To characterize the function of a novel Src homology 3 (SH3) adapter proteintermed NESH, we have established transfectants stably expressing NESH.We observed that every clone of NESH transfectants caused a marked reduction in motility, although the clones exhibited no significant differences in intrinsic cell growth compared with the control cells in vitro. The NESH transfectants also exhibited significant reduction in tumor metastatic potential in vivo. We found that NESH expression is frequently lost in invasive cancer cell lines despite its ubiquitous expression in normal tissues. The SH3 domain of NESH seems to interact with p21-activated kinase (PAK), which is involved in regulation of cell motility. Furthermore, a significant decrease in PAK phosphorylation at 402Thr was found in NESH transfectants. Taken together, these results suggest that NESH inhibits ectopic metastasis of tumor cells as well as cell migration through interaction with intracellular molecules such as PAK that are essential for cell motility.

One of the characteristics of tumor cells is their ability to metastasize. Tumor metastasis, which is the movement of malignant cells from a primary tumor to form colonies at other organs, remains a leading cause of death for cancer patients. Metastatic progression seems a complex process involving hormonal and genetic factors. Some cytokines such as vascular endothelial growth factor, tumor necrosis factor, and IFN can regulate cell motility and metastasis. Alterations of several genes have been identified to regulate metastasis (1, 2), some of which have also been proposed as molecular markers to help in making prognoses. Despite the above identification of factors and genes, the molecular mechanisms of the metastasis of cancer cells remain to be elucidated, and the genetic events that induce or suppress the tumor metastatic process are incompletely understood. Accordingly, identification of regulatory molecules in the process of metastasis is one of the major goals of cancer research.

NESH is an adapter protein that possesses SH34 -domain and proline-rich regions and that was initially identified by the reverse transcription-PCR procedure using oligonucleotide primers derived from conserved regions of SH3 (3). From amino-acid sequence and structural similarity, NESH is thought to be a member of E3B1/ArgBP/Avi2/NESH family. It has recently been shown that the members of this family are involved in membrane ruffling and lamellipodia formation (4, 5), which suggests an involvement in the mechanism of cell motility; therefore, we tried to elucidate the NESH function on cell motility. In this report, we will show that the NESH is involved in the process of cell migration and metastasis.

Cell and Cell Culture.

COS7 cells, Huvec cells (6), U87 MG cells (7), v-Src transformed NIH3T3 (SRD) cells (8) and the derived cells were cultured in DMEM with 10% fetal bovine serum and antibiotics.

Expression Vector, Transfection, and Antibodies.

Full-length wild-type human NESH cDNA was inserted into the EcoRI and XhoI sites of pcDNA3 or pHis-cDNA3 (Invitrogen). For introduction of NESH, COS7 cells were transiently transfected, and SRD cells and U87 MG cells were stably transfected with the NESH cDNAs. Polyclonal anti-NESH antibodies were prepared in rabbits immunized with the NH2-terminal region of human NESH (region N: amino acids 1–145) or the COOH-terminal region of human NESH (region C: amino acids 201–365) expressed as GST fusion proteins in Escherichia coli. The serum was applied to an antigen-immobilized column, and the bound antibodies were eluted with glycine buffer (pH 2.5). Polyclonal anti-PAK1, anti-PAK2, phospho-specific anti-PAK2, and anti-Sos1 antibodies were purchased from Santa Cruz Biotechnology. Monoclonal anti-His antibody was from Qiagen. Secondary antibodies linked to horseradish peroxidase (used for Western blotting) were from BIO SOURCE and Amersham Pharmacia. Secondary antibodies linked to fluorescein and Rhodamine (used for immunofluorescence microscopy) were from BIO SOUCE.

GST Pull-down Assay.

Cells were washed with ice cold PBS and then lysed on ice in 0.7 ml of TNE buffer [10 mm Tris (pH 7.6), 150 mm NaCl, 1 mm EDTA, 1% NP-40) containing 1 mm PMSF and aprotinin (10 μg/ml)]. The lysates were centrifuged at 1000 g for 15 min at 4°C, and the resulting supernatants were subjected to GST-pull down analysis. GST-fusion proteins (20 μg) containing NESH-SH3 domain (residues 296–366) were immobilized on glutathione beads and then mixed with various protein samples such as cell lysates. After incubation for 2 h with rotation, the beads were washed with TNE buffer and subjected to immunoblot analysis with indicated antibodies.

Immunofluorescence.

Cells seeded on glass coverslips were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min and incubated for 30 min in TBST [10 mm Tris-HCl (pH7.4), 0.9% NaCl, 0.05% Tween 20] containing 7% FBS. The cells were then incubated with primary antibodies such as anti-PAK2 or anti-NESH for 1 h at room temperature. After washing, they were incubated with secondary antibody linked to fluorescein. To visualize actin filaments, rhodamine-conjugated phalloidin was also added during the incubation with secondary antibodies. After 30 min incubation, coverslips were washed and mounted on slide glasses. They were observed with a confocal laser scanning microscope (Bio-Rad; model MRC 1024).

Cell Motility Assay.

Cells were assayed for their motility by a computer-assisted modification of the phagokinetic assays with gold colloid-coated glass plates described previously (9). Briefly, cells (2 × 103 cells/3.5-cm plate) were seeded on colloidal gold particle-coated glass coverslips and incubated for 24 h. After fixation with 4% paraformaldehyde, the coverslips were mounted onto glass microscope slides, and photographs were taken by a computer-assisted digital camera (model HS-300; Olympus, Tokyo, Japan) connected to a microscope. The areas of swept particles in which cells moved around during incubation were measured by NIH image (version 1.62) and statistically analyzed by Stat View (version 4.51). For the assay, 12 fields per sample were randomly selected and five to six cells per field were examined.

Cell Invasion Assay.

Invasive ability was measured as described previously (10) with some modifications. Briefly, polycarbonate filters, 8 μm pore size (Costar), were coated with an extract of basement membrane component (Matrigel, 5 μg/filter; Collaborative Research Co.), dried, and reconstituted with DMEM. The coated filters were placed in blind-well Boyden chambers. The cells to be tested (2 × 105 cells/chamber) were plated in the upper compartment of the chamber. SRD- or U87 MG-conditioned media were used as chemoattractants in the lower compartment of the chamber. After incubation for 4 h (SRD) or 6 h (U87 MG) at 37°C, the noninvasive cells were removed with a cotton swab. The cells that had migrated through the membrane and stuck to the lower surface of the membrane were fixed with methanol and stained with hematoxylin. For quantification, cells were counted under a microscope in five predetermined fields at ×400.

Zymography.

Cells (1 × 106 cells/6-cm plate) were incubated in serum-free DMEM at 37°C for 18 h, and the conditioned medium was collected. After clarification by centrifugation (10 min at 1000 rpm), the medium was diluted in 4× nonreducing sample buffer [0.27 m Tris (pH 6.8), 8% SDS, 40% glycerol, and 0.04 mg/ml bromphenol blue] and electrophoresed in 10% SDS polyacrylamide gels containing 0.1% (w/v) gelatin for MMP detection. Gels were washed repeatedly with 2.5% Triton X-100 for 30 min at room temperature and then were incubated with substrate buffer [50 mm Tris (pH 7.4), 0.02% NaN3, 10 mm CaCl2 (MMP detection)] for 16 h at 37°C. The gels were then stained with Coomassie Brilliant Blue and destained until white zones on a dark background appeared.

Cell Proliferation Assay.

Cell proliferation was measured by cellular uptake of MTT (Sigma Chemical Co.). For the cell proliferation assay, transfectant cells and control cells (2.5 × 103 cells/well) were prepared in 96-well plates in a serum-containing medium and were cultured for 6 days. MTT (0.5 mg/ml) was added to each well. After incubation for 4 h at 37°C, the supernatants were aspirated, and 100 μl of 0.04 n HCl in isopropanol were added. The color reaction was quantitated using an automatic plate reader, EAR 340 AT (SLT), at 590 nm with a reference filter of 620 nm as described previously (11).

Metastasis Assays.

Male BALB/c nude mice were obtained at 5 weeks of age. The indicated cells were collected by trypsinization, and were washed with HBSS. To produce experimental lung metastasis, 0.5 × 106 cells were injected into the fat pad or the lateral tail veins of the male athymic nude mice. After about 3 weeks, the mice were killed and analyzed. Lungs were removed, rinsed in ice-cold PBS, and fixed in Bouin’s solution to quantitate surface lesions.

The other basic methods such as SDS-PAGE or Western blot have been previously described elsewhere (3, 4, 5).

The schematic structure of NESH is shown in Fig. 1,A. A construct termed His-NESH was tagged with oligo-histidine at the NH2 terminus of NESH. We first checked the size of the protein expressed in COS7 cells by Western blot analysis with NESH-specific antibody. The recognized two proteins of Mr 50,000 and 52,000 in COS7 cells transfected with expression constructs of NESH cDNA were similar to the endogenous NESH in Huvec cell lysates (Fig. 1,B). Phosphatase digestion of NESH proteins suggested that the slow-migrated product was a phosphorylated form of the fast-migrated protein (Fig. 1,C). We could not detect the bands with antiphosphotyrosine antibody; therefore, NESH might be phosphorylated at the Ser/Thr residues. Immunoblot analysis also showed that almost all of the cancer cell lines examined, including highly metastatic murine fibroblast transformed by v-Src (SRD) and human glioblastoma cells (U87 MG), scarcely expressed NESH, except for Huvec cells (Fig. 1,D). To further examine the behavior of NESH in cancer cells, we transfected NESH expression plasmids into SRD and U87 MG cells. Subsequent neomycin selection resulted in numerous resistant colonies from the cell lines with stable NESH expression. SRD cells and U87 MG cells provided an ideal starting point for these studies, because immunoblotting of these cell lysates demonstrated almost no expression of NESH, and these cells were known to have highly metastatic potentials. We also established NESH transfectants using NIH3T3 and 3Y1 cells, which have no metastatic potential (data not shown). The absence of NESH in these cells led us to pursue potential roles for NESH in tumor behavior. We next examined the intracellular localization of NESH. As shown in Fig. 1, confocal microscopy confirmed the cytoplasmic localization of NESH in SRD, U87 MG (Fig. 1,D) and Huvec cells (Fig. 3 F).

Taking advantage of the available NESH transfectants, we investigated whether tumor cells expressing NESH have any change in their effect on cell migration compared with the parental cells. We assayed it by using glass plates coated with colloidal gold particles. In the area in which cells migrated during incubation, gold particles were removed by the cells, making the levels of cell motility visible. The parental cells and vector transfectants exhibited marked migratory activity whereas the NESH transfectants (NESH/SRD) showed significantly restricted motility (Fig. 2,A). Similar results were obtained with the use of U87 MG and NESH/U87 MG cells. We also examined whether NESH expression could repress cell invasion in vitro. The controls and NESH transfectants were assayed for their ability to migrate through polycarbonate membranes in a Boyden chamber. As shown in Fig. 2,B, the control cells showed a marked migratory response, whereas the NESH transfectant cells again showed a significantly reduced response. Similar results were also obtained with the use of U87 MG and NESH/U87 MG cells in this system. These results indicate that NESH expression is correlated with a reduction in motile response. To rule out the possibility that NESH expression had nonspecific toxicity to cell functions, we examined the effect of NESH on cell growth. The MTT assay showed that cell proliferation activities were not affected by NESH expression, either in SRD or in U87 MG as shown in Fig. 2 C. Similar results were also obtained with the use of NIH3T3, 3Y1, and the NESH transfectants (data not shown). These results showed no significant effect of NESH on cell growth.

Because small GTPases were reported to be involved in cell motility (12, 13), we checked whether activation of those molecules was affected by NESH expression to examine the molecular mechanisms underlying NESH-mediated effects on cell properties. However, neither significant activation nor suppression was found in Cdc42, Rac, or Ras (Fig. 3,A) nor in Rho (data not shown). The cells retained viability in serum and were routinely cultured in serum containing the same medium, which indicated that the lack of motility was not caused by some toxicity in the medium. Moreover, several independent clonal lines of NESH transfectants showed similar results (no significant effect on GTPases) compared with control transfectants or parental cells (data not shown). We next focused on the AKT and MAPK of signaling effectors, because these mediators are involved in a lot of cellular functions including proliferation and invasion (12, 14). However, no phosphorylational changes were observed either in AKT (Fig. 3,B) or in MAPK (data not shown). Because MMPs were reported to be involved in cell-invasion, we examined the MMP2, one of the well-known MMPs related to invasion (15), with zymographic analysis. Although clear proteolytic activity of MMP2 was observed, there were no significant differences between parental cells and NESH transfectants (Fig. 3,C). Two-hybrid screening, using the SH3 region of NESH as bait, identified Sos1 as a binding candidate to NESH. E3B1, a member of NESH family, had previously been shown to bind to Sos1 at the SH3 domain (4, 16). We could easily confirm the E3B1-Sos1 association in vitro and in vivo; however, we could not detect the NESH-Sos1 association in vitro (data not shown). We presume that the affinity of NESH-Sos1 may be very low. We next noticed that PAK was involved in cell migration and had proline-rich sequences, suggesting its interaction with certain SH3 domains (17, 18). As shown in Fig. 3,D, we found that the SH3 region of NESH as well as E3B1 could bind to PAK1 and PAK2 in vitro. Moreover, a marked reduction in phosphorylation at 402Thr of PAK2 was noted on the cells, using commercially available phospho-specific antibody (Fig. 3,E). The decrease of PAK2-phosphorylation did not differ among the NESH transfectants. Furthermore, analysis with confocal microscopy clearly indicated that PAK2 and NESH colocalized at the leading edge of the cells (Fig. 3 F). However, we could not detect the association of those endogenous molecules under the conditions in vivo. We suspect that the association may be weak or indirect and that some other molecule might bridge the interaction.

Cell motility has been shown in several studies to play an essential role in metastatic dissemination of tumor cells (19). The finding that NESH transfection resulted in decreased migration ability of the cells suggested a novel potential mechanism for the effects of NESH expression on reducing tumor metastasis. Accordingly, it was logical to examine the potential role of NESH in tumor biology. In particular, it would be of interest to investigate the effects of NESH on cell metastasis. Parental SRD and three independent clones from NESH transfectants were implanted into immunocompromised athymic nude mice to evaluate the role of NESH in tumor metastasis. All of the mice with NESH transfectants survived for longer than 1 month, whereas all of the mice in the control groups died of tumor growth within 3 weeks. The primary s.c. tumors were almost the same in size as shown in Fig. 4, A1 and A2. In the lung, however, rates of metastasis of NESH transfectants were lower than for parental SRD and did not change among the transfectants. Note that a few tumors developed in mice with NESH/SRD were of similar size compared with the tumors of parental cells (Fig. 4, A3 and A4 ). Analysis of the resulting lung metastatic tumors of NESH/SRD by immunoblotting showed that NESH expression disappeared in almost all of the tumors after metastasis, which suggests that cells lacking NESH expression were easy to metastasize (Fig. 4,B). Similar results were obtained when SRD or U87 MG cells were injected into the lateral tail veins or into the abdominal cavity (data not shown). Because some of the metastasis tumors exhibited NESH expression, we then checked the NESH cDNAs in them. Sequencing revealed that point mutations had occurred at the COOH terminal region of NESH (Fig. 4,C). Furthermore, the mutated SH3 region showed decreased affinity to the PAK1 (data not shown) and the PAK2 in vitro (Fig. 4 C). The data shown in the study suggested that intact NESH preferentially suppressed cell metastasis of SRD cells. Accordingly, these results showed metastasis-inhibitory activity of NESH in metastatic process in vivo that was consistent with decreased cell-motility in vitro observed above.

Interestingly, deletional mutations resulting from the locus including the NESH gene were observed in metastatic prostatic carcinomas (20). NESH may function as a suppresser of tumor metastasis in some circumstances. However, the precise cellular mechanisms and potential biochemical consequences by which the NESH protein may modulate cell motility and metastatic phenotype have not been well determined. Because the ability of tumor cells to invade beyond homeostatic boundaries is a central means by which the host succumbs to tumor, it is important to ascertain whether NESH should contribute to metastasis. Further insights into the molecules and cellular processes that are influenced by the function of NESH protein may have previously unexpected implications for our understanding of the signaling pathways that regulate tumor cell motility. It is also important to investigate the relationship between the other NESH family proteins and tumor cell motility or metastasis. Future studies will address the precise biological role of NESH and NESH family proteins on this point.

Fig. 1.

Structure, expression. and localization of NESH protein. A, schematic structure of the human NESH protein. It was tagged with oligo-histidine at the NH2 terminus in His-NESH. B, expression of NESH in SRD (Lane 1), COS7 (Lane 2), Huvec (Lane 3), NESH/SRD (Lane 4), and NESH/COS7 cells (Lane 5) detected by Western blot analysis with anti-NESH pAbs. In all of the panels, each Lane contains an equal amount of total protein. C, phosphatase digestion of NESH protein. The immunoprecipitants with anti-NESH pAbs were digested with or without calf intestinal alkaline phosphatase (AP) in the presence or absence of 50 mm NaF and 20 mm sodium PPi (Inhibitors), and analyzed by immunoblotting. D, detection of NESH expression in various cell lines. Western blot analysis using anti-NESH pAb was performed with 100 μg of total cell lysate from His-NESH/SRD, NESH/SRD, SRD, Qg90, N0M1, PC3, Huvec, MK45, NIH3T3, U87 MG, NESH/U87 MG, His-NESH/U87 MG corresponding to Lane 1–12, respectively (upper panel). Western blot using anti-Erk2 specific antibody is also shown as controls (lower panel). Intracellular localization of NESH in U87 MG (1), NESH/U87 MG (3), SRD (4), and NESH/SRD (6). Phalloidin stainings (2, 5) for actin fiber are also shown as controls.

Fig. 1.

Structure, expression. and localization of NESH protein. A, schematic structure of the human NESH protein. It was tagged with oligo-histidine at the NH2 terminus in His-NESH. B, expression of NESH in SRD (Lane 1), COS7 (Lane 2), Huvec (Lane 3), NESH/SRD (Lane 4), and NESH/COS7 cells (Lane 5) detected by Western blot analysis with anti-NESH pAbs. In all of the panels, each Lane contains an equal amount of total protein. C, phosphatase digestion of NESH protein. The immunoprecipitants with anti-NESH pAbs were digested with or without calf intestinal alkaline phosphatase (AP) in the presence or absence of 50 mm NaF and 20 mm sodium PPi (Inhibitors), and analyzed by immunoblotting. D, detection of NESH expression in various cell lines. Western blot analysis using anti-NESH pAb was performed with 100 μg of total cell lysate from His-NESH/SRD, NESH/SRD, SRD, Qg90, N0M1, PC3, Huvec, MK45, NIH3T3, U87 MG, NESH/U87 MG, His-NESH/U87 MG corresponding to Lane 1–12, respectively (upper panel). Western blot using anti-Erk2 specific antibody is also shown as controls (lower panel). Intracellular localization of NESH in U87 MG (1), NESH/U87 MG (3), SRD (4), and NESH/SRD (6). Phalloidin stainings (2, 5) for actin fiber are also shown as controls.

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Fig. 2.

NESH inhibits cell migration but not cell growth. A, effect of NESH expression on cell migration of SRD and U87 MG detected with colloidal gold-coated glass (bottom panels). Representative photographs of the cell migration are also shown (top panels). The gold colloid-free areas were measured in parental cells (S), empty-vector transfectants (V), two clones of NESH transfectant (c.2, c.9), and His-NESH transfectant (His-NESH). The data are mean average ± SD of gold colloid-free areas. All of the differences between control cells (▪) and NESH transfectants (□) are significant (P < 0.001). B, effect of NESH expression on invasive ability of SRD and U87 MG detected with a modified Boyden chamber method (bottom panels). Representative results of the invasion assay are shown (top panels). Numbers of cells penetrated the membrane, which appeared as black spots, in SRD cells compared with the NESH transfectants. The penetrated cells were counted in parental cells (S), empty-vector transfectants (V), NESH transfectant (NESH), and two clones of His-NESH transfectant (c.14, c.19). The data are mean average ± SD of penetrated cells. All of the differences between control cells (▪) and NESH transfectants (□) are significant (P < 0.001). C, effect of NESH expression on cell proliferation of SRD (left panel) and U87 MG (right panel) detected by MTT assay. No significant differences on cell growth were detected. These results (A, B, and C) were confirmed by additional experiments using more than four clones independently.

Fig. 2.

NESH inhibits cell migration but not cell growth. A, effect of NESH expression on cell migration of SRD and U87 MG detected with colloidal gold-coated glass (bottom panels). Representative photographs of the cell migration are also shown (top panels). The gold colloid-free areas were measured in parental cells (S), empty-vector transfectants (V), two clones of NESH transfectant (c.2, c.9), and His-NESH transfectant (His-NESH). The data are mean average ± SD of gold colloid-free areas. All of the differences between control cells (▪) and NESH transfectants (□) are significant (P < 0.001). B, effect of NESH expression on invasive ability of SRD and U87 MG detected with a modified Boyden chamber method (bottom panels). Representative results of the invasion assay are shown (top panels). Numbers of cells penetrated the membrane, which appeared as black spots, in SRD cells compared with the NESH transfectants. The penetrated cells were counted in parental cells (S), empty-vector transfectants (V), NESH transfectant (NESH), and two clones of His-NESH transfectant (c.14, c.19). The data are mean average ± SD of penetrated cells. All of the differences between control cells (▪) and NESH transfectants (□) are significant (P < 0.001). C, effect of NESH expression on cell proliferation of SRD (left panel) and U87 MG (right panel) detected by MTT assay. No significant differences on cell growth were detected. These results (A, B, and C) were confirmed by additional experiments using more than four clones independently.

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Fig. 3.

Biochemical characterization of NESH transfectants. A, levels of GTP-bound form of GTPases in the presence or absence of NESH expression. Lysates in SRD (−) and NESH/SRD (+) cells were incubated with agarose beads coupled to GST-PAK-CRIB for Rac and for Cdc42, or GST-Raf for Ras. Then, total cell lysates (TLC) and the beads were examined by Western blotting using specific antibodies against Cdc42 (top panel), Rac (middle panel), and Ras (bottom panel), so that GTP-bound GTPases could be visualized. B, no significant effect of NESH expression on AKT phosphorylation. Western blotting using phospho-AKT-specific antibody (top panel) was performed in the lysate of SRD, NESH/SRD, His-NESH/SRD, U87 MG, NESH/U87 MG, and His-NESH/U87 MG cells corresponding to Lanes 1–6, respectively. Expressions of AKT (middle panel) and NESH (bottom panel) are also shown. C, zymographic detection of endogenous MMP2-secretion measured in parental SRD (S), empty-vector transfectants (V), and three clones of NESH transfectant (c.2, c.7, and c.9). MMP2 gelatinase secreted into the cell culture supernatant is shown with gelatin-embedded zymography. Bands of lysis representing gelatinolytic activity appear white against a dark background (top panel). Pictures shown are representative of at least three independent experiments. Expression of NESH are shown by Western blotting (bottom panel). D, in vitro binding of NESH-SH3 to PAKs. Lysates of SRD were incubated with GST alone (Lane 2), GST fused to SH3 region of E3B1 (Lane 3), GST fused to SH3 region (aa 296–366) of NESH (Lane 4), or GST fused to NH2 terminus (aa 1–145) of NESH (Lane 5). The pulled-down beads and total cell lysate of SRD (TCL) were analyzed by Western blotting using antibodies against PAK1 (top panel) and PAK2 (bottom panel). E, effect of NESH expression on PAK2 phosphorylation. Western blotting using phospho-PAK2-specific antibody (top panel) was performed in the lysate of SRD (S) and three clones of NESH transfectants (c.2, c.5, and c.7). Expressions of PAK2 (middle panel) and NESH (bottom panel) are also shown. F, colocalization of NESH with PAK2 in Huvec cells. Confocal microscopy revealed endogenous NESH (top panel) colocalizing with PAK2 (middle panel) in Huvec cells; bottom panel, the merging picture of NESH and PAK2.

Fig. 3.

Biochemical characterization of NESH transfectants. A, levels of GTP-bound form of GTPases in the presence or absence of NESH expression. Lysates in SRD (−) and NESH/SRD (+) cells were incubated with agarose beads coupled to GST-PAK-CRIB for Rac and for Cdc42, or GST-Raf for Ras. Then, total cell lysates (TLC) and the beads were examined by Western blotting using specific antibodies against Cdc42 (top panel), Rac (middle panel), and Ras (bottom panel), so that GTP-bound GTPases could be visualized. B, no significant effect of NESH expression on AKT phosphorylation. Western blotting using phospho-AKT-specific antibody (top panel) was performed in the lysate of SRD, NESH/SRD, His-NESH/SRD, U87 MG, NESH/U87 MG, and His-NESH/U87 MG cells corresponding to Lanes 1–6, respectively. Expressions of AKT (middle panel) and NESH (bottom panel) are also shown. C, zymographic detection of endogenous MMP2-secretion measured in parental SRD (S), empty-vector transfectants (V), and three clones of NESH transfectant (c.2, c.7, and c.9). MMP2 gelatinase secreted into the cell culture supernatant is shown with gelatin-embedded zymography. Bands of lysis representing gelatinolytic activity appear white against a dark background (top panel). Pictures shown are representative of at least three independent experiments. Expression of NESH are shown by Western blotting (bottom panel). D, in vitro binding of NESH-SH3 to PAKs. Lysates of SRD were incubated with GST alone (Lane 2), GST fused to SH3 region of E3B1 (Lane 3), GST fused to SH3 region (aa 296–366) of NESH (Lane 4), or GST fused to NH2 terminus (aa 1–145) of NESH (Lane 5). The pulled-down beads and total cell lysate of SRD (TCL) were analyzed by Western blotting using antibodies against PAK1 (top panel) and PAK2 (bottom panel). E, effect of NESH expression on PAK2 phosphorylation. Western blotting using phospho-PAK2-specific antibody (top panel) was performed in the lysate of SRD (S) and three clones of NESH transfectants (c.2, c.5, and c.7). Expressions of PAK2 (middle panel) and NESH (bottom panel) are also shown. F, colocalization of NESH with PAK2 in Huvec cells. Confocal microscopy revealed endogenous NESH (top panel) colocalizing with PAK2 (middle panel) in Huvec cells; bottom panel, the merging picture of NESH and PAK2.

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Fig. 4.

Expression of NESH prevented rates of metastasis from rising. Five-week-old BALB/c nude mice received injections in the right hind flank either with SRD (A1 and A3) or with NESH/SRD (A2 and A4). Effect of NESH on primary tumor growth (A1 and A2) at day 14 after infection and on metastatic tumors in the lung (A3 and A4) at day 20 after infection in nude mice were shown. Arrows, tumors. These results were confirmed by the additional experiments using more than 20 mice; representative pictures were shown. B, NESH expression in the lung-metastatic tumors injected with NESH/SRD. Independent tumors from six mice were assayed by Western blotting with NESH pAbs. Only tumors 6, 11, and 23 remained to express NESH; Lane P, NESH transfectant as a positive control. C, the mutations found by sequencing the NESH-cDNAs from tumors 6, 11, 23 (top panel). The mutants of NESH-SH3 were then assayed for PAK2 binding in vitro. Pull-down assay with GST alone (G) or GST fused to the indicated mutants of SH3 (6, 11, 23) was performed as described in Fig. 3 D (bottom panel).

Fig. 4.

Expression of NESH prevented rates of metastasis from rising. Five-week-old BALB/c nude mice received injections in the right hind flank either with SRD (A1 and A3) or with NESH/SRD (A2 and A4). Effect of NESH on primary tumor growth (A1 and A2) at day 14 after infection and on metastatic tumors in the lung (A3 and A4) at day 20 after infection in nude mice were shown. Arrows, tumors. These results were confirmed by the additional experiments using more than 20 mice; representative pictures were shown. B, NESH expression in the lung-metastatic tumors injected with NESH/SRD. Independent tumors from six mice were assayed by Western blotting with NESH pAbs. Only tumors 6, 11, and 23 remained to express NESH; Lane P, NESH transfectant as a positive control. C, the mutations found by sequencing the NESH-cDNAs from tumors 6, 11, 23 (top panel). The mutants of NESH-SH3 were then assayed for PAK2 binding in vitro. Pull-down assay with GST alone (G) or GST fused to the indicated mutants of SH3 (6, 11, 23) was performed as described in Fig. 3 D (bottom panel).

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by a Grant-in-Aid for scientific research on priority areas and for COE Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, a Grant under the Monbukagakusho International Scientific Research Program, and in part by a Grant from the Aichi Cancer Research Foundation.

4

The abbreviations used are: SH3, Src homology 3; MMP, matrix metalloproteinase; PAK, p21-activated kinase; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; pAb, polyclonal antibody.

We thank Sachi Kozawa for her excellent technical assistance.

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