Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein

Article metrics

Abstract

The Nf2 tumor suppressor codes for merlin, a protein whose function is largely unknown. We have previously demonstrated a novel interaction between merlin and TRBP, which inhibits the oncogenic activity of TRBP. In spite of the significance of their functional interaction, its molecular mechanism still remains to be elucidated. In this report, we investigated how merlin inhibits the oncogenic activity of TRBP in association with cell growth conditions. In the human embryonic kidney 293 cell line, the level of endogenous merlin increased, whereas that of endogenous TRBP significantly decreased along with the increase in cell confluence. We demonstrated that the carboxyl-terminal region of TRBP was responsible for this phenomenon using stable cell lines expressing deletion mutants of TRBP. The overexpression of merlin decreased the protein level of TRBP, and the ubiquitin-like subdomain of merlin's FERM domain was important for this activity. We also demonstrated that TRBP is ubiquitinylated and the ubiquitinylated forms of TRBP are accumulated by ectopically expressed merlin or cell confluence in the presence of MG132, a proteasome inhibitor. Furthermore, we showed that the regulation of TRBP in response to cell confluence was abolished upon knockdown of merlin expression by specific small interfering RNA. Finally, we showed that ectopically expressed merlin restored cell–cell contact inhibition in cells stably expressing TRBP but not in TRBPΔc. These results suggest that merlin is involved in the regulation of TRBP protein level by facilitating its ubiquitination in response to such cues as cell–cell contacts.

Introduction

Neurofibromatosis type 2 (NF2) is a predominantly inherited disorder characterized by the development of schwann cell tumors and other brain tumors (Evans et al., 1992a, 1992b). Mutations or the loss of heterozygosity of the Nf2 locus has been detected in various tumors of the nervous system, such as schwannomas, meningiomas and ependymomas (Gusella et al., 1999). In addition, inactivation of Nf2 specifically in the schwann cells leads to the development of schwannomas and schwann cell hyperplasia in mice (Giovannini et al., 2000).

The Nf2 gene encodes merlin, which was named for its structural similarity to the ERM family proteins (moesin, ezrin and radixin) (Gusella et al., 1996; Tsukita and Yonemura, 1997; Bretscher et al., 2002; Sun et al., 2002). Merlin and ERM protein share the FERM domain, which is composed of three structural modules (F1, F2 and F3) that together form a compact clover-shaped structure (Pearson et al., 2000). Especially, F1 is very similar to the structure of ubiquitin (Vijay-Kumar et al., 1987).

Recent work has demonstrated that the protein level and phosphorylation states of merlin are affected by the growth conditions of cells, such as confluence, loss of adhesion or serum deprivation (Gutmann et al., 1999). The phosphorylation of merlin at serine 518 can be induced by an active form of Rac or cdc42, but not of Rho (Shaw et al., 2001). The Rac/cdc42-induced phosphorylation of merlin at serine 518 is mediated by a p21-activated kinase, Pak (Kissil et al., 2002; Xiao et al., 2002). Such phosphorylation can disrupt the intramolecular interaction of merlin and its association with the actin cytoskeleton, and this induces a change in the subcellular localization of merlin in LLC-PK1 cells (Shaw et al., 2001; Kissil et al., 2002). On the other hand, merlin inhibits the activation of Pak1 through direct binding to the Pak1 PBD, and it thereby interferes with the Pak1 recruitment to focal adhesions (Kissil et al., 2003).

TRBP belongs to the family of double-stranded RNA (dsRNA)-binding proteins, and it was originally identified as an HIV-1 TAR RNA-binding protein with two clearly defined dsRNA-binding domains (dsRBDs) and a C-terminal basic region (St Johnston et al., 1992; Gatignol et al., 1993; Bass et al., 1994; Kharrat et al., 1995; Daher et al., 2001). TRBP dsRBD2 binds TAR with a higher affinity than dsRBD1 does, because the former contains a KR-helix motif (St Johnston et al., 1992; Bannwarth et al., 2001).

The murine homolog of TRBP binds the 3′ untranslated region of Prm1 protamine RNA, represses its translation and plays a physiological role in spermatogenesis (Lee et al., 1996; Zhong et al., 1999). TRBP directly binds the dsRNA-activated protein kinase (PKR), which has antiviral and antiproliferative effects. TRBP inhibits the ability of PKR to phosphorylate eucaryotic translation initiation factor 2 (eIF-2), leading to its inactivation (Park et al., 1994, Benkirane et al., 1997). In relation to the inhibition of PKR activity, TRBP was demonstrated to play a growth-promoting role and it has oncogenic potential (BenKirane et al., 1997). However, it has not been elucidated how TRBP's function is regulated in cells.

In our previous study, we identified TRBP as a merlin-binding protein in a yeast two-hybrid screen and carboxyl-terminal regions of each protein as a domain responsible for their interaction (Lee et al., 2004). The consequences of merlin–TRBP interaction include inhibitory effects on TRBP-mediated cell proliferation, anchorage-independent cell growth, oncogenic transformation and tumor development in nude mice (Lee et al., 2004). Therefore, it may be possible to suggest that merlin has a role as a cellular regulator of TRBP function through certain mechanisms.

In this report, we aimed to understand the mechanism by which merlin inhibits the oncogenic activity of TRBP. We found that TRBP is degraded by ubiquitination under the high-confluence cell growth conditions and this is facilitated by merlin.

Materials and methods

Plasmids and adenovirus

The plasmids for wild-type merlin and full-length human TRBP and its derivatives were described in previous reports (Lee et al., 2004). The adenovirus expressing merlin was kindly gifted by Dr Jeun. The virus was propagated in the human embryonic kidney 293 (HEK293) cell line, and the viral titers were determined by a limiting-dilution bioassay in HEK293 cells.

Cell culture and transient transfection

NIH3T3, A172 and HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All the cell lines were maintained in DMEM that was supplemented with 10% FBS and antibiotics. The Gene Porter2 reagent (Gene Therapy System Inc.) and Lipofectamine 2000 (Invitrogen, San Diego, CA, USA) were used for transfection according to the manufacturer's recommendations. For the same amount of total transfected DNA, the pcDNA3.1 plasmid was used in all the transfections.

Construction of stable cell lines

NIH3T3 cells were plated at a density of 3 × 105?cells per 35?mm dish and they were transfected with plasmids containing full-length human TRBP DNA or the derivatives of human TRBP DNA. At 24?h after transfection, the cells were replated at the ratios of 3:1, 9:1, 27:1 and 50:1, and the G418-resistant colonies were selected with 400?μg/ml G418 for 2 weeks. Before each experiment using the constructed stable cell lines, Western blot analyses were performed to certify the expression of the integrated TRBP.

Antibodies, immunoprecipitation and Western blotting

The antibodies for merlin (sc-331, sc-28247 and a specific batch of sc-332, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), V5-epitope (Invitrogen, San Diego, CA, USA) and β-actin (Sigma Inc.) were obtained from commercial sources. Polyclonal anti-TRBP antibody was produced in a rabbit using a KLH-conjugated peptide (RSPPMELQPPVSPQQSECNPVGALQ) as an epitope (Peptron Inc., Daejeon, Korea), and the antibody product was tested by ELISA and Western analysis (data not shown).

For immunoprecipitation or Western blotting, the cells were lysed in RIPA-B buffer (0.5% Nonidet P-40, 20?mM Tris, pH 8.0, 50?mM NaCl, 50?mM NaF, 100?μ M Na3VO4, 1?mM DTT and 50?μg/ml PMSF) for 1?h on ice. The insoluble materials were removed by centrifugation at 12?000?r.p.m. for 20?min at 4°C. The supernatant was then subjected to SDS–PAGE and Western blotting. The blots were blocked in phosphate-buffered saline containing 5% skim milk and 0.05% Tween 20, and the blots were then incubated sequentially with the primary antibody and HRP-conjugated secondary antibody. Detection was performed according to the enhanced chemiluminescence protocol (Amersham, Arlington Heights, IL, USA).

Enrichment of ubiquitinated protein

Polyubiquitin affinity beads, in which the ubiquitin-associated domain of Rad23 was immobilized, and the control beads were obtained from Calbiochem-Novabiochem. The whole-cell extracts from HEK293 cells were obtained using a lysis buffer consisting of 50?mM HEPES (pH 7.5), 5?mM EDTA, 150?mM NaCl, 1% Triton X-100 and complete protease inhibitor (Roche Applied Science). A 1?mg portion of each whole-cell extract was incubated with 40?μl of beads in the lysis buffer for 4?h at 4°C, with constant mixing. The beads were then washed three times with the same buffer, and the bound proteins were extracted in SDS buffer and analysed by Western blotting using anti-ubiquitin antibody (Santa Cruz Biotechnology Inc.) or anti-TRBP antibody.

RNA interference to Nf2

Stealth™ small interfering RNA (siRNA) duplex oligonucleotides were designed based on the sequences specific to human Nf2 cDNA (5′-IndexTermGGACAAGAAGGUACUGGAUCAUGAU-3′ and 5′-IndexTermAUCAUGAUCCAGUACCUUCUUGUCC-3′). Stealth™ RNAi Negative Control Med GC was used as an experimental control. The antisense and sense siRNA oligonucleotides with a dTdT 3′-overhang were synthesized and annealed by Invitrogen Inc. (San Diego, CA, USA). Transfection of HEK293 cells with the siRNA duplexes was performed according to the manufacturer's instructions using Lipofectamine™ 2000.

Results

Downregulation of endogenous TRBP by confluency

We have previously reported that merlin interacts with TRBP and inhibits its oncogenic activity such as proliferation, anchorage-independent growth and tumorigenesis in a nude mouse model (Lee et al., 2004). However, the mechanism used for doing so has not been elucidated. Based on the function of TRBP and merlin as a putative oncogene and a tumor suppressor, respectively, we were interested in whether their functional interaction could induce changes in the level of each protein owing to the growth condition of cells such as the state of cell confluence and the cell-to-cell contact (Fisher and Yeh, 1967). To test this hypothesis, we first examined the cellular levels of endogenous TRBP and merlin by performing immunoblotting on HEK293 cells that were plated at increasing cell densities (Figure 1a). As shown in Figure 1b, the levels of merlin increased along with the increase in cell confluency, and this was particularly prominent when the majority of cells had established cell-to-cell contacts (upper panel). This is a well-known phenomenon (Sherman et al., 1997; Shaw et al., 1998a, 1998b). Strikingly, the levels of TRBP decreased along with increasing confluency, which showed an opposite result to that of merlin (Figure 1b, middle panel). Considering the interaction between merlin and TRBP, these data may suggest that merlin is involved in the downregulation of TRBP in response to cell confluence.

Figure 1
figure1

High cell density reduces TRBP protein level in HEK293 cells. (a) The phase-contrast images of HEK293 cells plated in 60?mm dishes at increasing cell densities as described. The next day, the cells were used for immunoblotting as shown in lanes 1–4 of (b). (b) Immunoblot analysis of merlin, TRBP and β-actin using the total cell lysates of HEK293 cells from (a). The representative results shown are from three independent experiments.

The carboxyl-terminal region of TRBP is responsible for TRBP decrease according to cell confluency

To further support downregulation of TRBP level by cell confluency, we investigated which region of TRBP was responsible for this. Several stable NIH3T3 cell lines were constructed, with each expressing wild-type TRBP or its two deletion mutants (TRBPΔR and TRBPΔC; Figure 2a) or EGFP. Each stable cell line was plated at increasing degrees of confluency; the protein levels of ectopically expressed TRBP and EGFP, and the endogenous merlin, were examined by immunoblotting. In all cases, the levels of endogenous merlin increased along with increasing cell confluency (Figure 2b–e). The level of the N-terminal deletion form of TRBP (TRBPΔR) decreased along with increasing confluency, similarly to that of the wild-type TRBP (Figures 2b and d). However, the levels of the C-terminal deletion form of TRBP and the negative control of EGFP did not show any change with respect to increasing cell confluency (Figures 2c and e). These results indicated that the confluency-induced TRBP decrease is a specific process, and it depends on the carboxyl-terminal region of TRBP.

Figure 2
figure2

The carboxyl-terminal region of TRBP is responsible for its decrease depending upon cell confluence. Immunoblot analysis of merlin and TRBP proteins in NIH3T3 cells stably expressing TRBP-V5 (b), TRBPΔC-V5 (c), TRBPΔR-V5 (d) and EGFP (e). The anti-V5 epitope antibody was used to detect TRBP proteins, and β-actin was detected as an internal control. The confluences of cells in each lane are as follows: lane 1, 2.5 × 105; lane 2, 5 × 105; and lane 3, 1 × 106 in a 60?mm dish. The TRBP constructs are depicted in (a). TRBP contains three domains: two dsRNA-binding domains (dsRBD1 and dsRBD2) and a C-terminal region. Deletion notation: TRBPR lacks dsRBD1; TRBPΔC lacks the C-terminal region.

Merlin plays a role in TRBP decrease

The above results showed that the levels of merlin and TRBP inversely correlated with cell confluency. Previously, we have also shown the specific interaction and functional counteraction between merlin and TRBP (Lee et al., 2004). Therefore, we assumed that the decrease in the level of TRBP according to cell confluency might be caused by the increase in the level of merlin. To prove this hypothesis, an increasing amount of merlin expression plasmid was cotransfected with a given amount of TRBP expression plasmid and 0.5?μg of pcDNA3.1-LacZ into NIH3T3 cells, and their cellular levels were examined. As shown in Figure 3a, the level of overexpressed TRBP decreased along with the increase in merlin levels. To see whether the merlin effect also occurs with endogenous TRBP, the adenoviruses expressing merlin (Ad-NF2) were infected to A172, a human glioblastoma cell line, and the level of endogenous TRBP was examined. As shown in Figure 3b, the level of endogenous TRBP was decreased by Ad-NF2 in a dose-dependent manner, but it was not decreased by the GFP-expressing adenovirus (Ad-GFP) in A172 cells. These results strongly suggest that the overexpression of merlin decreases the TRBP protein level.

Figure 3
figure3

Merlin is responsible for the decrease of TRBP. (a) Immunoblot analysis for merlin, TRBP and LacZ in NIH3T3 cells transfected with the indicated amounts of TRBP and merlin expression plasmids with 0.5?μg pcDNA-lacZ-his plasmid. (b) Immunoblot analysis of merlin and TRBP in A172 cells infected with the merlin-expressing adenovirus at the indicated MOIs. (c) Immunoblot analysis of merlin and TRBP in HEK293 cells transiently transfected with the siRNA to merlin (si-NF2) or a control siRNA (si-control). Mock represents the nontransfected control. In order to ensure confluent status, all cell lysates were prepared at 2 days after transfection or infection.

To further support the direct role of merlin, endogenous merlin was knocked down by a siRNA against merlin expression (si-NF2), and the TRBP protein level was examined. As shown in Figure 3c, the endogenous TRBP protein level specifically increased when si-NF2 was transfected. Taken together, these results suggest that merlin plays a role as a regulator of the TRBP protein level.

Ubiquitin-like subdomain of merlin's FERM domain is responsible for TRBP decrease, and phosphorylation of merlin at S518 does not affect it

To further support the specific role of merlin, we performed a domain analysis of merlin to determine which region of merlin has the ability to induce the decrease of TRBP. Various deletion mutants of merlin were constructed (Figure 4a). Wild-type merlin and its deletion mutants were each expressed in HEK293 cells together with V5 epitope-tagged TRBP and pcDNA3.1-lacZ. Subsequent Western blotting with anti-V5 antibody revealed that the wild-type and M3 deletion mutants of merlin clearly decreased the level of ectopically expressed TRBP and the M2 and M4 mutants of merlin showed relatively weak effects. Notably, the level of ectopically expressed TRBP was not affected by the expression of M1 mutant, which lacks the ubiquitin-like subdomain (F1 subdomain) of merlin's FERM domain. Additionally, the level of ectopically expressed TRBP was moderately decreased by M1 and M3 mutant coexpression (Figure 4b, upper panel). Western blotting with anti-merlin antibody revealed that the wild-type and deletion mutants of merlin were expressed at comparable levels (Figure 4b, middle panels). When comparing the domains in the deletion mutants, this result suggested that the ubiquitin-like subdomain of merlin's FERM domain is important for the decrease in TRBP level by merlin overexpression. We have quantified TRBP-V5 levels from three independent experiments (Figure 4c) and arrived at the same conclusion.

Figure 4
figure4

Ubiquitin-like subdomain of merlin's FERM domain is responsible for the decrease of TRBP. (a) Schematic presentation of wild-type and deletion mutants of merlin. (b) Immunoblot analysis of merlin and TRBP in NIH3T3 cells transiently transfected with the expression plasmids for merlin proteins described in (a) and/or TRBP-V5. (c) Three independent immunoblotting images of TRBP were scanned with GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Richmond, CA, USA) and analysed by Quantity One 4.2.2 program. Statistical analysis was performed with Student's t-test (*P<0.05 and **P<0.01 versus TRBP only). (d) Immunoblot analysis for TRBP and merlin proteins in NIH3T3 cells transfected with the plasmids expressing TRBP-V5, and the wild-type or mutant (S518A and S518D) merlin proteins. In order to ensure confluent status, all cell lysates were prepared at 2 days after transfection. The asterisks indicate putative depredation products of each protein.

It was previously shown that phosphorylation of merlin at serine 518 leads to relocalization and inactivation of merlin, whereas a merlin mutant of S518A functions as a constitutive active form (Shaw et al., 2001). To examine the effect of merlin phosphorylation at serine 518, we observed the amount of exogenous TRBP in NIH3T3 cells coexpressing each of wild-type, nonphosphorylatable mutant type (S518A) and phosphorylation mimicking mutant type (S518D) merlin proteins. As shown in Figure 4d, the exogenous TRBP protein level was decreased by all three forms of merlin, indicating that merlin phosphorylation at serine 518 does not affect its ability to induce TRBP decrease.

Ubiquitinylation of endogenous TRBP

As described above, TRBP is dramatically decreased by merlin expression and it interacts with merlin, as was previously demonstrated by us (Lee et al., 2004). Additionally, the ubiquitin-like domain of merlin's FERM domain is responsible for merlin-induced TRBP decrease. Taken together, if merlin immediately regulates TRBP level at the protein level, one of the possible mechanisms could be the ubiquitin–proteasome pathway. In our first attempt to elucidate this, we examined whether TRBP by itself is ubiquitinylated in HEK293 cells. After treatment of cells for 15?h with MG132, a proteasome inhibitor, the endogenous TRBP level increased according to MG132 concentration (Figure 5a, middle panel). Moreover, a ladder of high molecular weight endogenous TRBP bands, a characteristic of polyubiquitinylated proteins, was detected by TRBP immunoblotting and this was also increased in amount by MG132 treatment (Figure 5a, upper panel).

Figure 5
figure5

Ubiquitination of TRBP in HEK293 cells. (a) HEK293 cells were left untreated or treated with MG132, a specific proteasome inhibitor, as indicated. The total cell extracts were immunoblotted with anti-V5 epitope antibody to detect the tagged TRBP. Both the long- and short-exposure images are presented to show the ubiquitination and stabilization of TRBP by MG132, respectively. (b) Equivalent amounts of whole-cell extracts of HEK293 cells treated as above were applied to the polyubiquitin affinity beads or to the control beads. The pulled down proteins were analysed by Western blotting using anti-TRBP antibody. (c) HEK293 cell lysates were immunoprecipitated (IP) with anti-TRBP antibody or preimmune serum (PIS), and then immunoblotted (IB) with anti-ubiquitin antibody. All cell lysates were prepared at confluent status.

To demonstrate that the high molecular weight ladder of TRBP was due to ubiquitinylation, we used polyubiquitin affinity beads to pull the proteins down from the whole-cell extracts of HEK293 cells that were treated with MG132. Subsequent Western blotting with anti-TRBP antibody revealed a ladder of polyubiquitinylated TRBP bands from the pulled down proteins (Figure 5b). The ubiquitinylated TRBP bands were shifted up with similar size intervals, and the lowest band was about 6?kDa apart from the original position. These bands might have corresponded to the conjugation of one, two, three and four ubiquitins (Figure 5b, asterisks).

To further support TRBP ubiquitination, the cell lysates of HEK293 cells treated with MG132 were first immunoprecipitated with anti-TRBP antibody. The subsequent immunoblotting with anti-ubiquitin antibody revealed a smear band of polyubiquitinylated TRBP bands with high molecular weights (Figure 5c). Taken together, these results suggest that TRBP is ubiquitinylated and MG132, a specific proteasome inhibitor, can stabilize TRBP and also its ubiquitinylated forms.

Merlin facilitates TRBP degradation via the ubiquitin–proteasome pathway

We demonstrated in the above experiments that merlin decreases the amount of TRBP protein and that TRBP can be ubiquitinylated. To address merlin's function in TRBP ubiquitinylation, HEK293 cells were transfected with an increasing amount of merlin expression plasmids, and the cells were treated with 5?μ M MG132 for 15?h to inhibit the proteasomal degradation of TRBP. Subsequently, cell lysates were immunoprecipitated with anti-TRBP antibody and then immunoblotted with anti-ubiquitin antibody to show the ubiquitinylated TRBP. In cells expressing exogenous merlin, ubiquitinylated high molecular mass proteins were detected in the anti-TRBP immunoprecipitates and their amounts were enhanced by the increase in merlin expression (Figure 6a).

Figure 6
figure6

Overexpressed merlin increases the ubiquitinylated TRBP in HEK293 cells. (a) Immunoblot analysis of exogenously expressed merlin (top panel) and ubiquitinylated TRBP (middle panel) in HEK293 cells transfected with increasing amounts of merlin-expressing plasmid (pNF2). (b) Immunoblot analysis of endogenous ubiquitinylated TRBP in HEK293 cells transfected with wild-type, M3 and M4 merlin-expressing plasmid (right panel). Immunoblot analysis of exogenously expressed wild-type, M3 and M4 deletion mutant of merlin with anti-merlin C-term (left panel) and anti-merlin N-term antibody (center panel). The cells were treated with 5?μ M MG132 for 15?h at 1 day after transfection. All the cell lysates were immunoprecipitated (IP) with anti-TRBP antibody and immunoblotted (IB) with anti-ubiquitin antibody. In order to ensure confluent status, all cell lysates were prepared at 2 days after transfection or infection. The asterisks indicate the positions of the merlin mutants expected from their calculated molecular weights.

To further confirm merlin-induced TRBP ubiquitinylation, the same experiment was performed with M3 and M4 deletion mutants of merlin. As shown in Figure 6b, endogenous ubiquitinylated TRBP was detected in wild-type merlin and in the M3 deletion mutant. However, the M4 deletion mutant did not increase ubiquitinylated TRBP. Given the above observations, this result indicated that merlin may facilitate TRBP ubiquitinylation via the ubiquitin-like F1 subdomain of merlin.

Merlin mediates confluency-dependent TRBP regulation

To elucidate if TRBP ubiquitinylation is regulated by cell confluence via merlin, we investigated TRBP ubiquitinylation in HEK293 cells with various cell confluences. As shown in Figure 7a, endogenous ubiquitinylated TRBP was increased along with the increase in cell confluence. This result suggested that TRBP was regulated by cell confluence.

Figure 7
figure7

Confluency-dependent TRBP regulation is mediated by merlin-dependent ubiquitinylation of TRBP. (a) Immunoprecipitation-coupled immunoblot analysis of endogenous ubiquitinylated TRBP in HEK293 cells that show deferent confluency. The confluences of cells in each lane are as follows: lane 1, 7.5 × 105; lane 2, 1.5 × 106; lane 3; and lane 4, 3 × 106 in a 100?mm dish. (b) Immunoblot analysis of endogenous merlin (upper panel), TRBP (middle panel) and β-actin (bottom panel) in HEK293 cells that were transfected with si-control and si-NF2 RNA. The confluences of cells in each lane are as follows: lane 1, 2.5 × 105; lane 2, 5 × 105; and lane 3, 1 × 106 in a 60?mm dish.

Furthermore, we investigated confluency-dependent TRBP regulation in merlin knockdown cells. As shown in Figure 7b, si-control-transfected HEK293 cells showed confluency-dependent TRBP regulation. However, merlin knockdown cells did not show confluency-dependent TRBP regulation and also basal TRBP level was significantly higher than si-control-transfected NIH3T3 cells. Taken together, these data suggested that TRBP protein level was controlled by confluency via merlin.

Merlin overexpression restores contact inhibition in TRBP-overexpressing NIH3T3 cells

Previously, we demonstrated that NIH3T3 cells overexpressing TRBP exhibit transformed phenotypes, such as the loss of contact inhibition, the increase of cell proliferation, anchorage-independent growth and tumorigenesis in nude mouse (Benkirane et al., 1997; Lee et al., 2004). To elucidate the biological function of merlin, we investigated contact inhibition in TRBP-, TRBPΔc- and EGFP-overexpressing NIH3T3 cells that were infected with merlin-expressing adenovirus or not.

As shown in Figure 8, the TRBP- and TRBPΔc-expressing stable cell line (TRBP, TRBPΔc) showed significantly higher growth rates than the control cells (EGFP). Additionally, cell–cell contact inhibition was observed in EGFP- and TRBP-expressing cells, but the TRBP-expressing cell line had greater cell number than the EGFP-expressing cell line. In contrast, the TRBPΔc-expressing cell line showed less contact inhibition than other cell lines. Merlin-expressing adenovirus infection restored cell–cell contact inhibition in the TRBP stable cell line. In contrast, merlin-expressing adenovirus infection did not show a significant effect on TRBPΔc-expressing and EGFP-expressing cell lines. TRBPΔc, which cannot bind to merlin, is a stable protein, regardless of cell confluency (Figure 2c). These data suggest that merlin can rescue contact inhibition in TRBP-overexpressing cells.

Figure 8
figure8

Merlin overexpression rescued loss of contact inhibition by TRBP overexpression in NIH3T3 cells. NIH3T3 stable cell lines expressing the indicated proteins were plated on six-well plates with 2 × 105?cells/well. Merlin-expressing adenovirus was infected with 100?MOI at 1 day after seeding. The cell number was counted on indicated days. The results shown are the average of three independent experiments. Statistical analysis was performed with Student's t-test (*P<0.05 versus each mock infection).

Discussion

After positional cloning of the Nf2 gene, its encoded protein, merlin, was revealed as a member of a family of cytoskeleton:membrane linkers, and it functionally plays a role both as a tumor suppressor and metastasis suppressor. However, the molecular basis for the growth-suppressing function of merlin still remains rather elusive. We recently reported that merlin interacts specifically with TRBP via its carboxyl-terminal region, and it consequently inhibits the oncogenic activities of TRBP that induce cell proliferation, anchorage-independent cell growth, oncogenic transformation and tumor development in nude mice (Lee et al., 2004). However, the regulation mechanism and downstream events of their interaction remain to be elucidated. We now provide several lines of evidence suggesting a novel function of merlin to induce ubiquitination and degradation of TRBP in response to the signals by cell confluency.

Previous study has demonstrated that the cellular level of merlin was increased at high cell confluency, and that merlin was differentially phosphorylated by such growth conditions as cell confluency (Shaw et al., 1998a, 1998b). Furthermore, cell confluency influences merlin's function such as its interaction with Pak1, resulting in the inhibitory effect on Pak1 activation (Kissil et al., 2003). Given the correlation of merlin's function and cell confluency, we were interested to know whether growth inhibition by high cell confluency might cause a change in the cellular level of TRBP. Our data showed that the level of endogenous TRBP is decreased by increasing confluency, and it is inversely correlated with the level of merlin in HEK293, a human kidney cell line (Figure 1). Furthermore, we also showed that the carboxyl-terminal region of TRBP is responsible for this phenomenon using stable NIH3T3 cell lines expressing deletion mutants of TRBP (Figure 2). Notably, we described in a previous report (Lee et al., 2004) that the merlin–TRBP interaction depends on the same carboxyl-terminal region of TRBP. Therefore, it could be speculated that the merlin–TRBP interaction may be necessary for the confluency-induced downregulation of TRBP. Although we did not directly see the importance of their interaction, we proved the role of merlin in decreasing TRBP using transient expression systems and siRNA against merlin (Figure 3). Taken together, it would be reasonable to suggest that increased merlin expression either from exogenous or endogenous sources induced the decrease in TRBP level.

On the domain analysis of merlin (Figure 4b), ubiquitin-like F1 subdomain of merlin was responsible for the downregulation of TRBP level. It is interesting that this domain was also shown in our previous report to be responsible for the merlin-dependent downregulation of Mdm2 level (Kim et al., 2004). Therefore, the F1 subdomain of merlin appears to play role(s) in the downregulation of some of its target proteins in cells. The TRBP level was apparently decreased by the M3 mutant of merlin lacking the TRBP-binding region (Lee et al., 2004). It may be possible that endogenous merlin and other ERM protein are involved in this phenomenon. Merlin and ERM protein form homo- or hetero-oligomer in normal cells (Bretscher et al., 2000). Therefore, if the M3 mutant formed an oligomer with endogenous merlin, TRBP may bind with the M3–merlin oligomer.

Phosphorylation of merlin can represent one mechanism of regulating the function of merlin by modulating its folding states, being inactive to inhibit cell growth when phosphorylated (Shaw et al., 1998a, 1998b; Rong et al., 2004; Surace et al., 2004). The phosphorylation state of merlin at serine 518 was recently suggested to be very important for its function in suppressing cell growth and motility. The mutation of serine 518 to alanine (S518A) resulted in the constitutive active form of merlin, while its mutation to glutamate (S518D), mimicking a ‘phosphorylated’ form of merlin, abolished its activities (Surace et al., 2004). In this report, we also examined the function of these two mutations of merlin (S518A and S518D), but they showed no effect on TRBP downregulation (Figure 4d). However, our previous results demonstrated that hypophosphorylated merlin preferentially binds to TRBP (Lee et al., 2004). In addition, phospho-amino-acid analyses have demonstrated that merlin is phosphorylated on multiple serine and/or threonine residues (Shaw et al., 1998a, 1998b). Therefore, we cannot exclude the possibility that other merlin phosphorylation sites can affect its function to downregulate TRBP.

We demonstrated that TRBP was ubiquitinylated at the physiological level (Figure 5), and exogenous merlin and M3 mutant expression can induce the ubiquitinylation of endogenous TRBP in HEK293 cells (Figure 6). Furthermore, we showed TRBP ubiquitinylation was increased by cell confluency (Figure 7a) and merlin expression was responsible for confluency-dependent TRBP regulation (Figure 7b). Finally, we demonstrated that ectopically expressed merlin can restore the contact inhibition in cells stably expressing TRBP (Figure 8). This line of evidence suggested that merlin facilitates ubiquitinylation and proteasomal degradation in response to cell confluency.

We cannot directly show merlin-dependent TRBP ubiquitinylation using an in vitro ubiquitinylated assay because E3 ubiquitin ligase for TRBP has not yet been identified. However, we provided the following evidence to exclude the possibility that the decrease in TRBP is simply a secondary result of merlin's growth inhibitory effect. First, we showed that TRBPΔc, which cannot bind to merlin, is a stable protein regardless of cell confluency (Figure 2). Furthermore, ectopically expressed merlin cannot restore contact inhibition in cells stably expressing this merlin-resistant TRBPΔc mutant, although it can do so in cell expressing wild-type TRBP cells (Figure 8). Finally, we showed that downregulation of TRBP in response to cell confluence was abolished upon knockdown of merlin expression by a specific siRNA (Figure 7b). These results show that TRBP degradation is not simply a secondary effect of cell confluency. It requires both a functional merlin and TRBP–merlin interaction.

Considering the growth-promoting role of TRBP, it is not so strange that the cellular TRBP level is tightly regulated by the ubiquitin–proteasome degradation pathway. In terms of merlin, it has been recently reported that a deletion mutant of merlin frequently observed in NF2 patients is efficiently degraded by the ubiquitin–proteasome pathway (Gautreau et al., 2002). However, this is the first report that associates merlin with the function of inducing ubiquitination and degradation of target proteins.

Although the molecular machinery for merlin inducing proteasomal degradation of TRBP is still largely unknown, the data presented here represent a first step toward understanding how merlin, a tumor suppressor, regulates the growth-promoting activity of TRBP. Taken together, our results suggest that merlin may serve its tumor suppressor role in response to the signals from increased cell-to-cell contact at least in part by inducing the proteasomal degradation of TRBP.

Abbreviations

NF2:

neurofibromatosis type 2

TRBP:

trans-activation-responsive RNA-binding protein

dsRBD:

double-stranded RNA-binding domain

References

  1. Bannwarth S, Talakoub L, Letourneur F, Duarte M, Purcell DF, Hiscott J et al. (2001). J Biol Chem 276(52): 48803–48813.

  2. Bass BL, Hurst SR, Singer JD . (1994). Curr Biol 4(4): 301–314.

  3. Benkirane M, Neuveut C, Chun RF, Smith SM, Samuel CE, Gatignol A et al. (1997). EMBO J 16(3): 611–624.

  4. Bretscher A, Chambers D, Nguyen R, Reczek D . (2000). Annu Rev Cell Dev Biol 16: 113–143.

  5. Bretscher A, Edwards K, Fehon RG . (2002). Nat Rev Mol Cell Biol 3(8): 586–599.

  6. Daher A, Longuet M, Dorin D, Bois F, Segeral E, Bannwarth S et al. (2001). J Biol Chem 276(36): 33899–33905.

  7. Evans DG, Huson SM, Donnai D, Neary W, Blair V, Newton V et al. (1992a). J Med Genet 29(12): 847–852.

  8. Evans DG, Huson SM, Donnai D, Neary W, Blair V, Teare D et al. (1992b). J Med Genet 29(12): 841–846.

  9. Fisher HW, Yeh J . (1967). Science 155(762): 581–582.

  10. Gatignol A, Buckler C, Jeang KT . (1993). Mol Cell Biol 13(4): 2193–2202.

  11. Gautreau A, Manent J, Fievet B, Louvard D, Giovannini M, Arpin M . (2002). J Biol Chem 277(35): 31279–31282.

  12. Giovannini M, Robanus-Maandag E, van der Valk M, Niwa-Kawakita M, Abramowski V, Goutebroze L et al. (2000). Genes Dev 14(13): 1617–1630.

  13. Gusella JF, Ramesh V, MacCollin M, Jacoby LB . (1996). Curr Opin Genet Dev 6(1): 87–92.

  14. Gusella JF, Ramesh V, MacCollin M, Jacoby LB . (1999). Biochim Biophys Acta 1423(2): M29–36.

  15. Gutmann DH, Sherman L, Seftor L, Haipek C, Hoang Lu K, Hendrix M . (1999). Hum Mol Genet 8(2): 267–275.

  16. Kharrat A, Macias MJ, Gibson TJ, Nilges M, Pastore A . (1995). EMBO J 14(14): 3572–3584.

  17. Kim H, Kwak NJ, Lee JY, Choi BH, Lim Y, Ko YJ et al. (2004). J Biol Chem 279(9): 7812–7818.

  18. Kissil JL, Johnson KC, Eckman MS, Jacks T . (2002). J Biol Chem 277(12): 10394–10399.

  19. Kissil JL, Wilker EW, Johnson KC, Eckman MS, Yaffe MB, Jacks T . (2003). Mol Cell 12(4): 841–849.

  20. Lee JY, Kim H, Ryu CH, Kim JY, Choi BH, Lim Y et al. (2004). J Biol Chem 279(29): 30265–30273.

  21. Lee K, Fajardo MA, Braun RE . (1996). Mol Cell Biol 16(6): 3023–3034.

  22. Park H, Davies MV, Langland JO, Chang HW, Nam YS, Tartaglia J et al. (1994). Proc Natl Acad Sci USA 91(11): 4713–4717.

  23. Pearson MA, Reczek D, Bretscher A, Karplus PA . (2000). Cell 101(3): 259–270.

  24. Rong R, Surace EI, Haipek CA, Gutmann DH, Ye K . (2004). Oncogene 23: 8447–8454.

  25. Shaw RJ, Henry M, Solomon F, Jacks T . (1998a). Mol Biol Cell 9(2): 403–419.

  26. Shaw RJ, McClatchey AI, Jacks T . (1998b). J Biol Chem 273(13): 7757–7764.

  27. Shaw RJ, Paez JG, Curto M, Yaktine A, Pruitt WM, Saotome I et al. (2001). Dev Cell 1(1): 63–72.

  28. Sherman L, Xu HM, Geist RT, Saporito-Irwin S, Howells N, Ponta H et al. (1997). Oncogene 15(20): 2505–2509.

  29. St Johnston D, Brown NH, Gall JG, Jantsch M . (1992). Proc Natl Acad Sci USA 89(22): 10979–10983.

  30. Sun CX, Robb VA, Gutmann DH . (2002). J Cell Sci 115(Part 21): 3991–4000.

  31. Surace EI, Haipek CA, Gutmann DH . (2004). Oncogene 23(2): 580–587.

  32. Tsukita S, Yonemura S . (1997). Curr Opin Cell Biol 9(1): 70–75.

  33. Vijay-Kumar S, Bugg CE, Cook WJ . (1987). J Mol Biol 194(3): 531–544.

  34. Xiao GH, Beeser A, Chernoff J, Testa JR . (2002). J Biol Chem 277(2): 883–886.

  35. Zhong J, Peters AH, Lee K, Braun RE . (1999). Nat Genet 22(2): 171–174.

Download references

Acknowledgements

We thank Dr BH Choi for the invaluable advice and Dr CY Choi for the helpful comments and all the other members of our laboratory for their valuable assistance. This study was supported by a grant of the Korean Health 21 R&D Project, Ministry of Health Welfare, Republic of Korea (00-PJ3-PG6-GN02-0002, 01-PJ3-PG6-GN07-0004).

Author information

Correspondence to Y H Kim.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, J., Moon, H., Lee, W. et al. Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein. Oncogene 25, 1143–1152 (2006) doi:10.1038/sj.onc.1209150

Download citation

Keywords

  • Nf2
  • merlin
  • tarbp2
  • TRBP
  • ubiquitination
  • degradation

Further reading