Homotypic and heterotypic interaction of the neurofibromatosis 2 tumor suppressor protein merlin and the ERM protein ezrin. (1/103)

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 each other 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. We have studied the ability of merlin to self-associate and bind ezrin. Ezrin was coimmunoprecipitated 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 molecules can 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 differential regulation of merlin from ERM proteins.  (+info)

Merlin: the neurofibromatosis 2 tumor suppressor. (2/103)

In recent years, it has become clear that the ERMs occupy a crucial position as protein linkers that both respond to and participate in reorganization of membrane-cytoskeletal interactions. With the identification of new binding partners, the ERMs are also implicated in linked regulation of the activities of particular membrane proteins. Thus, they reside at a junction in a complex web of interactions that must respond to stimuli from both outside and inside the cell. As expected from its structural motifs, merlin behaves in a manner similar to the ERM proteins, but with some notable differences. Chief among these is the absence of intramolecular interaction to mask intermolecular interaction domains in isoform 2. The full range of merlin's intermolecular interactions remains to be delineated, but it can be expected from the comparison to ERMs that merlin also sits within a web of interactions that may involve multiple partners and signaling pathways, some of which it shares with the ERMs. Defining merlin's tumor suppressor function will likely require identifying those differences that are peculiarly important in the target cell types of NF2. However, the fact that inactivation of merlin in the mouse by targeted mutagenesis produces a variety of malignant tumors with a high rate of metastasis [33] suggests that merlin's suppression of tumor formation may involve different partners and pathways in different cell types and genetic backgrounds. Consequently, the disruptions due to merlin inactivation in the progression of malignant mesothelioma may represent a tumor suppressor role operating by a different pathway than that in schwannoma or meningioma.  (+info)

NF2 gene mutations and allelic status of 1p, 14q and 22q in sporadic meningiomas. (3/103)

Formation of meningiomas and their progression to malignancy may be a multi-step process, implying accumulation of genetic mutations at specific loci. To determine the relationship between early NF2 gene inactivation and the molecular mechanisms that may contribute to meningioma tumor progression, we have performed deletion mapping analysis at chromosomes 1, 14 and 22 in a series of 81 sporadic meningiomas (54 grade I (typical), 25 grade II (atypical) and two grade III (anaplastic)), which were also studied for NF2 gene mutations. Single-strand conformational polymorphism analysis was used to identify 11 mutations in five of the eight exons of the NF2 gene studied. All 11 tumors displayed loss of heterozygosity (LOH) for chromosome 22 markers; this anomaly was also detected in 33 additional tumors. Twenty-nine and 23 cases were characterized by LOH at 1p and 14q, respectively, mostly corresponding to aggressive tumors that also generally displayed LOH 22. All three alterations were detected in association in seven grade II and two grade III meningiomas, corroborating the hypothesis that the formation of aggressive meningiomas follows a multi-step tumor progression model.  (+info)

Novel alternatively spliced isoforms of the neurofibromatosis type 2 tumor suppressor are targeted to the nucleus and cytoplasmic granules. (4/103)

We cloned novel splice variants Mer150, Mer151 and Mer162 of the neurofibromatosis 2 (NF2) tumor suppressor, which demonstrate a tissue-specific and development-specific expression pattern. Isoform Mer150 is created by cryptic splicing from exon 8 to 14 and represents an N-terminal truncation of 259 residues. Mer151 is characterized by in-frame splicing out of several exons and a modified C-terminus due to a frameshift in exons 13+14 and premature termination. Mer162 represents a head-to-tail isoform resulting from in-frame skipping of exons 5-16. As a common feature, the alpha-helical domain and a variable proportion of the ERM homology domain are spliced out in these isoforms. To investigate differences in subcellular localization, we expressed epitope-tagged cDNA constructs of the wild-type NF2 as well as of the three alternatively spliced transcripts in NIH 3T3 cells by nuclear microinjection or lipid-mediated transfection. Subcellular localization of Mer151 in filopodia and ruffling membranes was similar to the wild-type NF2. Mer151, however, was targeted to the nucleus, which was not observed for wild-type NF2, Mer150 or Mer162. A putative nuclear localization signal created by alternative splicing was identified in Mer151. In contrast to Mer151, Mer150 and Mer162 were not found in regions of the plasma membrane, but localized to a granular intracellular compartment. The results suggest that the recently described actin-binding domain in exon 10, but not the presence or absence of exons 2+3, is relevant for subcellular targeting. Although the NF2 protein is known as a cytoskeletal linker, additional functions in a cytoplasmic compartment and in the nucleus may exist.  (+info)

Molecular genetic analysis of ependymal tumors. NF2 mutations and chromosome 22q loss occur preferentially in intramedullary spinal ependymomas. (5/103)

Ependymal tumors are heterogeneous with regard to morphology, localization, age at first clinical manifestation, and prognosis. Several molecular alterations have been reported in these tumors, including allelic losses on chromosomes 10, 17, and 22 and mutations in the NF2 gene. However, in contrast to astrocytic gliomas, no consistent molecular alterations have been associated with distinct types of ependymal tumors. To evaluate whether morphological subsets of ependymomas are characterized by specific genetic lesions, we analyzed a series of 62 ependymal tumors, including myxopapillary ependymomas, subependymomas, ependymomas, and anaplastic ependymomas, for allelic losses on chromosome arms 10q and 22q and mutations in the PTEN and NF2 genes. Allelic losses on 10q and 22q were detected in 5 of 56 and 12 of 54 tumors, respectively. Six ependymomas carried somatic NF2 mutations, whereas no mutations were detected in the PTEN gene. All six of the NF2 mutations occurred in ependymomas of WHO grade II and were exclusively observed in tumors with a spinal localization (P = 0.0063). These findings suggest that a considerable fraction of spinal ependymomas are associated with molecular events involving chromosome 22 and that mutations in the NF2 gene may be of primary importance for their genesis. Furthermore, our data suggest that the more favorable clinical course of spinal ependymomas may relate to a distinct pattern of genetic alterations different from that of intracerebral ependymomas.  (+info)

Tight association of loss of merlin expression with loss of heterozygosity at chromosome 22q in sporadic meningiomas. (6/103)

Mutations of NF2, the gene for neurofibromatosis 2, are detected in 20-30% of sporadic meningiomas, and almost all mutations lead to loss of merlin expression. However, loss of heterozygosity (LOH) at chromosome 22q is found at a much higher frequency, up to 50-70%, and the possibility of another tumor suppressor gene in this region has not been excluded. Furthermore, a recent report proposed that abnormal activation of a protease micro-calpain can be an alternative pathway for merlin loss in meningiomas and schwannomas. To determine the correlation of merlin loss with NF2 genetic alteration or micro-calpain activation, we performed a molecular genetic analysis of 50 sporadic meningiomas and also examined the expression status of merlin and active form micro-calpain. LOH assay of five microsatellite markers franking NF2 revealed LOH in 22 cases, and single-strand conformation polymorphism assay detected six frameshift mutations, two splicing mutations, one nonsense mutation, and one missense mutation, all accompanied by 22q LOH. In addition, a multiplex PCR assay indicated homozygous deletion of NF2 in two cases. Interestingly, a marked decrease of merlin expression was seen exclusively in the 22 cases with 22q LOH. Activated micro-calpain expression was observed in 28 cases at various levels but showed no correlation with merlin status. These data strongly support the notion that NF2 is the sole target of 22q LOH in meningiomas and that loss of merlin expression is always caused by genetic alteration of NF2, following the classic "two hit" theory.  (+info)

The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation. (7/103)

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. To understand the basic cellular functions of Merlin, we are investigating a Drosophila Neurofibromatosis-2 homologue, Merlin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in another Protein 4.1 superfamily member in Drosophila, expanded. Because of the phenotypic and structural similarities between Merlin and expanded, we asked whether Merlin and Expanded function together to regulate cell proliferation. In this study, we demonstrate that recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other. Consistent with this genetic interaction, we determined that Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones revealed that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members.  (+info)

Functional analysis of the neurofibromatosis type 2 protein by means of disease-causing point mutations. (8/103)

Despite intense study of the neurofibromatosis type 2 (NF2) tumor-suppressor protein merlin, the biological properties and tumor-suppressor functions of merlin are still largely unknown. In this study, we examined the molecular activities of NF2-causing mutant merlin proteins in transfected mammalian cells, to elucidate the merlin properties that are critical for tumor-suppressor function. Most important, we found that 80% of the merlin mutants studied significantly altered cell adhesion, causing cells to detach from the substratum. This finding implies a function for merlin in regulating cell-matrix attachment, and changes in cell adhesion caused by mutant protein expression may be an initial step in the pathogenesis of NF2. In addition, five different mutations in merlin caused a significant increase in detergent solubility of merlin compared to wild type, indicating a decreased ability to interact with the cytoskeleton. Although not correlated to the cell-adhesion phenotype, four missense mutations decreased the binding of merlin to the ERM-interacting protein EBP-50, implicating this interaction in merlin inhibition of cell growth. Last, we found that some NF2 point mutations in merlin most closely resembled gain-of-function alleles in their cellular phenotype, which suggests that mutant NF2 alleles may not always act in a loss-of-function manner, as had been assumed, but may include a spectrum of allelic types with different phenotypic effects on the function of the protein. In aggregate, these cellular phenotypes provide a useful assay for identifying the functional domains and molecular partners necessary for merlin tumor-suppressor activity.  (+info)