Activation of the tumor suppressor merlin modulates its interaction with lipid rafts. (65/243)

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by bilateral schwannomas of the eighth cranial nerve. The NF2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane/F-actin linkers. Merlin resists solubilization by the detergent Triton X-100 (TX-100), a property commonly attributed to association with the cytoskeleton. Accordingly, NF2 patient mutations that encode merlins with enhanced TX-100 solubility have been explained previously in terms of loss of cytoskeletal attachment. However, here we present data to suggest that the detergent resistance of merlin is a result of its constitutive residence in lipid rafts. Furthermore, when cells are grown to high density, merlin shifts to a more buoyant lipid raft fraction in a density gradient. This shift is mimicked in subconfluent cells treated with cytochalasin D, suggesting that the shift results from merlin dissociation from the actin cytoskeleton, but not from lipid rafts. Intramolecular NH(2)- and COOH-terminal binding, which occurs when merlin transitions to the growth-suppressive form, also brings about a similar change in buoyant density. Our results suggest that constitutive residence of merlin in lipid rafts is crucial for its function and that as merlin becomes growth suppressive in vivo, one significant molecular event may be the loss of interaction with the actin cytoskeleton. To our knowledge, merlin is the first tumor suppressor known to reside within lipid rafts, and the significance of this finding is underscored by known loss-of-function NF2 patient mutations that encode merlins with enhanced TX-100 solubility.  (+info)

Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity. (66/243)

The neurofibromatosis type 2 gene-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeleton-associated proteins. Recent studies suggest that the loss of neurofibromatosis type 2 function contributes to tumor development and metastasis. Although the cellular functions of merlin as a tumor suppressor are relatively well characterized, the cellular mechanism whereby merlin controls cell proliferation from membrane locations is still poorly understood. During our efforts to find potential merlin modulators through protein-protein interactions, we identified transactivation-responsive RNA-binding protein (TRBP) as a merlin-binding protein in a yeast two-hybrid screen. The interaction between TRBP and merlin was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization experiments. The carboxyl-terminal regions of each protein were responsible for their interaction. Cells overexpressing TRBP showed enhanced cell growth in cell proliferation assays and also exhibited transformed phenotypes, such as anchorage-independent cell growth and tumor development in mouse xenografts. Merlin efficiently inhibited these oncogenic activities of TRBP in our experiments. These results provide the first clue to the functional interaction between TRBP and merlin and suggest a novel mechanism for the tumor suppressor function of merlin both in vitro and in vivo.  (+info)

Many ribosomal protein genes are cancer genes in zebrafish. (67/243)

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.  (+info)

A NHERF binding site links the betaPDGFR to the cytoskeleton and regulates cell spreading and migration. (68/243)

The Na(+)/H(+) exchanger regulatory factor, NHERF, is a multifunctional adapter protein involved in a wide range of physiological activities. NHERF associates with merlin and the ezrin/radixin/moesin (MERM) family of membrane-actin cytoskeletal linker proteins through its C-terminus and is capable of interacting via its PDZ1 domain to the betaPDGF receptor (betaPDGFR). Thus, NHERF, potentially links the betaPDGFR to the actin cytoskeleton through its interaction with MERM proteins. In the present study, we have examined whether abolishing the interaction of betaPDGFR with NHERF results in actin cytoskeletal rearrangements. We have stably expressed a wild-type betaPDGFR, a mutant betaPDGFR (L1106A) that is incapable of interacting with NHERF, as well as a kinase defective mutant receptor (K634R), in PDGFR-deficient mouse embryonic fibroblasts. Our observations indicate that cells expressing betaPDGFR (L1106A) were impaired in their ability to spread and migrate on fibronectin compared with wild-type and K634R cells. L1106A mutant cells also revealed an increased number of focal adhesions, a condensed F-actin ring at the cell periphery and a decrease in total focal adhesion kinase (FAK) tyrosine phosphorylation. Further, we show that NHERF and MERM proteins could act as intermediary bridging proteins between betaPDGFR and FAK. Thus, the interaction of betaPDGFR with NHERF may provide an essential link between the cell membrane and the cortical actin cytoskeleton independent of receptor activity.  (+info)

Targeted gene expression in the chicken eye by in ovo electroporation. (69/243)

PURPOSE: The chicken embryo lens is a classical model system for developmental and cell biology studies. To understand the molecular mechanisms that underlie the morphological changes that occur during lens development, it is important to develop an effective gene transfer method that permits the analysis of gene functions in vivo. In ovo electroporation has been successfully used for introducing DNA into neural and mesenchymal tissues of chicken embryos. In this study, we explored the possibility of using this technique to manipulate gene expression in lens epithelial and fiber cells, as well as in other cells of the chicken eye. METHODS: Two DNA constructs were used in this study. pCAX contains a chicken beta-actin promoter fused to the CMV IE enhancer to drive enhanced green fluorescent protein (EGFP) expression. pMES-cNf2 uses the same chimeric promoter to drive the expression of the chicken neurofibromatosis 2 (cNf2) and EGFP proteins in the same cell. Plasmid DNA was injected into the lumen of the lens vesicle in chicken embryos at stage 15. For corneal epithelial and retinal cell electroporation, DNA was placed near the surface ectoderm in the eye region or injected into the vitreous cavity, respectively. Electroporation was performed with one electrode above the eye and the other underneath the head of the embryo. Chicken embryos were harvested at different time points for EGFP expression analysis by immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) incorporation assays were used to evaluate the effects of cNf2 on lens epithelial cell proliferation. RESULTS: A strong EGFP signal can be detected in lens cells 4 h after electroporation. The transfected cells maintain high levels of EGFP expression for at least 5 days. Overexpressing cNf2 in lens epithelial cells significantly inhibits cell proliferation. Ectopic expression of EGFP in corneal epithelial and retinal cells was also achieved by in ovo electroporation. CONCLUSIONS: We have demonstrated that exogenous DNA can be effectively introduced into lens, corneal and retinal cells in the living embryo by in ovo electroporation. In comparison to viral infection and transgenic mouse approaches, in ovo electroporation offers an easier and quicker way to manipulate gene expression during embryonic development. This technique will be a useful tool for exploring the molecular mechanisms of lens and eye development.  (+info)

Neurofibromatosis 2 (NF2) tumor suppressor merlin inhibits phosphatidylinositol 3-kinase through binding to PIKE-L. (70/243)

Neurofibromatosis 2 (NF2) is a tumor suppressor, although the molecular mechanism accounting for this effect remains unknown. Here, we show that merlin exerts its activity by inhibiting phosphatidylinositol 3-kinase (PI3-kinase), through binding to PIKE-L. Wild-type merlin, but not patient-derived mutant (L64P), binds PIKE-L and inhibits PI3-kinase activity. This suppression of PI3-kinase activity results from merlin disrupting the binding of PIKE-L to PI3-kinase. In addition, merlin suppression of PI3-kinase activity as well as schwannoma cell growth is abrogated by a single PIKE-L point mutation (P187L) that cannot bind merlin but can still activate PI3-kinase. Knocking down PIKE-L with RNA interference abolishes merlin's tumor-suppressive activity. Our data support the hypothesis that PIKE-L is an important mediator of merlin growth suppression.  (+info)

Erbin regulates mitogen-activated protein (MAP) kinase activation and MAP kinase-dependent interactions between Merlin and adherens junction protein complexes in Schwann cells. (71/243)

Biallelic mutations in the neurofibromatosis 2 (NF2) gene are linked to schwannoma and meningioma tumorigenesis. Cells with NF2 mutations exhibit elevated levels of phosphorylated extracellular signal-regulated kinase (ERK) and aberrant cell-cell and cell-matrix contacts. The NF2 gene product, merlin, associates with adherens junction protein complexes, suggesting that part of its function as a tumor suppressor involves regulating cell junctions. Here, we find that a novel PDZ protein, called erbin, binds directly to the merlin-binding partner, EBP0, and regulates adherens junction dissociation through a MAP kinase-dependent mechanism. Reducing erbin expression using a targeted siRNA in primary cultures of Schwann cells results in altered cell-cell interactions, disruption of E-cadherin adherens junctions, increased cell proliferation, and elevated levels of phosphorylated ERK, all phenotypes observed in cells that lack merlin. Reduction of erbin expression also results in the dissociation of merlin from adherens junction proteins and an increase in the levels of phosphorylated merlin. These phenotypes can be rescued if cells with reduced levels of erbin are treated with a pharmacological inhibitor of ERK kinase. Collectively, these data indicate that erbin regulates MAP kinase activation in Schwann cells and suggest that erbin links merlin to both adherens junction protein complexes and the MAP kinase signaling pathway.  (+info)

Sensitive detection of deletions of one or more exons in the neurofibromatosis type 2 (NF2) gene by multiplexed gene dosage polymerase chain reaction. (72/243)

Mutation detection in the neurofibromatosis type 2 (NF2) gene is challenging because when combining mutation detection methods such as single-strand conformational polymorphism and heteroduplex analysis, denaturing gradient gel electrophoresis, and direct sequencing of aberrant polymerase chain reaction (PCR) fragments only 30 to 60% of the constitutional mutations are detected. Because large deletions and complete chromosome rearrangements are also described methods such as microarray-comparative genomic hybridization and fluorescence in situ hybridization are also used. The one type of mutation often missed corresponds to deletions encompassing one or few exons. To detect this type we have developed a swift and reliable method. We perform a gene dosage analysis with two fluorescent multiplex PCR assays that amplify 15 of the 17 NF2 exons. The labeled PCR products are quantified and gene dose is calculated with respect to controls. We tested the reliability of this method with DNA from eight NF2 patients with known heterozygous NF2 deletions, eight controls and four unknown NF2 patients. In all of the patients with known heterozygous deletions we found in several exons a reduction of gene dosage to 50 to 69%. In one NF2 patient with previously unknown mutation and a severe phenotype we found the gene dosage of two exons reduced by 50% indicating a deletion of these two exons on one allele. This finding was validated by reverse transcriptase-PCR on fibroblast and schwannoma cell cultures of this patient and cDNA sequencing. Our gene dosage assay will detect deletions of one or more exons as well as gross deletions of the whole coding region of the gene. It can complement the existing screening methods because it is faster and easier.  (+info)