Putative domain structure, protein sequence alignment, and expression pattern of NGB. (A) Schematic representation of domain structure of NGB protein, which is composed of a GTP binding domain at the N terminus and a coiled-coil region at the C terminus. (B) Alignment of amino acid sequence of NGB from human, mouse, Drosophila, C. elegans, and yeast cells. Conserved residues are indicated by asterisks. (C) Sequence comparison of GTP-binding domain between NGB, R-Ras, RAP2, and rasl. Conserved amino acids are boxed. (D) Northern blot analysis. A human multiple-tissue mRNA blot was hybridized with [³²P]dCTP-labeled NGB, NF2, and β-actin cDNA probes. M., muscle.

Interaction between NF2 and NGB. (A and B) NGB binds to NF2/merlin in HEK293 cells cotransfected with HA-NF2 and Flag-NGB. After 36 h of transfection, cells were lysed, immunoprecipitated with anti-Flag (α-Flag), and detected with anti-HA (α-HA) antibody (A) or vice versa (B). Bottom panels show expression of transfected plasmids. (C and D) Specificity of anti-NGB antibody. HeLa cells were transiently transfected with Flag-NGB, lysed, and then subjected to immunoblotting (C) or IP-Western blotting analysis (D) with indicated antibodies. (E and F) NGB interacts with NF2 at physiological protein levels. 82HTB rhabdomyosarcoma cells were lysed and immunoprecipitated with anti-NF2 (α-NF2) antibody or the antibody was preincubated with GST-NF2 antigen or preimmune serum. The immunoprecipitates were detected with anti-NGB (α-NGB) antibody (E). Conversely, the NGB immunoprecipitates were blotted with anti-NF2 antibody (F). (G) GST-NGB pull down of merlin. A GST pull-down assay was performed by incubation of GST and GST-NGB with HeLa cell lysate. After being washed four times, pull-down products were immunoblotted with anti-NF2 antibody (top). The bottom panel is an SDS-PAGE gel stained with Coomassie blue (CBS). (H) NGB colocalizes with NF2 in the perinuclear region. 82HTB rhabdomyosarcoma cells were cultured on glass coverslips, probed with anti-NGB and -NF2 antibodies, incubated with fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies, and analyzed by confocal microscopy. (I and J) Yeast two-hybrid mapping of interaction domains between NF2 and NGB. EGY191 yeast was cotransformed with plasmids encoding a fusion protein between the GAL4 activation domain and full-length or NF2 deletion constructs and plasmids encoding a fusion protein between LexA-DNA binding domain and full-length or deletion NGB constructs. All yeast clones grew on SD-Ura/Trp/His medium containing Gal. The positive interaction was indicated by blue. (K) Identification of the binding site(s) of NGB. HEK293 cells were transfected with the indicated plasmids and lysed, and then the protein was immunoprecipitated with anti-GFP (α-GFP) antibody and detected with anti-HA (α-HA) antibody (top). The middle and bottom panels show expression of transfected plasmids.

NGB inhibits JS1 cell proliferation and tumor growth in xenograft mouse model. (A) Immunoblotting (IB) analysis of NGB and NF2 stably transfected JS1 schwannoma clonal cell lines with anti-NGB (α-NGB, left panel), anti-HA (α-HA, middle panel) and indicated antibodies (right panel). (B) NGB inhibits cell growth. Stable NGB- and NF2-transfected cells as well as pcDNA3 vector-transfected cells were seeded in a 48-well plate at 0.2 × 10⁵/well. The cell number was counted daily for 3 days. The experiment was performed three separate times. (C) NGB inhibits DNA synthesis. The stably transfected cells were labeled with [³H]thymidine (3 plates/clone cell/time point) and assayed as described in Materials and Methods. Tumor growth (D) and tumor weight (E) were reduced by NGB. The stably transfected clonal cells were subcutaneously injected into nude mice (3 × 10⁶ cells/mouse). Tumor volume was measured every 2 days, and tumor weight was recorded at time of euthanasia. Data are representative of two independent experiments carried out with 24 mice each (8 mice/clonal cell line). The number above each column represents the proliferative index revealed by Ki-67 immunostaining. Mean values ± standard errors of the proliferative index are given as the counts of Ki-67-positive tumor cell nuclei in 10 consecutive high-power (magnification, ×400) fields.

NGB is down-regulated in glioma cells, and reconstitution of NGB induces cell growth arrest but not cell death. (A-C) Immunoblotting analysis of doxycycline (Doxy)-inducible NGB HeLa cells (A), tumor cell lines (B), and stably transfected NGB H4 clonal cell lines (C) with indicated antibodies. Right panels of panel B show mRNA levels of NGB in glioma cell lines examined by reverse transcription-PCR. (D and E) NGB inhibits cell growth. HeLa Tet-on/NGB and H4-NGB cells were seeded in a 48-well plate in triplicate and treated with (+) or without (−) doxycycline (D) as indicated on the figure. The cell number was counted daily for 3 days. The experiment was performed three separate times. (F) NGB does not sensitize cells to chemotherapeutic drug-induced apoptosis. The cells were seeded and treated as described for panels D and E and then administered VP16 (5 μM), taxol (100 nM), or doxorubicin (Doxo., 2 μM). After 12 h of incubation, cells were assayed for caspase-3 activity using the EnzChek Caspase-3 assay kit.

Ectopic expression of NGB reduces cell mobility, attachment, and aggregation. (A) Reduced cell mobility was observed in cells overexpressing NGB. An MICS assay was used to directly measure cell migration. The migration is normalized to values obtained with pcDNA3 vector controls and is expressed in μM/h. Standard deviations for each cell line are shown. (B) NGB inhibits cell adhesion. NGB-59 and pcDNA3-rat schwannoma cells were incubated in the presence or absence of 100 μM zinc chloride overnight and then harvested by trypsinization. Equal numbers of cells were added to laminin-, fibronectin-, collagen IV-, and collagen I-coated 96-well plates. After 1 h, the number of adherent cells was determined using crystal violet staining and a spectrophotometric assay. NF2-transfected cells (NF2-19) were used as controls. The experiment was repeated three times. OD450, optical density at 450 nm. (C) JS1 cell aggregation was inhibited by NGB. Single-cell suspensions (500 μl; 5 × 10⁴ cells/ml in DMEM containing 0.5% BSA) were plated into each well of a 24-well low-binding plate. The plate was incubated at 4°C or 37°C on a rotating platform for 30 min. Photographs were taken using a Nikon N6006 camera (magnification, ×100).

Mutation of NGB in a human glioma and loss of tumor suppressor activity of NGB-P561R and NGB-K395/R394A. (A) Single-strand conformation polymorphism analysis of exon 16 of the NGB gene. An abnormal band shift (arrow) was detected in tumor DNA of patient 2 (T2) but not matched DNA (N2). (B) Sequence analyses revealed that a mutation from guanine to cytosine occurs in tumor DNA (arrow between middle and bottom panels), resulting in a change of amino acid from proline to serine. (C) Immunoblotting analysis of H4 cells, which were transfected with wild-type NGB, a naturally occurring mutant NGB-P561R, or the binding site mutant NGB-K395/R394A, with indicated antibodies. (D and E) NGB-P561R and NGB-K395/R394A failed to inhibit cell growth (D) and DNA synthesis (E). H4-NGB-2 is a wild-type NGB stably transfected clonal cell line (see Fig. 4C).

NGB possesses GTP-binding and GTPase activities that are not regulated by merlin. (A) NGB specifically binds to GTP. NGB (1 μg) or BSA (1 μg) was incubated with [³⁵S]GTPγS (1 μM) at 22°C for the indicated times (left panels) or for 1 h in the absence or presence of the indicated nucleotides at concentration of 20 μM (right panels). The amount of [³⁵S]GTPγS binding was determined in a filter binding assay as described in Materials and Methods. Each value is the average of the results from duplicate incubations and is representative of three separate experiments. (B and C) [α-³²P]GTP hydrolysis by NGB and merlin does not affect NGB GTPase activity. BSA (control) and NGB were incubated with [α-³²P]GTP (10 μM; 2 Ci/mmol). Aliquots were removed from the reaction mixture at the indicated times, analyzed by poly(ethyleneimine) thin-layer chromatography plates, and exposed to film for 5 min. (D) Merlin does not regulate the GTP-binding activity of NGB. A GTP-binding assay was performed as described above except that incubation of NGB was in the presence or absence of merlin.

NGB up-regulates merlin through inhibition of merlin ubiquitination. (A) The protein level of merlin is increased by ectopic expression of NGB. HEK 293 cells were transfected with increasing amounts of Flag-NGB. After 48 h of incubation, cells were lysed and immunoblotted with indicated antibodies. (B) NGB does not regulate merlin at the transcriptional level. Total RNA was isolated from NGB-transfected HEK293 cells and subjected to Northern blot analysis with radiolabeled NF2 cDNA as a probe (top). The bottom panel depicts the ethidium bromide-stained gel showing equal loading of total RNA. (C) NGB inhibits degradation of merlin. Pulse-chase assays were carried out in NIH 3T3 cells transfected with NGB or pcDNA3 vector alone. After 60 min of [³⁵S]methionine labeling, merlin was immunoprecipitated, separated by SDS-PAGE, exposed (top and medium) and quantified with a PhosphorImager (bottom). (D) Knockdown of NGB decreases merlin expression and results in cell growth. NGB-59 JS1 cells were transfected with NGB-RNAi or control RNAi. After 48 h of incubation, cells were lysed and immunoblotted with indicated antibodies (upper). The bottom panel shows the cell growth curve. (E) Knockdown of NGB increases merlin ubiquitination and MG132 inhibits merlin degradation resulting from NGB knockdown. HeLa cells were transfected with Myc-ubiquitin and NGB-RNAi and then treated with MG132 (10 μM) for 6 h. Cells were lysed, immunoprecipitated with anti-merlin antibody, and detected with anti-ubiquitin antibody (top). Panels 2 to 4 show expression of NGB, merlin, and actin. (F and G) Reconstitution of NF2 in Nf2-deficient MEFs does not affect NGB expression, and knockdown of NGB decreases the expression of reintroduced merlin. Results of Western blot analysis of Nf2^−/− MEFs, which were transfected with NF2 (F) or NF2/NGB-RNAi (G), with indicated antibodies are shown. (H) Knockdown of NGB largely abrogated merlin-inhibited DNA synthesis. Nf2-null MEFs were transfected with indicated plasmids and/or NGB-RNAi. After 36 h culture, [³H]thymidine incorporation was assayed as described in Materials and Methods.

NGB reduces cyclin D1 expression and inhibits cell growth via merlin. (A) Protein and mRNA levels of cyclin D1 are inhibited by NGB. JS1 cells were transfected with increasing amounts of Flag-NGB and immunoblotted with indicated antibodies (panels 1 to 3). Total RNA was isolated from the cells and subjected to semiquantitative reverse transcription-PCR using primers specific for cyclin D1 and β-actin (panels 4 and 5). (B) Knockdown of NGB increases cyclin D1 level. HeLa cells were transfected with NGB-RNAi using Lipofectamine. Following 72 h of incubation, cells were lysed and immunoblotted with anti-NGB (top), -cyclin D1 (middle), and -actin (bottom) antibodies. (C) NGB-reduced cyclin D1 is abrogated by knockdown of merlin. JS1/NGB-59 cells were infected with lentivirus pLKO.1-shRNA/Nf2 and pLKO.1-puro vector and immunoblotted with indicated antibodies. (D and E) Merlin mediates NGB-inhibited cell growth and tumorigenicity. Indicated cells were seeded in 48-well plates at 0.2 × 10⁵/well. The cell number was counted daily for 3 days. The experiment was performed in triplicate (D). The cells were subcutaneously injected into nude mice (3 × 10⁶ cells/mouse). Tumor volume was measured every 2 days. Data shown are representative of results from two independent experiments carried out with 16 mice each (8 mice/cell line) (E).

Overexpression of cyclin D1 largely overrides the tumor suppressor functions of NGB and merlin. (A) Immunoblotting analysis of JS1/NGB-59, JS1/NF2-19, and JS1/pcDNA cells, which were infected with adeno-cyclin D1 or adeno-vector viruses, with anti-cyclin D1 (upper) and -β-actin (bottom) antibodies. (B and C) Ectopic expression of cyclin D1 significantly reduced the inhibitory effects of NGB and merlin on cell proliferation and DNA synthesis. Cells were seeded in 24-well plates (0.8 × 10⁵/well) in triplicate. Following 24 h of incubation, cell proliferation (B) and DNA synthesis (C) were examined by MTS and [³H]thymidine incorporation assays. (D) Cyclin D1 overrode NGB- and merlin-inhibited cell anchorage-independent growth. Colony formation assays were performed in 100-mm soft agar plates. The number of colonies was counted after 21 days.