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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
PMCID: PMC2810124
NIHMSID: NIHMS167932

Akt Phosphorylation of Merlin Enhances its Binding to Phosphatidylinositols and Inhibits Tumor Suppressive Activities of Merlin

Abstract

The NF2 tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin, which belongs to the band 4.1 family of cytoskeleton-associated proteins that link cell surface glycoproteins to actin cytoskeleton. Merlin suppresses PI 3-kinase/Akt signaling through directly binding and inhibiting PIKE-L ‘s stimulatory activity on PI 3-kinase. Akt feeds back and phosphorylates merlin and provokes its polyubiquitination and degradation. Here we show Akt phosphorylation and PI(3,4,5)P3 binding mediate the tumor suppressive activity of merlin. The extreme N-terminus of merlin directly interacts with phosphatidylinositols, for which the unfolded conformation is required. Moreover, Akt phosphorylation enhances merlin binding affinity to phosphatidylinositols and inhibits its pro-apoptotic actions. Further, Akt phosphorylation and phosphatidylinositols increase merlin binding to CD44. EGF treatment and Akt phosphorylation provoke merlin to aggregate in the ruffled plasma membrane and promote cell migration. Thus, these results suggest that PI 3-kinase signaling regulates merlin’s tumor suppressive activity via both Akt phosphorylation and phosphatidylinositol lipids binding to merlin.

Keywords: NF2, merlin, Akt, phosphorylation, phosphatidylinositols

Introduction

The NF2 tumor suppressor protein merlin is structurally related to the Protein 4.1 family of molecules and, specifically, a subgroup including ezrin, radixin and moesin (ERM proteins). Like the ERM proteins, merlin contains an N-terminal domain (NTD, residues 1–302), which is highly conserved among all members of the Protein 4.1 family and is thought to mediate interactions with the cytoplasmic tail of cell surface glycoproteins such as glycophorin C and CD44 (1). The second half of the molecule contains a predicted α-helical domain (residues 303–478) and a unique C-terminus (CTD, residues 479–595). ERM proteins link the actin cytoskeleton to cell surface glycoproteins. Merlin can associate with polymerized actin in vitro by virtue of an amino (N-) terminal actin binding domain including residues 178–367. Merlin actin binding is not affected by natural NF2 patient mutations or alternatively spliced isoforms (2). Phosphatidylinositol lipids have been implicated in ERM protein activation. For example, PI(4,5)P2 enhances the ability of full-length radixin, but not its N-terminal domain, to bind the cytoplasmic tail of CD44. However, the tail of CD44 does not bind (4,5)P2, suggestive of an activating effect on full-length radixin (3). Analysis of recombinant moesin T588D, which mimics phosphorylated moesin, suggests an auxiliary role for (4,5)P2 in unmasking the C-terminal F-actin binding site. Recent work using either unphosphorylated or T558 phosphorylated moesin purified from platelets revealed a dual requirement for T558 phosphorylation and the presence of a detergent and a phospholipid, such as (4,5)P2, for F-actin binding (4). In support of a two-step activation process in which (4,5)P2 renders moesin susceptible to C-terminal phosphorylation, transfection studies have suggested that enhanced phosphatidylinositol 4-phosphate 5-kinase activity results in enhanced phosphorylation of the C-terminal threonine (5).

Merlin is phosphorylated on both serine and threonine residues, and the phosphorylation status of merlin varies in response to growth conditions (6). PAK2 phosphorylates S518 and abolishes merlin N-term/C-term binding. PAK2 also impairs the ability of merlin to bind to two interacting proteins, CD44 and HRS, both critical for merlin growth suppression (7). In addition to phosphorylation of S518, PKA has recently been showed to phosphorylate S10 and affect actin cytoskeletion, mediating cell migration (8). Most Recently, we show that Akt phosphorylates merlin on residues T230 and S315, which abolishes merlin intramolecular NTD/CTD interaction and merlin binding to PIKE-L and other binding partners, leading to merlin degradation by polyubiquitination (9). Phosphorylation and (4,5)P2 binding coordinately mediate the activation of ERM proteins (5, 10). However, whether the dual regulatory machinery also controls merlin activity remains elusive. In this report, we show that the NTD of merlin directly associates with phosphatidylinositols, for which the “open” conformation is required. Moreover, Akt phosphorylation enhances the interaction between merlin and phosphatidylinositols. Akt phosphorylation blocks its pro-apoptotic activity. Finally, we show that Akt phosphorylation of merlin substantially inhibits its pro-apoptotic activity and blocks its tumor suppressive activity on cell migration.

Materials and Methods

Cells and reagents

Both 54 and 67, which are RT4-D6P2T schwannoma cells stably transfected with wild-type merlin, merlin T230DS315D and T230AS315A stably expressed cells were maintained in DMEM including 10% FBS and 100 units penicillin-streptomycin, 500 μg/ml G-418 and 1 μg/ml puromycin. L64P, S518A and S518D cells are patient-derived mutant (L64P) and S518 phosphorylation mutant stably transfected RT4-D6P2T cells. Merlin proteins were induced by 1 μg/ml doxycycline treatment and then incubated for 24 h. HCT116 and HCT116 PTEN−/− cells were maintained McCoy’s 5A in including 10% fetal bovine serum (FBS) and 100 units penicillin-streptomycin. All cells were maintained at 37°C with 5% CO2 atmosphere in a humidified incubator. Wortmannin and LY294002 and GST-HRP were from Sigma. Akt 1/2 inhibitor was from VWR (cat# 124018) and PAK inhibitor IPA-3 was from Sigma (cat# I2285). Phosphatidylinositol-conjugated beads, and various phosphatidylinositols diC16 were from Echelon, Inc. (Logan, Utah).

Cosedimentation of merlin recombinant proteins with Lipid Vesicles

The analysis of merlin–lipid interactions by cosedimentation of proteins with multilamellar liposomes was performed as described (11). Multilamellar liposomes were prepared from PC and PI(3,4,5)P3, (4,5)P2, PI(3,4)P2 and PI in a buffer containing 20 mM Hepes, pH 7.4, 0.2 mM EGTA. Proteins were subsequently incubated for 15 min at 25°C in the presence of 130 mM KCl and in the absence of liposomes, followed by a further incubation in the absence or presence of liposomes for 15 min. The mixtures were subsequently centrifuged for 30 min at 100,000 g, 4°C. The pellets were solubilized in 50–100 μl sample buffer. After SDS-PAGE, the amount of protein present in pellets and supernatants was quantified by scanning the bands of the Coomassie blue-stained gels. The amount of merlin sedimented in the absence of liposomes was subtracted from that sedimenting in the presence of lipid.

In vitro binding assay between GST-CD44-CTD and merlin in the presence of lipid micelles

The glutathione beads binding to GST-CD44 CTD or GST alone were pretreated with RIPA buffer (0.1% SDS, 0.5% deoxycholic acid, 1% NP40, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 20 min at room temperature. They were washed with 20 Vol of buffer containing 10 mM Hepes, pH 7.5, 1 mM MgCl2, 40 mM NaCl. After brief centrifugation, the beads were resuspended in 130 μl buffer containing 50 μg/ml phosphatidylserine (PS), and a variety of 5μg/ml phosphatidylinositols. The mixture was sonicated and the resulting vesicles were mixed with the glutathione beads and 2.6 μg purified merlin. The reaction mixture was incubated at room temperature with constant rotation, followed by immunoblotting.

Wound healing assays

RT4 schwannoma cell stably transfected with inducible T230DS315D and T230AS315A mutant constructs. Three independent clones for each mutant were employed for the migration assay. The cells were induced by doxycycline and split into 10 cm dishes and incubated until 90–100% confluent. After dishes were scratched by blue tip respectively (0 h), cells were incubated for different time courses. The pictures were taken at 0, 6, 12 and 18 h, respectively. Phase contrast images were taken by OLYMPUS IX71. Measurement of the would healing gap distance was performed using the computer program image J. Results were expressed as mean ± S.E.M. P ≤ 0.05 was considered significant (Student’s t-test).

Results

The N-terminus of merlin binds phosphatidylinositols

To examine whether phosphatidylinositol lipids are involved in binding to merlin, we performed in vitro binding assay. PI(3,4,5)P3 selectively interacted with both NTD and full-length merlin but not CTD or negative control GST. By contrast, (4,5)P2 weakly associated with both NTD and CTD but not full-length merlin (Figure 1A). Truncation assay demonstrated the extreme N-terminal 1-82 and 1-132 fragments associated with PI(3,4,5)P3; in contrast, all the other truncates lacking the N-terminal 1-132 region failed to bind PI(3,4,5)P3 (Figure 1B, top panel).

Figure 1
N-terminus of merlin binds to phosphatidylinositol lipids

As a measure for bilayer association, cosedimentation of proteins with liposomes containing 20% a variety of phosphatidylinositol lipids and 80% PC was analyzed. Control values of protein sedimenting in the absence of lipid were subtracted. Lipid interactions were measured under physiological ionic strength conditions as described in the Experimental Section. GST-merlin NTD strongly cosedimented with PI(3,4,5)P3 liposomes (25 ± 5%). It revealed comparable affinity to both (4,5)P2 and PI(3,4)P2 with 18 ± 4% and 20 ± 3%, respectively. However, its affinity to PI(3)P was substantially decreased to 6 ± 2%. By contrast, GST-merlin CTD almost did not bind to 3′ phosphorylated phosphatidylinositol lipids, but it potently cosedimented with (4,5)P2 (26 ± 6%). GST alone revealed negligible cosedimentation activity to all liposomes (Figure 1C). Therefore, these findings support merlin NTD possesses much stronger PI(3,4,5)P3 binding affinity than its CTD, which, instead, reveals tighter affinity to (4,5)P2.

The unfolded conformation is required for the association between merlin and phosphatidylinositols

The N-terminus of ezrin reveals more potent binding activity to (4,5)P2 than full-length ezrin in the presence of physiological ionic condition, suggesting the folding conformation mediates its binding affinity to phosphatidylinositol lipids (11). To explore whether merlin folding conformation mediates its binding effect to phosphatidylinositol lipids, we employed 3 forms of well-characterized merlin mutants: L64P, S518D and S518A. The patient-derived L64P mutant displays an “open” inactive conformation, so does S518D. However, S518A possesses a folded active conformation. We examined the binding activity in RT4-D6P2T schwannoma cell lines stably transfected with inducible wild-type and mutated merlins. The parental RT4-D6P2T schwannoma cell line expresses negligible amount of merlin (7). Interestingly, no any binding activity was observed with S518A. Surprisingly, S518D robustly associated with phosphatidylinositol lipids (Figure 2, left 2nd panel). Both wild-type and L64P merlin interacted with (4,5)P2, PI(3,4)P2, and PI(3,4, 5)P3. However, wild-type merlin in 54 cells displayed the strongest binding activity to (4,5)P2. By contrast, L64P revealed the most prominent interaction with PI(3,4,5)P3. These observations strongly support the notion that PI(3,4,5)P3 or PI(3,4)P2 robustly bind to “open” merlin, while (4,5)P2 preferentially interacts with native merlin.

Figure 2
The unfolded conformation is required for the association between merlin and phosphatidylinositols

Akt phosphorylation of merlin increases its affinity to phosphatidylinositols

To explore whether Akt phosphorylation of merlin mediates its binding activity to phosphatidylinositols, we conducted an in vitro binding assay. Under control condition, the NTD strongly bound to PI(3,4,5)P3, whereas full-length merlin barely associated with PI(3,4,5)P3. In the presence of active Akt, both the NTD and full-length merlin displayed substantially elevated binding affinities to PI(3,4,5)P3 (Figure 3A). To examine the effect of Akt phosphorylation on merlin’s binding behaviors to phosphatidylinositols in intact cells, we infected 54 schwannoma cells with adenovirus expressing plasma membrane localized active myristoylated Akt (Akt-myr) and shRNA of Akt1, respectively. Under control condition (Figure 3B, bottom left panel), wild-type merlin exhibited the binding effect to various phosphatidylinositols with the strongest activity to (4,5)P2, a phenomena similar to what was observed in Figure 2A; However, when Akt1 was depleted, merlin lost its binding activity to both PI(3,4)P2 and PI(3,4,5)P3, but it still retained the affinity to (4,5)P2, indicating that Akt phosphorylation is not required for merlin to bind (4,5)P2 (Figure 3B, middle left panel). In the presence of active Akt, merlin displayed obviously increased binding effect to both PI(3,4,5)P3 and PI(3,4)P2, while merlin remained comparable affinity to (4,5)P2 as the control condition (Figure 3B, top left panel), supporting that Akt phosphorylation provokes merlin binding to 3′ phosphorylated phosphatidylinositols. Both GSK3β and merlin S315 phosphorylation tightly correlated with Akt1 expression and activation profiles (Figure 3B, right panels). Compared to wild-type NTD, purified recombinant GST-merlin 1-332 (T230DS315D) revealed much stronger binding activity to PI(3,4,5)P3 (Figure 3C). We have made the similar observation with mammalian cells transfected with GFP-merlin. The phosphorylation mimetic merlin (T230DS315D) displayed markedly higher affinity to both (4,5)P2 and PI(3,4,5)P3 than wild-type merlin. By contrast, unphosphorylated merlin (T230AS315A) failed to bind either lipid (Figure 3D), similar to S518A. These data further support that Akt phosphorylation is indispensable for merlin to interact with PI(3,4,5)P3. Thus, Akt plays a critical role in dictating merlin’s binding to phosphatidylinositols.

Figure 3
Phosphorylation of merlin by Akt enhances its lipid binding activity

PI(3,4,5)P3 lipid enhances merlin binding affinity to CD44

To explore whether phosphatidylinositols regulate the interaction between merlin and CD44, we conducted an in vitro binding assay in the presence of PS or PIPx/PS micelles. Under control condition (50 μg/ml PS), GST-CD44 CTD but not GST selectively interacted with merlin (Figure 4A, lane 9 and 10). PI did not significantly elevated the interaction between merlin and CD44-CTD, while PI(3,4)P2 and PI (4,5)P2 notably augmented the association (Figure 4A). Remarkably, PI(3,4,5)P3 elicited the strongest binding affinity between CD44-CTD and merlin (lane 8), indicating that PI 3-kinase might upregulate merlin/CD44 association.

Figure 4
Akt phosphorylation and phosphatidylinositols enhance merlin binding to CD44

Inactivating mutations of PTEN tumor suppressor gene are found in a wide range of human cancers. PTEN is a lipid phosphatase that converts the second messenger PI(3,4,5)P3 to PI(3,4)P2 (12). To further test whether PI(3,4,5)P3 mediates the interaction between CD44 and merlin in intact cells, we employed PTEN knockout HCT116 colon cell line, a previously described isogenic set of HCT116 cells in which PTEN genes had been deleted (13). We cultured the wild-type and PTEN-deleted cells at high density (100% confluence) and low density (50% confluence) and immunoprecipitated merlin. CD44/merlin association was substantially enhanced at low density compared to high density in wild-type HCT116 cells. Interestingly, significantly more CD44 was coprecipitated with merlin in highly confluent PTEN-null HCT116 cells than low density cells (Figure 4B, left panels). Akt phosphorylation was augmented in low density HCT116 cells compared to high density cells. As expected, Akt was highly phosphorylated in PTEN-null cells no matter whether the cells were confluent or not. Accordingly, merlin S315 phosphorylation tightly correlated with Akt activation pattern (Figure 4B, right panels). These data suggest that Akt phosphorylation and PI(3,4,5)P3 lipid regulate the association between CD44 and merlin.

Since PTEN antagonizes PI 3-kinase signaling by hydrolyzing PI(3,4,5)P3 back into (4,5)P2, we wondered whether the elevated concentrations of PI(3,4,5)P3 and hyperactive Akt in PTEN knockout cells would affect merlin subcellular distribution. In wild-type HCT116 cells, endogenous merlin mainly localized in the cytoplasm and the cell-cell contact region. EGF treatment relocalized merlin to the whole cytoplasm and the concentration in the cell-cell contact was significantly decreased (Figure 4C). This effect was substantially blocked by PI 3-kinase inhibitor Wortmannin pretreatment (data not shown), indicating that Akt phosphorylation and PI(3,4,5)P3 binding might contribute to the cytoplasm redistribution of merlin. However, in HCT116 PTEN-null cells, endogenous merlin distributed in the whole cells, resembling the patterns observed in EGF-treated wild-type cells. It was worth noting that merlin was highly enriched in the microvilli and small protrusions (Figure 4D, upper panel, white arrows). By contrast, the actin filament was enriched in the cell boundaries. Strikingly, EGF provoked tremendous number of small protrusions on the plasma membrane, where merlin and F-actin were aggregated and colocalized (Figure 4D, lower panels, white arrows). These results support that PI(3,4,5)P3 binding and Akt phosphorylation synergistically facilitate merlin relocation and regulate its cellular effect on F-actin organization. Immunostaining with anti-phospho-S315 antibody revealed the similar results (data not shown).

Akt phosphorylation of merlin inhibits its pro-apoptotic activity

Transduction of merlin into human schwannoma cells decreases cell proliferation by inducing apoptosis (14). In Drosophila, Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs (15). Thus, merlin somehow implicates in triggering apoptosis. To explore whether Akt phosphorylation of merlin mediates its pro-apoptotic effect, we cotransfected a variety of GFP-tagged merlin constructs into HEK293 cells with constitutively active Akt (CA) or kinase-dead Akt (KD) and monitored cell apoptosis. Compared to control, overexpression of wild-type merlin provoked significant apoptosis, which was further elevated by unphosphorylate mutant (T230AS315A). By contrast, apoptosis was substantially decreased with the mutant (T230DS315D). Cotransfection of active Akt with wild-type merlin markedly diminished its pro-apoptotic actions. The apoptosis activity was recovered when merlin was cotransfected with Akt-KD (Figure 5A). We also monitored cell death with Trypan blue assay (Figure 5B). PARP cleavage and caspase-3 activation were in alignment with the quantitative apoptotic results, (Figure 5C), underscoring that merlin phosphorylation by Akt is required to block its pro-apoptotic action.

Figure 5
Akt phosphorylation of merlin blocks its pro-apoptotic activity

The extreme N-terminus of merlin is implicated in binding phosphatidylinositol lipids. It remains unknown whether this interaction is implicated in merlin’s pro-apoptotic action. Wild-type merlin and phosphorylated/mutated merlin like S518A and S518D display different binding affinities to PI(3,4,5)P3 and two different PIP2. To address whether lipids binding by merlin plays any role in mediating its pro-apoptotic action, we monitored cell survival activity. When merlin is in “open” conformation like S518D, the pro-apoptotic action of merlin was substantially diminished as in T230DS315D transfected cells; in contrast, when merlin is in “close” conformation like S518A, it displayed more potent apoptotic action than wild-type merlin. Interestingly, truncation of 1-133 in merlin had no effect on merlin’s pro-apoptotic action (Figure 5D). PARP cleavage and caspase-3 activation correlated with apoptosis results (data not shown). Hence, PIP2 or PI(3,4,5)P3 binding is not essential for merlin’s pro-apoptotic activity.

Merlin phosphorylation by Akt affects its subcellular distribution and cell migration

To explore whether Akt phosphorylation alone regulates merlin subcellular residency, we transfected HEK293 cells with Myc-merlin constructs. T230DS315D mainly accumulated in the small protrusion structures on the ruffled membrane, long cell extension and in the cytoplasm in most of the transfected cells. Actin filament strongly colocalized with merlin T230DS315D in the long cell extension (white arrow). In contrast, merlin mutant (T230AS315A) predominantly resided on the smooth plasma membrane without the aggregation in the protrusion (Figure 6A). EGF also provoked Myc-merlin accumulation in the ruffle membrane, which was block by Wortmannin (Figure 6B, top panels), underscoring that Akt phosphorylation of merlin dictates its subcellular distribution. Immunostaining with anti-phospho-merlin S315 confirmed the observation (Figure 6B, middle panel). To further test the notion that Akt phosphorylation regulates merlin aggregation in the ruffled plasma membrane, we cotransfected GFP-merlin into HEK293 cells with constitutive active Akt-CA and Akt-KD. GFP-merlin was strongly phosphorylated by Akt-CA but not KD, and accumulated on the disheveled plasma membrane (Figure 6C). We made the similar observation in GFP-merlin and RFP-Akt cotransfected cells (Supplemental Figure 1). Therefore, Akt phosphorylation plays an essential role in regulating merlin subcellular distribution.

Figure 6
Merlin phosphorylation by Akt regulates its subcellular distribution and cell migration

To explore whether Akt phosphorylation regulates merlin’s effect in cell motility, we conducted a wound-healing assay with RT4 schwannoma cells. The stable clones were transfected with inducible T230DS315D and T230AS315A mutants. Both cell lines transfected with wild-type merlin (54 and 67) loosely filled the wound. However, T230DS315D cells migrated faster and completely sealed the gap. Nevertheless, T230AS315A cells moved much slower than wild-type controls, and only about half of the gap was filled (Figure 6D, left panels). These data support that Akt phosphorylation of merlin plays a critical role in mediating its effect on cell migration, fitting with our previous observation in Metrigel Boyden chamber (9).

Both Akt and PAK implicate in phosphorylating merlin and regulate its binding to phosphatidylinositol lipids, but it remains unknown which signaling is essential for dictating merlin’s effect in cell migration. To quantitatively analyze this effect, we conducted a time course cell migration assay. In Supplemental Figure 2, we show that merlin S518D cell migrated much faster than 54 cells. Blocking Akt by Akt 1/2 or Wortmannin inhibitor largely diminished both wild-type 54 and S518D cell migration; by contrast, inhibiting PAK3 markedly decreased wild-type but not S518D cell migration. This finding suggests that PI3K/Akt signaling is critical for schwannoma cell migration, for which merlin phosphorylation by Akt might be critical. On the other hand, S518 phosphorylation by PAK is also implicated in this event. Compared to merlin lacking V1 schwannoma control cells, induction of wild-type merlin markedly decreased cell migration in 54 cells in a time-dependent manner. Notably, induction of S518A further inhibited cell migration. As expected, phosphorylation of S518 significantly abolished merlin’s inhibitory effect in cell migration as the migratory speed was substantially increased in S518D cells (Figure 6D, right panel).

Discussion

In this report, we show that merlin directly binds phosphatidylinositols, and this action is mediated by merlin folding conformation and Akt phosphorylation. Wild-type merlin reveals a stronger affinity to (4,5)P2 than PI(3,4,5)P3, whereas the patient-derived L64P, which possesses an unfolded conformation, exhibits a more potent affinity to PI(3,4,5)P3 than (4,5)P2. This observation was further supported by the finding with S518D, which also displays an open structure. By contrast, the folded S518A mutant fails to bind any of the phosphatidylinositols, underscoring that the open inactive conformation is required for merlin to bind PI(3,4,5)P3. Moreover, we found that the interaction between merlin and PI(3,4,5)P3 was evidently enhanced by Akt phosphorylation. Nevertheless, Akt phosphorylation is dispensable for merlin to associate with (4,5)P2. Although binding to phosphatidylinositol lipids require merlin to maintain an inactive status, the functional relevance of this interaction remains obscure. Truncation of 1-133 in the N-terminus, which is implicated in binding to PI(3,4,5)P3, does not alter merlin’s pro-apoptotic action (Figure 5D), suggesting that phosphatidylinositol binding to merlin is dispensable for this effect.

Previously, it has been reported that hypophosphorylated merlin, but not ezrin or moesin, binds the cytoplasmic tail of CD44 at high cell density. At low cell density, ezrin, moesin, and the phosphorylated form of merlin are associated with CD44, and this CD44/merlin association is likely to be indirect through ERM proteins (16). Consistently, we observed the increased interaction between CD44 and merlin in low density of HCT116 cells compared to high density cells. Strikingly, the strongest CD44/merlin association occurred in high density in PTEN–null cells, which was slightly decreased when the cell density was reduced (Figure 5B). It remains unclear why in PTEN-null cells, the association between CD44 and merlin was attenuated in low density compared to high density. Although PI 3-kinase/Akt signaling cascade is markedly increased, when PTEN is depleted, other cell signalings that are dictated by cell/cell contact remain intact in PTEN-null cells. For instance, contact inhibition can be triggered by the addition of cell membrane preparations to dividing cells in vitro (17, 18). Adhesion molecules, including particular cadherins and integrins, induce cell cycle arrest upon contact with specific components of the extracellular matrix or with neighboring cells (19, 20). Presumably, these mechanisms operate in concert or in hierarchy to mediate cellular responses to contact with extracellular matrix and with other cells in addition to PI3-kinase/Akt. This notion is indirectly supported by the cell migration observation that PI3K inhibitor wortmannin and Akt inhibitor Akt 1/2 slightly but significantly decreased HCT116-PTEN cell migration; by contrast, PAK inhibitor exhibited negligible effect (Supplemental Table 1). The weak inhibitory effect by Akt inhibitors might be due to the hyperactivity of Akt in PTEN null cells.

To address the functional consequences of the enhanced binding affinity by Akt-phosphorylated merlin to CD44, we knocked down CD44 with its siRNA in T230DS315D stably transfected cells, but we failed to observe any significant effect on cell migration as compared to control siRNA (data not shown). This finding indicates that the increased interaction between CD44 and Akt phosphorylated-merlin is not implicated in cell migration. This reault is consistent with the previous finding that CD44 is not required for hyaluronan-induced vascular smooth muscle cell migration, which is dependent on PI3K-mediated Rac activation (21).

(4,5)P2 strongly binds full-length wild-type merlin from schwannoma 54 cells (Figure 3), whereas it fails to interact with the purified recombinant full-length merlin (Figure 1A). This discrepancy might result from posttranslational modification of merlin in 54 cells. For example, phosphorylation of merlin might alter its folding conformation, unveiling the binding motif for (4,5)P2 lipid. Unlike other ERM proteins, both the NTD and CTD of merlin bind to (4,5)P2, however, PI(3,4,5)P3 selectively associates with the NTD of merlin. Under subconfluent condition, a portion of merlin might be phosphorylated by PAK on S518 in the CTD, leading to an unfolded conformation of merlin. (4,5)P2 binds merlin and docks it to the plasma membrane. When growth factors are introduced, Akt is immediately activated and translocated to the plasma membrane, where its PH domain binds to newly generated PI(3,4,5)P3 by PI 3-kinase, the active Akt then attacks the NTD of merlin and phosphorylates it, resulting in a fully opened conformation. PI(3,4,5)P3 subsequently tightly binds to the NTD, which in turn further enhances its phosphorylation by Akt and many other kinases including PKA and PAK etc, leading to the full inactivation of merlin.

Merlin directly associates with PIKE-L GTPase that binds PI 3-kinase and enhances its kinase activity (22, 23). Merlin exerts its growth inhibitory effect, at least in part, through inhibiting PI 3-kinase/Akt signaling. This suppressive activity was through blocking PIKE-L’s stimulatory effect on PI 3-kinase (23). Recently, we show that Akt feeds back and robustly phosphorylates merlin on both T230 and S315 residues and unfolds merlin, leading to its polyubiquitination and degradation. Although either Akt or PAK phosphorylating merlin results in its binding to phosphatidylinositol lipids, merlin S518 phosphorylation by PAK can not provoke merlin polyubiquitination and degradation (9). This finding suggests the unique aspect of the regulatory role of Akt in mediating merlin tumor suppressor. Nonetheless, it remains unclear whether phosphatidylinositol binding plays any role in triggering merlin degradation or plasma membrane residency. Here, we provide biochemical evidence revealing that PI(3,4,5)P3, an essential second messenger generated by active PI 3-kinase, strongly binds Akt phosphorylated merlin. Conceivably, this phosphatidylinositol lipid binding action might coordinately inactivate merlin with Akt. The dual regulation of merlin by phosphatidylinositol binding and Akt phosphorylation provides a novel mechanism explaining how the oncogenic PI 3-kinase signaling crosstalk with the tumor suppressor merlin.

Supplementary Material

supplemental figures

Acknowledgments

This work is supported by a Research-Initiated Award from Department of Defense (W81XWH0610286) and RO1 (CA117872) from NIH to K. Ye.

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