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Originally published In Press as doi:10.1074/jbc.M313916200 on February 23, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18559-18566, April 30, 2004
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Cyclic AMP-dependent Protein Kinase Phosphorylates Merlin at Serine 518 Independently of p21-activated Kinase and Promotes Merlin-Ezrin Heterodimerization*

Kaija Alfthan{ddagger}§, Leena Heiska{ddagger}, Mikaela Grönholm{ddagger}, G. Herma Renkema¶, and Olli Carpén{ddagger}||

From the {ddagger}Biomedicum Helsinki, Department of Anatomy and Pathology, Neuroscience Program, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland and Institute of Medical Technology, University of Tampere and Tampere University Hospital, FIN-33014 Tampere, Finland

Received for publication, December 19, 2003 , and in revised form, February 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrin-radixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKA-induced phosphorylation. In vitro [{gamma}-33P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growth-suppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inactivation of the neurofibromatosis 2 (NF2) tumor suppressor gene leads to development of multiple benign tumors of the nervous system, in particular schwannomas and meningiomas. The NF2 gene encodes for merlin (schwannomin), which exists as two major isoforms. The 595-residue isoform 1 and the 590-residue isoform 2 result from alternative splicing of the last two exons and differ in their C-terminal sequences and, apparently, in their tumor suppressor activity. It has been suggested that isoform 1 is the tumor-suppressive form of merlin (1). The overall structure of merlin is similar to that of ERM1 (ezrin-radixin-moesin) proteins (2, 3). They are members of the band 4.1 superfamily based on the presence of a shared FERM domain in the N terminus. In cells, ERM proteins provide a regulated link between plasma membrane proteins and the cortical cytoskeleton, and they also participate in signal transduction pathways. Like the ERM proteins, merlin can be divided into three apparent structural domains: the globular N-terminal FERM domain, an extended {alpha}-helical region, and a short C-terminal domain. Both inter- and intramolecular associations can regulate the functions of merlin and its interactions with other proteins (4–6). ERM proteins are negatively regulated by an intramolecular association between the N- and C-terminal domains, and changes in the conformation are needed to "open" or "activate" the molecules for interaction with other proteins. Recent data provide evidence that both phosphorylation of a conserved threonine residue (Thr-567 in ezrin, Thr-564 in radixin, Thr-558 in moesin) in the C terminus and binding of phospholipids regulate the activity of the ERM proteins (7). Rho kinase (8) and two different isoforms of protein kinase C (9, 10) can phosphorylate this conserved threonine residue.

Merlin has been shown to be phosphorylated on both serine and threonine residues, and the phosphorylation status of merlin varies in response to growth conditions (11). At low cell density merlin is phosphorylated, whereas high cell density, serum starvation, or loss of adhesion results in dephosphorylation of merlin. Furthermore, phosphorylation of merlin is induced by activated forms of Rac and Cdc42 but not by activated Rho. These findings link merlin to Rac/Cdc42-dependent signaling (12). The site for the Rac-induced phosphorylation was determined to be the serine 518 residue in the C terminus of merlin (12). Serine 518 is phosphorylated by p21-activated kinases (PAKs) (13, 14), which are common downstream targets of both Rac and Cdc42. Moreover, a recent study shows that merlin interacts directly with the Cdc42/Rac binding domain of PAK1 and inhibits PAK1 activity (15). The data suggest that merlin both is regulated by Rac/Cdc42 signaling pathway and can serve as a negative regulator of this pathway.

Cyclic AMP (cAMP) regulates a number of key cellular processes such as cell growth and differentiation, gene transcription, and ion channel conductivity. This second messenger mediates most of its cellular effects by activating the cAMP-dependent protein kinase A (PKA). In neural systems cAMP/PKA signaling represents an important pathway for gene expression, synaptic plasticity, learning, and memory (16, 17). In Schwann cells activation of the cAMP/PKA pathway promotes cell growth and cell cycle progression (18), and cAMP/PKA is also required for myelin formation (19). PKA has a number of physiological substrates, and, recently, ezrin also has been reported to be phosphorylated by PKA (20). Because PKA is involved in a number of parallel signaling cascades, proper localization and timing are prerequisites for an efficient substrate selection and catalytic activation. The intracellular targeting and compartmentalization of PKA is achieved through association with protein kinase A-anchoring proteins (AKAPs) (21–23). We have recently shown that merlin binds to a regulatory subunit (RI{beta}) of PKA and may function as an AKAP for RI{beta}-containing PKA both in the central nervous system and in cultured neuronal cells (24). Here, we provide evidence that merlin is also a substrate for PKA in vitro and in vivo. The site of PKA-induced phosphorylation was determined to be serine at position 518. Furthermore, our results show that phosphorylation of the serine 518 of merlin promotes interaction between merlin and ezrin both in vitro and in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The human merlin isoform 1 cDNA in pcDNA3 vector (Invitrogen) has been described earlier (25). The plasmid encoding the catalytic subunit (C) of human PKA (PKAc) (26) was kindly provided by Dr. Kjetil Taskén (University of Oslo, Norway). The constitutively active PAK2 construct containing the T402E mutation was cloned into the pEBB-ME vector, which is the pEF-BOS vector (27) containing an extended polylinker and a C-terminal Myc-His6-HA tag. A PAK2 N-terminal domain (aa 1–248) construct with a CRIB-motif mutation (H82L,H85L) was cloned in the same pEBB vector with a C-terminal Myc-His6 tag. The recombinant GST-merlin N terminus (aa 1–314), {alpha}-helical domain (aa 314–477), and C terminus (aa 492–595) were obtained by PCR amplification and subcloned into the EcoRI site of the pGEX4TI vector (Amersham Biosciences). T576A and S518A mutations of merlin in pcDNA3 and the GST fusion proteins in pGEX4TI were made by site-directed mutagenesis using the QuikChange Kit (Stratagene, La Jolla, CA). The recombinant GST-ezrin C terminus (aa 477–585) has been described (28), and the plasmid encoding the vesicular stomatitis glycoprotein (VSVG)-tagged N-domain of ezrin (aa 1–309) (29) was a kind gift of Dr. Monique Arpin (Institut Curie, Paris, France).

Antibodies—KF10 mAb, kindly provided by Dr. E. Zwarthoff (Erasmus University, Rotterdam, the Netherlands) (30) and sc-331 rabbit antiserum (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used to detect merlin. HM2175 antiserum (13), kindly provided by Dr. J. Kissil (Massachusetts Institute of Technology, Cambridge, MA), was used to detect merlin phosphorylated at serine 518. PKAc mAb (BD Biosciences) was used to detect the catalytic subunit of PKA, and Myc mAb (clone 9E10, Covance Research Products, Inc., Princeton, NJ) was used to recognize both the constitutively active PAK2 (T402E) and the N-terminal domain of PAK2 (aa 1–248/H82L,H85L). 3C12 mAb (31) and Ez9 antiserum were used to detect ezrin. The Ez9 antibody was raised in rabbits immunized with full-length ezrin, produced as a GST fusion in Escherichia coli, followed by cleavage of the GST part by thrombin (Amersham Biosciences). VSVG mAb (clone P5D4, Roche Diagnostics) was used to recognize the N-terminal domain of ezrin, and X63 mAb (ATCC, Manassas, VA) was used as a control.

Transfections and Immunoblotting—HEK293 cells grown in RPMI 1640 medium with 10% fetal calf serum were transfected using Fu-GENE transfection reagent (Hoffmann-La Roche). After 48 h the cells were rinsed with phosphate-buffered saline and lysed in 400 µl of lysis buffer 1 (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 10 mM NaPPi, 1.5 µM okadaic acid, 1 mM sodium orthovanadate, and protease inhibitors). For H89 treatment, cells were incubated with 20 µM H89 (Sigma) for 5 h before lysis. Lysed cells were scraped from the plates and centrifuged at 20,000 x g for 30 min at 4 °C. Total protein concentration of the supernatants was determined by Bio-Rad Protein Assay (Bio-Rad). Equal amounts of proteins were run in 8% SDS-PAGE, transferred to nitrocellulose filters, and analyzed by immunoblotting. Bound proteins were visualized by enhanced chemiluminescence (ECL).

Immunoprecipitation and Phosphatase Treatment—For immunoprecipitation of merlin, HEK293 cells cotransfected with merlin and PKAc were lysed, the lysate was centrifuged as described above, and the supernatant was incubated with merlin sc-331 antiserum overnight at 4 °C. Antibody-bound merlin was precipitated with protein G-Sepharose beads (Amersham Biosciences) for 4 h at 4 °C. Beads were washed three times with 400 µl of phosphate-buffered saline and two times with 400 µl of calf intestinal phosphatase (CIP) buffer (50 mM Tris-HCl, pH 7.9, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol) and resuspended in 80 µl of the CIP buffer. The immunoprecipitate was divided into two equal aliquots (40 µl), one of which was incubated with buffer alone and the other with 5 units of CIP (Finnzymes Oy, Espoo, Finland) for 30 min at 37 °C. The reaction was stopped by adding 10 µl of 5x Laemmli reducing buffer, and 15 µl of each sample was analyzed by immunoblotting.

In Vitro Phosphorylation of Merlin by PKA—GST-fusion proteins were expressed in E. coli and purified according the protocol provided by the manufacturer (Amersham Biosciences). 15 µl of glutathione-Sepharose beads carrying ~4 µg of fusion protein, as judged by Coomassie staining of serially diluted beads, were washed twice in PKA buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 7.4). The phosphorylation reaction was carried out in a 30-µl buffer volume including 10 µCi of [{gamma}-33P]ATP (PerkinElmer Life Sciences) and purified bovine catalytic subunit of PKA (Sigma-Aldrich) for 30 min at 30 °C. The reaction was stopped by washing the beads quickly in ice-cold PKA buffer and subsequently adding 30 µl of Laemmli reducing buffer. 15 µl of the boiled sample was resolved by 10% or 12% SDS-PAGE. The gel was fixed and stained by Coomassie Blue followed by drying and autoradiography.

Stimulation of Endogenous PKA in Attached and Nonattached HEK293 Cells—HEK293 cells were transfected with wild-type merlin in pcDNA3 vector alone or with the PAK2 construct (aa 1–248/H82L,H85L) in pEBB vector. After 24–29 h the cells were first rinsed with serum-free RPM1–1640 and then grown on culture plates overnight in serum-free medium. After serum starvation, forskolin (Sigma) and 3-isobutyl-1-methylxanthine (IBMX; Sigma) were added to the cells (at 25 and 50 µM, respectively) without or with 20 µM H89 (Sigma), and cells were incubated on the plates for different time periods. Whole cell lysates, prepared by adding 500 µl of 1x Laemmli reducing sample buffer on the plates, were used for immunoblotting.

To stimulate endogenous PKA in nonattached cells, merlin-transfected HEK293 cells were serum-starved and grown on culture plates as described above. After overnight serum starvation, cells were rinsed in phosphate-buffered saline, detached with trypsin, resuspended into serum-free medium, and collected in a sterile tube. After centrifugation for 5 min at 500 x g, cells were washed twice with serum-free RPMI 1640, resuspended into the same medium, and incubated in tubes with 25 µM forskolin alone or together with 20 µM H89 with gentle rotation for the indicated periods of time. Whole cell lysates of 300 µl were prepared for immunoblotting.

In Vitro Interaction between Merlin and Ezrin—Three aliquots of GST-merlin C (aa 492–595) coupled to glutathione-Sepharose beads (5 µg/aliquot) were washed twice in PKA buffer containing phosphatase inhibitors (50 mM NaF and 10 mM NaPPi). Two samples were phosphorylated in vitro in the presence of 200 µM ATP and purified bovine catalytic subunit of PKA for 60 min at 30 °C, and the third sample was incubated only with ATP. The beads were washed once in ice-cold PKA buffer and three times in CIP buffer. One of the phosphorylated samples was treated further with 10 units of CIP for 30 min at 37 °C, whereas the other two samples were incubated in CIP buffer only. Beads were washed once in ice-cold CIP buffer and twice in binding buffer (50 mM HEPES, pH 7.4, 150 mM NaCl). 50 mM NaF and 10 mM NaPPi were included in all steps (except CIP treatment) preceding the washes of the sample to be dephosphorylated. Beads carrying 5 µg of GST protein or GST-ezrin C (aa 477–585) fusion protein were used as a negative and a positive control for the binding (not shown).

HEK293 cells transfected with VSVG-tagged N-terminal ezrin construct were lysed in 750 µl of lysis buffer 2 (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, and protease inhibitors) and centrifuged at 15 000 x g for 10 min at 4 °C. 400 µl of the supernatant diluted 1:1 to the binding buffer was added to each bead aliquot, and the mixture was incubated for 2 h at 25 °C. Beads were washed twice in binding buffer and boiled in 30 µl of Laemmli reducing buffer. 10 µl of each sample was run on 12% SDS-PAGE followed by immunoblotting with anti-VSVG mAb. Another identical immunoblot of the samples was probed with antiserum HM2175 that has been raised against phosphoserine 518 of merlin (13). The blot was further stripped and reprobed with KF10 mAb to verify equal amounts of GST-merlin on the beads.

Coimmunoprecipitation of Merlin and Ezrin—HEK293 cells expressing endogenous ezrin were transfected with wild-type merlin in pcDNA3 vector. Subconfluent cells were lysed in 500 µl of lysis buffer 1, whereas confluent cells were lysed in the same buffer without phosphatase inhibitors. Lysates were centrifuged at 15.000 x g for 1 h at 4 °C. The supernatant was incubated with KF10 mAb, 3C12 mAb, or X63 mAb, together with protein G-Sepharose beads for 4 h at 4 °C. Immunoprecipitates were washed with lysis buffer 1-0.1% Nonidet P-40, and bound proteins were eluted from the beads by boiling in Laemmli nonreducing sample buffer and analyzed by immunoblotting. To study the role of phosphorylation of serine 518 in heterodimerization, subconfluent cells were transfected with wild-type or S518A mutant merlin. Cells were lysed as described above, ezrin was immunoprecipitated, and coprecipitating merlin was analyzed by immunoblotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vivo Phosphorylation of Merlin by PKA—We previously characterized merlin as an AKAP for the RI{beta} subunit containing PKA (24). In this work we studied whether merlin would also serve as a substrate for PKA. To make this assessment, HEK293 cells were transiently transfected with merlin alone or together with human PKAc, and after 48 h, cell lysates were analyzed by immunoblotting. Merlin migrates mainly as a doublet in HEK293 cells, but cotransfection of PKAc increased the amount of the slower migrating merlin species and induced the appearance of a third, even more slowly migrating band (Fig. 1A). To confirm that these shifts in migration were mediated by PKA, cells cotransfected with merlin and PKAc were treated with H89 before lysis of the cells. H89 is a competitive inhibitor of PKA that binds to the catalytic subunit of PKA (32) and is a selective although not a specific PKA inhibitor (33). As shown in Fig. 1A, treatment of HEK293 cells with H89 resulted in a shift toward faster migrating merlin forms, suggesting that the change in the electrophoretic mobility is PKA-dependent. Interestingly, the band pattern observed here resembled that obtained earlier by Rac/PAK-induced phosphorylation of merlin, where the three bands represent differentially phosphorylated forms of merlin (12–14).



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  		FIG. 1.
PKA catalyzes phosphorylation of merlin in vivo. A, immunoblot analysis of lysates from HEK293 cells transfected with either merlin alone or with merlin and PKAc. Cells expressing only merlin exhibit a doublet band, whereas coexpression of merlin and PKAc results in the appearance of an additional, third, more slowly migrating band. Treatment of the cells with the PKA inhibitor H89 shifts the bands toward the faster migrating form. Merlin was detected with KF10 mAb, and transfection of PKA was confirmed with PKAc mAb. B, lysate from HEK293 cells cotransfected with merlin and PKAc was precipitated with rabbit anti-merlin antiserum sc-331 and protein G beads. Equal aliquots of the immunoprecipitate were treated with CIP buffer alone or with CIP. CIP treatment eliminates both the slowest migrating form of merlin (visible after longer exposure (left panel)) and the middle form (visible after short exposure (right panel)). KF10 mAb was used to detect immunoprecipitated merlin. This is a representative of three independent experiments.

 
To investigate further the nature of the slower migrating merlin species observed in cotransfections by PKA, HEK293 cells were cotransfected with merlin and PKAc, and the lysate was immunoprecipitated with anti-merlin antibody. Treatment of the immunoprecipitate with CIP eliminated both slower migrating forms of merlin, which demonstrates that they represent phosphorylated forms of merlin (Fig. 1B). We designated the three different merlin species observed here as hypophosphorylated, phosphorylated, and hyperphosphorylated merlin, analogous with the merlin forms observed in Rac/PAK-induced phosphorylation.

Serine 518 of Merlin Is a Substrate for PKA—To investigate whether merlin is a direct substrate for PKA, we performed an in vitro phosphorylation reaction on recombinant GST-merlin fusion proteins, which contained the N-terminal FERM domain (aa 1–314), the {alpha}-helical domain (aa 314–477), and the C terminus (aa 492–595). Incubation of these fusion proteins with [{gamma}-33P]ATP and bovine catalytic subunit of PKA resulted in incorporation of 33P into both the N and the C termini but not into the {alpha}-helical domain or GST (Fig. 2A). Use of human recombinant catalytic subunit instead of that purified from bovine heart gave identical results (data not shown).



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  		FIG. 2.
In vitro phosphorylation of GST-merlin fusion proteins. Bacterially expressed GST-merlin fusion proteins were phosphorylated in vitro using [{gamma}-33P]ATP and the purified catalytic subunit of PKA and were separated on SDS-PAGE. A, N-terminal domain (N, aa 1–314), {alpha}-helical domain ({alpha}, aa 314–477), C terminus (C, aa 492–595), and GST. B, C terminus of wild-type sequence (WT), S518A mutation, T576A mutation, and GST. Similar amounts of the GST-merlin fusion proteins were used in the reaction as indicated by the Coomassie-stained gels (right), which were dried and exposed on x-ray film (left). Both N- and C-terminal fragments were phosphorylated, whereas {alpha}-helical domain and GST were not. S518A but not T576A mutation abolishes phosphorylation of the C-terminal construct. This is a representative of three independent experiments.

 
To identify the substrate residues for PKA in merlin, we analyzed the potential consensus kinase recognition motifs. A recent report demonstrates that gastric ezrin is a substrate for PKA and that serine 66 is phosphorylated by PKA (20). However, this serine is not conserved in merlin. Analysis of the amino acid sequence of merlin suggested five potential PKA phosphorylation sites of which the sequence KRLS518 has the highest prediction to be a consensus phosphorylation site (scansite.mit.edu) (34). To test whether the serine 518 is a substrate of PKA we performed in vitro phosphorylation on GST-merlin C-terminal fragments including wild-type and mutant S518A. Threonine 576 in merlin is analogous to the conserved threonine in the ERM proteins, which has been shown to be a target for both Rho kinase- and protein kinase C-induced phosphorylation (8–10). The sequence KHNT576 was among the predicted sites, although with lower probability than serine 518. Thus, mutant T576A also was included in the phosphorylation assay. Incorporation of 33P was detected in both wild-type merlin and T576A mutant but not in S518A substitution, indicating that serine 518 is a substrate to PKA (Fig. 2B). Interestingly, it has been shown that this residue is also phosphorylated by PAK1 and -2 (13, 14).

To verify that serine 518 also is phosphorylated by PKA in vivo, HEK293 cells were transfected with wild-type merlin or the S518A and T576A mutants with or without PKAc. Both wild-type merlin and T576A mutant migrated as doublets when transfected alone, and cotransfection with PKAc resulted in a mobility shift of the merlin bands toward the slower migrating phosphorylated form (Fig. 3). In contrast, conversion of serine 518 to alanine eliminated the slower migrating forms of merlin in control and PKAc expressing cells, and only the hypophosphorylated merlin was detected. These data are consistent with those obtained in the in vitro phosphorylation studies and indicate that serine 518 is the target for PKA-induced phosphorylation in vivo.



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  		FIG. 3.
Identification of PKA-induced phosphorylation site in vivo. Lysates from HEK293 cells transfected with wild-type merlin or with mutants T576A or S518A with or without PKAc were separated on SDS-PAGE, blotted onto nitrocellulose, and probed with merlin antibody KF10 mAb (top panel). Transfection of PKA was confirmed by PKAc mAb (middle panel). To verify equal loading and transfer of proteins, the filter was stripped and reprobed by ezrin antibody Ez9 (bottom panel). PKA induced the slower migrating form in wild-type and T576A merlin but not in S518A merlin. This is a representative of three independent experiments.

 
PAK Activity Is Not Required for PKA-induced Phosphorylation of Merlin—As serine 518 is phosphorylated by both PAK and PKA, we next examined the possibility of a direct functional link between PKA and PAK as reported in other experimental models (35). More specifically, we wanted to test whether PKA-induced phosphorylation of merlin in vivo is mediated by PAK or vice versa. To address this question, we transfected subconfluent HEK293 cells with merlin alone or together with the N-terminal domain of PAK2 (aa 1–248/H82L,H85L). This regulatory domain associates with the C-terminal kinase domain and inhibits the kinase activity of endogenous PAK2 in vivo (data not shown) analogous to the results reported earlier for PAK1 (36). Because of the sequence homology between the different PAKs, in addition to PAK2 PAK1 activity also is blocked with the PAK2 construct (data not shown). Furthermore, the PAK2 construct contains a mutation in the Rac/Cdc42-binding site (H82L,H85L), which disrupts the interaction of this fragment with the GTPases and thus leaves the GTPase pool in the cell unaffected. Coexpression of merlin and the PAK2 N terminus in HEK293 cells markedly decreased the amount of the slower migrating merlin species, suggesting that endogenous PAK activity was mainly responsible for phosphorylation of merlin in unstimulated sub-confluent HEK293 cells (Fig. 4A). As expected, coexpression of merlin and PKAc in cells resulted in an increase in the amount of both hyperphosphorylated and phosphorylated merlin. Inhibition of endogenous PAK activity did not change the electrophoretic pattern of merlin, indicating that PAK activity is not required for PKA-induced phosphorylation of merlin in vivo (Fig. 4A).



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  		FIG. 4.
PKA and PAK phosphorylate merlin independently in vivo. A, HEK293 cells were transfected with merlin alone or cotransfected with merlin, PAK2 (aa 1–248/H82L,H85L), and PKAc as indicated. The lysates were separated by SDS-PAGE and immunoblotted for merlin with the KF10 mAb (top panel). Expression of the PAK2 mutant and PKAc was confirmed by incubating the immunoblots with the anti-myc (middle panel) and anti-PKAc mAbs (bottom panel), respectively. B, HEK293 cells were transfected with merlin or cotransfected with merlin and the constitutively active PAK2 (T402E). The cells were treated with 20 µM H89 for 5 h to inhibit endogenous PKA. Cell lysates were analyzed by immunoblotting using the merlin mAb KF10 (top panel) and the anti-Myc mAb for detection of merlin and PAK2 (bottom panel), respectively. Inhibition of the PAK activity did not affect PKA-induced phosphorylation of merlin or vice versa. This is a representative of three independent experiments.

 
To rule out the possibility that PAK-induced phosphorylation of merlin is mediated via activation of PKA, we transfected HEK293 cells with merlin alone or with a constitutively active PAK2 (T402E) mutant. Endogenous PKA activity was then inhibited with H89. As shown in Fig. 4B, inhibition of PKA activity did not alter PAK2-induced phosphorylation of merlin as indicated by the unchanged pattern of merlin species obtained from untreated versus H89-treated cells. This result suggests that PAK phosphorylates merlin in a PKA-independent manner in vivo. Based on these observations we propose that PKA and PAK phosphorylate merlin independently in HEK293 cells.

Increase in Intracellular cAMP Induces Phosphorylation of Merlin in Vivo—To investigate whether merlin phosphorylation is induced in response to stimulation of endogenous PKA with cAMP, HEK293 cells were first cotransfected with merlin and PAK2 (aa 1–248/H82L,H85L) to block endogenous PAK activity (Fig. 4A). Transfected cells were serum-starved overnight and then left untreated or stimulated with a mixture of forskolin and IBMX for different times (<=30 min). In lysates prepared from unstimulated cells, merlin exists mainly as a single hypophosphorylated form with fastest electrophoretic mobility in SDS-PAGE. Treatment of the cells with forskolin + IBMX induced phosphorylation of merlin, which was abolished by treatment of the cells with H89 (Fig. 5A). This shows that cAMP stimulation of the cells in vivo results in PKA-dependent phosphorylation of merlin. No increase in the phosphorylation status of merlin was observed in stimulated serum-starved cells in which endogenous PAK activity was not blocked (Fig. 5B). To confirm the activation of PKA in this and other stimulation experiments, lysates of unstimulated and stimulated (±H89) cells were compared by immunoblotting using an antibody recognizing phosphorylated PKA substrate motif (data not shown).



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  		FIG. 5.
PKA activation results in merlin phosphorylation in attached cells with inhibited PAK activity. HEK293 cells were cotransfected with merlin and PAK2 (aa 1–248/H82L,H85L) constructs (A) or with merlin alone (B). Transfected cells were grown on plates, serum-starved, and either left untreated or treated with forskolin (F) + IBMX (I) (25 and 50 µM, respectively) ± 20 µM H89 for the indicated times to activate endogenous PKA. After incubation, cells were lysed in Laemmli buffer and analyzed by immunoblotting with merlin KF10 mAb (top panel) and anti-Myc mAb for the N-terminal domain of PAK2 (bottom panel in A), respectively. An increase in intracellular cAMP induces PKA-dependent phosphorylation of merlin in cells with decreased PAK activity. Phosphorylation of merlin was decreased when the cells were treated with H89. This is a representative of three independent experiments.

 
As another approach to monitoring cAMP-induced phosphorylation of merlin, we stimulated cells detached from the substratum, as loss of adhesion is known to result in dephosphorylation of merlin (11). Merlin-transfected HEK293 cells were serum-starved, detached by trypsin, and stimulated with forskolin. Consistent with earlier studies, loss of adhesion resulted in a nearly complete dephosphorylation of merlin (Fig. 6). Stimulation of the nonattached cells with forskolin increased the amount of phosphorylated merlin compared with unstimulated cells or cells treated with forskolin + H89. These results are consistent with findings on adherent HEK293 cells with suppressed PAK activity, indicating that an increase in intracellular cAMP concentration results in PKA-dependent phosphorylation of merlin in vivo.



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  		FIG. 6.
cAMP stimulates merlin phosphorylation in nonattached cells in vivo. HEK293 cells transfected with merlin were grown on culture plates, serum-starved, detached by trypsin, and incubated in serum-free medium in suspension without or with 25 µM forskolin ± 20 µM H89 for the indicated times. Whole cell lysates were prepared, separated in SDS-PAGE, immunoblotted, and detected for merlin with KF10 mAb. Treatment of the cells with forskolin induced phosphorylation of merlin when compared with unstimulated cells or cells treated with forskolin + H89, which expressed mainly hypophosphorylated merlin. This is a representative of three independent experiments.

 
Phosphorylation of Serine 518 Promotes Heterodimerization between Merlin and Ezrin—We investigated whether phosphorylation of serine 518 affects heterodimerization between merlin and ezrin in vitro and in vivo. Bead-bound C-terminal GST-merlin (aa 492–595) was phosphorylated by purified bovine catalytic subunit of PKA, and the beads were used to precipitate VSVG-tagged N-terminal ezrin (aa 1–309) from cell lysates. In this assay PKA-treated C-terminal merlin precipitated N-terminal ezrin markedly better than untreated C-terminal merlin (Fig. 7A). Furthermore, phosphatase treatment of the phosphorylated C terminus reduced heterodimerization. Immunoblotting with the phospho-Ser-518-specific merlin antibody HM2175 verified both PKA-induced phosphorylation and CIP-induced dephosphorylation of serine 518 (Fig. 7A, middle panel), the only residue in the C terminus phosphorylated by PKA (Fig. 2). The results indicate that the increase in intermolecular binding is dependent on phosphorylation of serine 518 in merlin.



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  		FIG. 7.
Phosphorylation of serine 518 promotes binding between merlin C terminus and ezrin N terminus. A, bead-bound GST-merlin C terminus (aa 492–595) was untreated, phosphorylated with purified catalytic subunit of PKA, or phosphorylated and subsequently treated with CIP. Beads were incubated with a lysate containing VSVG-tagged ezrin (aa 1–309) from transfected HEK293 cells. Bound proteins were separated by SDS-PAGE, and ezrin was visualized with anti-VSVG tag antibody (top panel). The PKA-phosphorylated merlin C terminus binds ezrin N terminus better than unphosphorylated merlin. The samples were also probed with phospho-Ser-518 (pS518) merlin antibody to verify the effect of PKAc and CIP treatment on serine 518 (middle panel). The bottom panel indicates that all samples contained equal amount of GST-merlin. B, HEK293 cells were transfected with merlin cDNA. Subconfluent (upper panels) or confluent cells (lower panels) were lysed, and merlin (mAb KF10) or ezrin (mAb 3C12) was immunoprecipitated (IP). mAb X63 was used as a control (contr). Western blot analysis shows that subconfluent but not confluent lysate contains phosphorylated merlin bands. In subconfluent lysates only, merlin coprecipitates with ezrin and vice versa. Furthermore, only the phosphorylated merlin coprecipitated with ezrin (upper panel, 3C12 immunoprecipitate). C, wild-type (WT) or S518A mutant merlin cDNA was transfected to HEK293 cells. Ezrin was immunoprecipitated, and coprecipitating merlin was visualized by immunoblotting. More wild-type than S518A merlin is bound to ezrin. Results represent three independent experiments.

 
To examine whether phosphorylation of serine 518 also promotes heterodimerization in vivo, we transfected merlin into subconfluent and confluent HEK293 cells expressing endogenous ezrin. In subconfluent cells merlin is phosphorylated by endogenous PAK, and all three different forms of merlin were detected in the lysate (Fig. 7B). Phosphorylation of serine 518 by PAK instead of PKA allowed us to eliminate the possible effects of PKA-induced phosphorylation of ezrin (20) on the heterodimerization. Immunoblotting of the beads revealed that under subconfluent conditions ezrin coimmunoprecipitates with merlin and vice versa. Further analysis showed that forms of merlin that coimmunoprecipitate with ezrin represent the slower migrating, i.e. phosphorylated, forms (Fig. 7B). In confluent cells lysed without phosphatase inhibitors, only hypophosphorylated merlin was observed (Fig. 7B). Under these conditions, no coimmunoprecipitation of the proteins was observed, which confirms that ezrin binds phosphorylated merlin. The role of phosphorylation of serine 518 in heterodimerization was further analyzed by transfecting subconfluent cells with either wild-type or S518A mutant merlin. In these experiments, more wild-type than S518A merlin coprecipitated with ezrin (Fig. 7C). Taken together, the in vitro and in vivo results suggest that phosphorylation of serine 518 regulates heterodimerization between merlin and ezrin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Whereas the function of merlin as a tumor suppressor is well established, the molecular and cell biological bases of the tumor suppressor mechanism of merlin are still poorly understood (37). Previous results, which because of low expression of endogenous merlin in cultured cells are mostly based on transfection experiments, suggest that post-translational modifications such as phosphorylation regulate the biological activity of merlin. It has been proposed that the dephosphorylated and presumably closed form of merlin is the growth-inhibitory or tumor-suppressive form, whereas phorphorylated merlin is growth-permissive (11, 12, 38).

In this report we show several lines of in vitro and in vivo evidence indicating that merlin serves as the substrate for PKA. We have shown earlier that the RI{beta} subunit of PKA, abundantly present in brain, binds to a conserved {alpha}-helical PKA-anchoring protein motif in merlin, suggesting a novel function for merlin as an AKAP (24). Taken together, these observations could indicate a role for PKA in regulating the intramolecular and/or intermolecular interactions of merlin via phosphorylation, whereas merlin could also, in part, shuttle PKA and modulate its activity. Although the number of identified physiological substrates of PKA is increasing, only a few of these also function as AKAPs. Notably, ezrin serves as an AKAP in gastric parietal cells by binding a regulatory subunit of PKA (RII{alpha}) and anchoring the kinase to critical regions in the canalicular target membranes during stimulation (39). Gastric ezrin is phosphorylated at the N terminus by PKA as a response to histamine stimulation in parietal cells (20). Another example, microtubule-associated protein 2 (MAP2), a neuronal AKAP that targets PKA to microtubules (40), is phosphorylated by PKA at three serines located in the C-terminal tubulin-binding region. Phosphorylation of the serine residues regulates MAP2 interactions with microtubules and actinbased cytoskeletal structures (41).

Our in vitro phosphorylation data indicate that merlin is phosphorylated by PKA both at the FERM domain and at the C terminus. Further experiments with nonphosphorylatable alanine mutants revealed that serine 518 in the C terminus also is phosphorylated by PKA in vivo. The potential N-terminal phosphorylation site was not mapped further, and the experimental design did not allow us to verify the result in vivo. Therefore, further investigations will be required to judge the biological significance of the in vitro results on phosphorylation of the merlin FERM domain. Recent studies have shown that the serine 518 phosphorylated by PKA is also the substrate of PAKs (13, 14). In addition, immunoblot analysis revealed that PKA induced apparently similar forms of phosphorylated merlin to those observed in earlier studies with PAKs (13, 14). Interestingly, the merlin isoform-2, which differs from isoform-1 in the very C terminus, was phosphorylated at the serine 518 but migrated as a single species in the gel (data not shown). This observation suggests that the last 16 amino acids in isoform-1 are directly or indirectly involved in the appearance of the differently migrating merlin species. It has been reported that isoform-2 does not display intramolecular interaction but instead exists constitutively in an open conformation (1).

Our observation that PKA phosphorylates the same residue as do PAKs suggests that phosphorylation of serine 518 is an important factor in the functional regulation of merlin. As PKA and PAKs are activated in response to different stimuli, this may indicate that merlin is involved in regulating signals from two or perhaps more different signal transduction pathways. Phosphorylation by multiple kinases in response to different extracellular signals may allow diverse modulation of important regulatory proteins. For example, phosphorylation of serine 9 in synapsin I, a neuronal protein, either by PKA or PAK, can regulate the activity of the Gz GTPase-activating protein (Gz GAP) (42). Interestingly, cAMP and MAP kinase signaling pathways are known to cross-talk, and depending on the cell type, PKA can either activate or inhibit the Raf-MEK-ERK kinase cascade (43). In NIH3T3 fibroblasts, detachment-dependent activation of PKA prevents anchorage-independent activation of MAP kinase by inactivating PAK (35). In this study we did not observe any direct functional relationship between the two enzymes, and our data suggest independence rather than a direct interplay in the merlin phosphorylation activity of the two kinases. Despite the constitutive activity of PAK in HEK293 cells, phosphorylation of merlin by PKA in vivo was obvious in conditions where PKA was overexpressed or the PAK activity was suppressed. The difficulty in detecting cAMP-induced merlin phosphorylation in attached cells without PAK inhibition is most probably because of the relatively high basal phosphorylation of merlin in the cells.

The finding that phosphorylation of the serine 518 promotes heterodimerization between merlin and ezrin is interesting and suggests a novel and important role for this residue. It has been shown earlier that the S518D mutation, which mimics the phosphorylation state, weakens self-association between the FERM domain and the C terminus of merlin (12). Thus, phosphorylation of serine 518 by PKA or PAK may not only open the molecule, but according to the interaction studies, it also enhances directly the association between merlin and ezrin. In these experimental conditions we did not detect self-association between the merlin N and C termini (not shown). This is probably because of both the lower affinity of the merlin C-domain to the merlin N-domain than to the ezrin N-domain (6) and the relatively short merlin C-terminal fragment used in the present study. Phosphorylation of merlin in HEK293 cells induced association between merlin and ezrin, and substitution of serine 518 with alanine reduced the association, suggesting that phosphorylation of the serine 518 also regulates heterodimerization in vivo. It has been shown that in growth-promoting conditions (low cell density) phosphorylated merlin exists in complex with ezrin, moesin, and CD44, whereas in growth-inhibitory conditions (high cell density) hypophosphorylated merlin interacts with the cytoplasmic tail of CD44 (38). Based on these results, a model has been proposed that suggests distinct functions for the two different merlin complexes in Ras signaling and cell proliferation (38, 44). Our finding that phosphorylation of merlin by PKA promotes heterodimerization is consistent with this model, and we have now extended the model to cAMP/PKA signaling (Fig. 8). Interestingly, our previous data showed that the regulatory subunit of PKA is bound to the hypophosphorylated, and probably the tumor suppressive form, of merlin (24). However, further studies are required to test the validity of the model presented and to understand the possible link between the different signaling pathways.



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  		FIG. 8.
A model of regulation of merlin activity by PKA-induced phosphorylation. Previous studies have shown that hypophosphorylated merlin inhibits cell growth, possibly by blocking receptor (e.g. CD44)-mediated signaling and Ras activation, whereas phosphorylated merlin permits growth (12, 38). In the growth-inhibitory complex, merlin may bind a regulatory subunit of inactive PKA (24). On the other hand, PKA signaling is known to promote the growth of Schwann cells (18). According to the model, which combines previous and current results, binding of a ligand (e.g. hormones, neurotransmitters) to G-protein-coupled receptor increases cAMP level, which results in the release of PKA catalytic subunits from regulatory subunits. Catalytic subunits phosphorylate merlin at serine 518, which promotes heterodimerization between merlin and ezrin. In this growth-promoting complex, phosphorylated ezrin is bound to the receptor resulting in signaling and cell proliferation. An analogous chain of events may result from activation of PAK. M, merlin; E, ezrin; GPCR, G-protein-coupled receptor; R, regulatory subunit of PKA; C, catalytic subunit of PKA; P, phosphate group.

 
Although merlin is expressed in a variety of tissues, high levels of expression are detected in neural cells such as Schwann cells. Mutations in the NF2 gene result in changes in Schwann cell morphology, loss of cell-cell contacts, and increased proliferation (45). The cytoskeletal abnormalities due to the absence of merlin can, however, be reversed by the introduction of merlin into the tumor-derived schwannoma cells (46). Interestingly, an earlier report has shown that PKA activity is required for Schwann cell proliferation and cell cycle progression (18). In this study, treatment of the cells with human glial growth factor-2 transiently increased the level of intracellular cAMP with the maximum increase at the G0/G1 stage of the cell cycle. Furthermore, treatment of the Schwann cells with the PKA inhibitor H89 inhibited the cell proliferation induced by growth factors and blocked the G0/G1 phase, which indicates an important role of PKA in cell growth. Activation of PKA by forskolin had an inhibitory effect on glial growth factor-2-induced MAP kinase activity, which suggests cross-talk in Schwann cells between the two signaling pathways initiated by receptor tyrosine kinase and cAMP. However, the mechanism underlying these observations remains unclear. As loss of functional merlin predisposes Schwann cells to tumor formation, it is tempting to speculate that PKA-dependent phosphorylation of merlin provides a means to control the antiproliferative activity of merlin. Therefore, further studies using other cell models such as Schwann cells will be required to understand the role of PKA-induced phosphorylation in the function of merlin and, perhaps ultimately, in tumor formation.


   FOOTNOTES
 
* This work was supported by United States Army Neurofibromatosis Research Grant DAMD17-00-0550 and by grants from the Academy of Finland and the Finnish Cancer Organizations. 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. Back

§ Present address: VTT Biotechnology, Tietotie 2, P. O. Box 1500, FIN-02044 VTT, Finland. Back

|| To whom correspondence should be addressed: Neuroscience Program, Rm. C524, Biomedicum, P. O. Box 63, Haartmaninkatu 8, University of Helsinki, FIN-00014 Helsinki, Finland. Tel.: 358-9-19125650; Fax: 358-9-47171964; E-mail: olli.carpen{at}helsinki.fi.

1 The abbreviations used are: ERM, ezrin-radixin-moesin; FERM, four-point-one ezrin-radixin-moesin; PKA, protein kinase A; PKAc, catalytic subunit of human PKA; PAK, p21-activated kinase; AKAP, protein kinase A-anchoring protein; GST, glutathione S-transferase; HEK293 cells, human embryonic kidney 293 cells; CIP, calf intestinal phosphatase; IBMX, 3-isobutyl-1-methylxanthine; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; mAb, monoclonal antibody; aa, amino acid(s); HA, hemagglutinin; MAP, microtubule-associated protein; VSVG, vesicular stomatitis virus glycoprotein G. Back


   ACKNOWLEDGMENTS
 
We thank Kjetil Taskén for PKAc construct, Monique Arpin for the VSVG-ezrin construct, Ellen Zwarthoff for the KF10 mAb, Joseph Kissil for the HM2175 antibody, Kalle Saksela for helpful comments, and Helena Ahola, Tuula Halmesvaara, and Hanne Ahola for skillful technical assistance.



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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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