is required for N-term/C-term domain association. NF2 patient mutations have been identified that dramatically reduce each of these interactions in vitro. Based on these findings, a model is proposed for merlin folding critical to its ability to function as a tumor suppressor protein (Gutmann, 1999).

Protein interactions of vertebrate merlin

NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, betaII-spectrin (also known as fodrin: see Drosophila alpha Spectrin) has been identified as a schwannomin-binding protein. Interaction occurs between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. In contrast, isoform 1 of schwannomin interacts weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitates with betaII-spectrin at physiological concentrations. The two proteins interact in vitro and co-localize in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations show reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides show alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signaling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types (Scoles, 1998).

In contrast to the ERM proteins, merlin lacks a conventional C-terminal actin binding site, but still localizes to the ruffling edge of plasma membranes. In this study, the ability of merlin to interact with actin through a nonconventional actin binding domain is examined. Merlin can associate with polymerized actin in vitro by virtue of an amino (N-) terminal actin binding domain, including residues 178-367. Merlin actin binding is not affected by several naturally-occurring NF2 patient mutations or by alternatively spliced isoforms. These results suggest that merlin, like other ERM proteins, can directly interact with the actin cytoskeleton. In addition, merlin associates with polymerized microtubules in vitro using a novel microtubule binding region in the N-terminal region of merlin that is masked in the full-length merlin molecule, such that wild-type functional merlin in the "closed" conformation fails to bind polymerized microtubules. These microtubule association results confirm the notion that merlin exists in 'open' and 'closed' conformations relevant to its function as a negative growth regulator (Xu, 1998).

Neurofibromatosis 2 (NF2) protein (known as merlin or schwannomin) is a tumor suppressor involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas. NF2-related ERM proteins function as membrane organizers and may act aslinkers between plasma membrane molecules, such as CD44 and ICAM-2, and the cytoskeleton. The distribution and effects of transfected NF2 protein were examined in COS-1, CHO and 293 cells, and endogenous NF2 protein in U251 glioma cells. The distribution was compared to ezrin, CD44 and F-actin. Both transfected and endogenous NF2 proteins localize underneath the plasma membrane in a pattern typical of an ERM protein. In COS-1 transfectants, NF2 protein typically codistributes with ezrin but, in cells with poorly developed actin cytoskeleton, it replaces ezrin in filopodia andruffling edges. NF2 protein colocalizes with CD44, which in transfected cells accumulates into restructured cell membrane protrusions. The association of CD44 and NF2 protein is further suggested by binding of CD44 from cellular lysates to recombinant NF2 protein. Interaction between NF2 protein and the actin-containing cytoskeleton is indicated by partial colocalization, by cytochalasin B-induced coclustering, and by retention of NF2 protein in the detergent-insoluble fraction. Transfected NF2 protein induces morphogenic changes. The cells contain restructuredmembrane extensions and blebs, and CHO cells expressing NF2 protein are more elongated than control transfectants. In conclusion, NF2 protein possesses functional properties of an ERM family member (Sainio, 1997).

The human homolog of a regulatory cofactor of Na(+)-H+ exchanger (NHE-RF) has been identified as a novel interactor for merlin, the neurofibromatosis 2 tumor suppressor protein. NHE-RF mediates protein kinase A regulation of Na(+)-H+ exchanger NHE3, to which it is thought to bind via one of its two PDZ domains. The carboxyl-terminal region of NHE-RF, downstream of the PDZ domains, interacts with the amino-terminal protein 4.1 domain-containing segment of merlin in yeast two-hybrid assays. This interaction also occurs in affinity binding assays with full-length NHE-RF expressed in COS-7 cells. NHE-RF binds to the related ERM proteins, moesin and radixin. Human NHE-RF has been localized to actin-rich structures such as membrane ruffles, microvilli, and filopodia in HeLa and COS-7 cells, where it co-localizes with merlin and moesin. These findings suggest that hNHE-RF and its binding partners may participate in a larger complex (one component of which might be an Na(+)-H+ exchanger) that could be crucial for the actin filament assembly activated by the ERM proteins and for the tumor suppressor function of merlin (Murthy, 1998).

Ezrin, radixin and moesin (ERM) are homologous proteins, which are linkers between plasma membrane components and the actin-containing cytoskeleton. The ERM protein family members associate with one another in a homotypic and heterotypic manner. The neurofibromatosis 2 (NF2) tumor suppressor protein merlin (schwannomin) is structurally related to ERM members. Merlin is involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas, but the tumor suppressor mechanism is poorly understood. The ability of merlin to self-associate and bind ezrin has been examined. Ezrin coimmunoprecipitates with merlin from lysates of human U251 glioma cells and from COS-1 cells transfected with cDNA encoding for merlin isoform I. The interaction was further studied and the association domains were mapped with the yeast two-hybrid system and with blot overlay and affinity precipitation experiments. The heterotypic binding of merlin and ezrin and the homotypic association of merlin involves interaction between the amino- and carboxy-termini. The amino-terminal association domain of merlin involves residues 1-339 and has similar features with the amino-terminal association domain of ezrin. The carboxy-terminal association domain cannot be mapped as precisely as in ezrin, but it requires residues 585-595 and a more amino-terminal segment. Unlike ezrin, merlin does not require activation for self-association but native merlin moleculescan interact with each other. Heterodimerization between merlin and ezrin, however, occurs only following conformational alterations in both proteins. These results biochemically connect merlin to the cortical cytoskeleton and indicate differentialregulation of merlin from ERM proteins (Gronholm, 1999).

The product of the neurofibromatosis type II (NF2) tumor suppressor gene,merlin, is closely related to the ezrin-radixin-moesin (ERM) family, a group ofproteins believed to link the cytoskeleton to the plasma membrane. Mutation inthe NF2 locus is associated with Schwann cell tumors (schwannomas). The twopredominant merlin isoforms, I and II, differ only in the carboxy-terminal 16residues and only isoform I is anti-proliferative. Merlin lacks an actin-bindingdomain conserved among ezrin, radixin and moesin. Because merlin, ezrin andmoesin are co-expressed in Schwann cells, and all homodimerize, the ability of merlin and ezrin to dimerize with one another has been examined. Both merlin isoforms have been shown, byimmunoprecipitation and yeast two-hybrid assays, tointeract with ezrin. The interaction occurs in a head-to-tail orientation, withthe amino-terminal half of one protein interacting with the carboxy-terminalhalf of the other. The two merlin isoforms behave differently in theirinteraction with ezrin. Isoform I binds only ezrin whose carboxy-terminus isexposed, whereas isoform II binds ezrin regardless of whether ezrin is in theopen or closed conformation. The heterodimerization of merlin is a much strongerinteraction than the interaction between either merlin isoform and ezrin, andcan inhibit merlin-ezrin binding. This suggests that, in vivo, merlindimerization could regulate merlin-ERM protein interaction, and could thusindirectly regulate other interactions involving ERM proteins (Meng, 2000).

The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved inNF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members,implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as atumor suppressor is unknown. In an attempt to shed light on schwannomin function, a novel coiled-coil protein,SCHIP-1, has been identified that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein ishomologous to human FEZ proteins and the related C. elegans gene product UNC-76. Immunofluorescentstaining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmicmembrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can beobserved only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and3, but not with the schwannomin isoform exhibiting growth-suppressive activity. These observations suggest that SCHIP-1interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslationalmodifications, alternative splicing, or mutations (Goutebroze, 2000).

Merlin, the neurofibromatosis 2 tumor suppressor protein, has two major isoforms with alternate C termini and is related to theERM (ezrin, radixin, moesin) proteins. Regulation of the ERMs involves intramolecular and/or intermolecular head-to-tailassociations between family members. A determination was made as to whether merlin undergoes similar interactions; the findingsindicate that the C terminus of merlin isoform 1 is able to associate with its N-terminal domain in a head-to-tail fashion.However, the C terminus of isoform 2 lacks this property. Similarly, the N terminus of merlin can also associate with Cterminus of moesin. The effect of merlin self-association on binding to the regulatory cofactor ofNa(+)-H(+) exchanger (NHE-RF), an interacting protein for merlin and the ERMs, was examined. Merlin isoform 2 captures more NHE-RFthan merlin isoform 1 in affinity binding assays, suggesting that in full-length merlin isoform 1, the NHE-RF binding site ismasked because of the self-interactions of merlin. Treatment with a phospholipid known to decrease self-association of ERMsenhances the binding of merlin isoform 1 to NHE-RF. Thus, although isoform 1 resembles the ERM proteins, which transitionbetween inactive (closed) and active (open) states, isoform 2 is distinct, existing only in the active (open) state and presumablyconstitutively more available for interaction with other protein partners (Gonzalez-Agosti, 1999).

Neurofibromatosis type 2, a disease characterized by the formation of multiple nervous system tumors, especiallyschwannomas, is caused by mutation in the gene-encoding merlin/schwannomin. The molecular mechanism by which merlinfunctions as a tumor suppressor is unknown, but is hypothesized to involve plasma membrane and cytoskeleton interaction.Several merlin antibodies were used to study merlin expression, localization, and protein association in primary cultures of ratsensory neurons, Schwann cells (SCs), and SCs grown with neurons (SC/N cultures) before and during differentiation intomyelinating cells. Western blot analysis has revealed that neurons predominantly express a 68-kD protein, but SCs expresstwo additional 88- and 120-kD related proteins. Extensive immunological characterization have demonstrated that the 88-kDprotein shares three domains with the 68-kD merlin protein. The majority of merlin and related proteins are soluble in isolated SCs and undifferentiated SC/N cultures,but become insoluble in myelinating SC/N cultures. Merlin translocatesfrom the perinuclear cytoplasm in undifferentiated SCs to the subplasmalemma in differentiating SCs and partially colocalizeswith beta1 integrin. Finally, beta1 integrin antibody coimmunoprecipitates 68-kD merlin from isolated SC and undifferentiatedSC/N cultures, but predominantly the 88-kD protein from differentiating SC/N cultures. Together, these results provideevidence that merlin interacts with beta1 integrin and that merlin localization changes from a cytosolic to cytoskeletalcompartment during SC differentiation (Obremski, 1999).

The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cellmembrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminalF-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neitherstress fibers nor cortical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter- and intramoleculardomain interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N-and C-terminal domains of merlin type 1 bind to each other. However, full-length merlin and its N- and C-terminal domains, aswell as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domaininteracts with itself. Merlin 1 function in cells may thus depend on intra- and inter-molecular interactions and their modulations,which include interactions with other members of this protein family (Huang, 1999).

CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. Glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bid to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin are narrowed downto their juxta-membrane 20-30-amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) are all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 are expressed in mouse L fibroblasts, they are co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, it is concluded that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain (Yonemura, 1998).

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. Murine merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypophosphorylated 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 (Morrison, 2001).

Evidence that three band 4.1 family members, merlin, ezrin, and moesin, are involved in a molecular switch that signalscellular growth or growth inhibition. Merlin expression is increased in normal rat Schwann cells at high cell density but is not regulated bycell density in a malignant rat schwannoma cell line. Increasing merlin expression in these transformed cells to a degree similar to thatobserved in confluent normal Schwann cells is sufficient to inhibit growth, but only at high cell density. Under these conditions, merlin isdephosphorylated and interacts with CD44. Ezrin and moesin are not detected in this complex. The association with CD44 is necessaryfor merlin action on cell proliferation. In logarithmically growing cells, however, merlin has no influence on cell growth, is phosphorylated, and is in a complex withezrin and moesin. This complex also is associated with the cytoplasmic tail of CD44. The CD44 ligand, HA, as well as antibodies that mimic CD44 ligands canimitate the effects of high cell density in logarithmically growing cells, rapidly inducing merlin dephosphorylation and inhibiting cell growth (Morrison, 2001).

A molecular switch that instructs a cell when intercellular or cell-matrix contact has occurred should exist in two conformations: a cell proliferation-inhibiting modeand a growth-permissive mode. It is proposed that merlin and CD44 together function as one such molecular switch and that the growth-inhibiting state of this switch isinduced by HA binding to CD44, resulting in merlin hypophosphorylation and direct association with the ERM-binding domain of the CD44 cytoplasmic tail.It is predicted that this switch is at least partly controlled by one or several phosphatases that are associated directly with the complex. In contrast, thegrowth-permissive protein complex consists of CD44 in association with ezrin, moesin, and phosphorylated merlin. It is conceivable that protein kinases thatmaintain CD44-ERM association and prevent merlin activation also would be associated with this complex. The function of ezrin alsowill likely depend on association with CD44 or a similar transmembrane protein. When the CD44 tail overexpressors are kept in low-density culture for several days(in contrast to cells in confluence), tail expression is invariably lost, suggesting that sequestering ezrin away from CD44 is not compatible with proliferation (Morrison, 2001).

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. A novel interaction is described between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as evidenced by the observation that the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 results in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis (Kissil, 2003).

The stability of p53 tumor suppressor is regulated by Mdm2 via the ubiquitination and proteasome-mediated proteolysis pathway. The c-Abl and PTEN tumor suppressors are known to stabilize p53 by blocking the Mdm2-mediated p53 degradation. This study investigated the correlation between p53 and merlin, a neurofibromatosis 2 (NF2)-related tumor suppressor, in association with the Mdm2 function. The results showed that merlin increases p53 stability by inhibiting the Mdm2-mediated degradation of p53, which accompanies the increase in the p53-dependent transcriptional activity. The stabilization of p53 by merlin appears to be accomplished through Mdm2 degradation, and the N-terminal region of merlin is responsible for this novel activity. This study also showed that overexpression of merlin induces apoptosis of cells depending preferentially on p53 in response to the serum starvation or a chemotherapeutic agent. These results suggest that merlin may be a positive regulator of p53 in terms of tumor suppressor activity, and provide the promising therapeutic means for treating tumors with non-functional merlin or Mdm2 overexpression (Kim, 2004).

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. The neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, has been identified as a binding partner for RIbeta. Merlin and RIbeta demonstrate a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins coprecipitate from brain lysates by cAMP-agarose and coimmunoprecipite from cellular lysates with specific antibodies. In vitro binding studies have verified that the interaction is direct. The interaction appears to be under conformational regulation and is mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling (Gronholm, 2003).

Signaling upstream of merlin

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton-membrane linkers. However, the molecular basis for the tumor- and metastasis-suppressing activity of merlin is unknown. This study places merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression (Shaw, 2001).

The results indicate that merlin is regulated by Rac. Although phosphopeptide mapping experiments indicate that merlin phosphorylation is complex, the existing data suggest that Rac-induced phosphorylation inactivates merlin. Phosphorylation of the Rac-responsive S518 residue inhibits merlin self-association, weakens its association with the cytoskeleton, and compromises its ability to inhibit Rac-mediated signaling. This suggests that the closed or oligomerized form of merlin is the active, growth suppressing form. This is consistent with the observations that the same amino acid residues necessary for self-association are also required for the growth- and motility- suppressing function of merlin in schwannoma cells. Importantly, Rac is activated by growth factor stimulation and matrix adhesion, conditions which also stimulate endogenous merlin phosphorylation. The finding that merlin is phosphorylated in response to activation of Rac/Cdc42 and not Rho would indicate that merlin and the ERMs are regulated in distinct ways. Together with the observation that merlin can heterodimerize with ERM proteins, this suggests that this family of proteins could serve to coordinate the activities of these GTPases (Shaw, 2001 and references therein.

The observation that merlin is phosphorylated in response to Rac activation might have suggested that merlin is an effector of Rac and thus required for some downstream functions of Rac. Instead, the results are consistent with a model wherein merlin normally acts to attenuate Rac signaling: (1) overexpression of merlin inhibits JNK activity as well as Rac-induced AP-1 activity and transformation; (2) Nf2-deficient cells exhibit phenotypes that are consistent with hyperactivation of Rac signaling including increased activation of JNK, AP-1, membrane ruffling, and motility. Notably, overexpression of merlin inhibits cell spreading and motility. Together these observations are consistent with a model in which merlin functions as a sensor of Rac signaling. Phosphorylation of merlin in response to Rac activation or other stimuli inactivates merlin, thereby potentiating Rac signaling; dephosphorylation would restore its inhibitory function (Shaw, 2001 and references therein).

Subcellular localization of vertebrate merlin

continued: Merlin Evolutionary homologs part 2/2 |

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

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