FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080708346> ?p ?o ?g. }

• W2080708346 endingPage "18566" @default.
• W2080708346 startingPage "18559" @default.
• W2080708346 abstract "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 [γ-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. 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 [γ-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. 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 (1Sherman L. Xu H.-M. Geist R.T. Saporito-Irwin S. Howells N. Ponta H. Herrlich P. Gutmann D.H. Oncogene. 1997; 15: 2505-2509Google Scholar). The overall structure of merlin is similar to that of ERM 1The 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. (ezrin-radixin-moesin) proteins (2Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Nature. 1993; 363: 515-521Google Scholar, 3Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Haase V.H. Ambrose C.M. Munroe D. Bove C. Haines J.L. Martuza R.L. MacDonald M.E. Seizinger B.R. Short M.P. Buckler A.J. Gusella J.F. Cell. 1993; 72: 791-800Google Scholar). 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 α-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 (4Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Google Scholar, 5Grönholm M. Sainio M. Zhao F. Heiska L. Vaheri A. Carpén O. J. Cell Sci. 1999; 112: 895-904Google Scholar, 6Nguyen R. Reczek D. Bretcher A. J. Biol. Chem. 2001; 276: 7621-7629Google Scholar). 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 (7Bretcher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 586-599Google Scholar). Rho kinase (8Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Google Scholar) and two different isoforms of protein kinase C (9Pietromonaco S.F. Simons P.C. Altman A. Elias L. J. Biol. Chem. 1998; 273: 7594-7603Google Scholar, 10Ng T. Parsons M. Hughes W.E. Monypenny J. Zicha D. Gautreau A. Arpin M. Gschmeissner S. Verveer P.J. Bastiaens P.I.H. Parker P.J. EMBO J. 2001; 20: 2723-2741Google Scholar) 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 (11Shaw R.J. McClatchey A.I. Jacks T. J. Biol. Chem. 1998; 273: 7757-7764Google Scholar). 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 (12Shaw R.J. Paez J.G. Curto M. Yaktine A. Pruitt W.M. Saotome I. O′Bryan J.P. Gupta V. Ratner N. Der C.J. Jacks T. McClatchey A.I. Dev. Cell. 2001; 1: 63-72Google Scholar). The site for the Rac-induced phosphorylation was determined to be the serine 518 residue in the C terminus of merlin (12Shaw R.J. Paez J.G. Curto M. Yaktine A. Pruitt W.M. Saotome I. O′Bryan J.P. Gupta V. Ratner N. Der C.J. Jacks T. McClatchey A.I. Dev. Cell. 2001; 1: 63-72Google Scholar). Serine 518 is phosphorylated by p21-activated kinases (PAKs) (13Kissil J.L. Johnson K.C. Eckman M.S. Jacks T. J. Biol. Chem. 2002; 277: 10394-10399Google Scholar, 14Xiao G.-H. Beeser A. Chernoff J. Testa J.R. J. Biol. Chem. 2002; 277: 883-886Google Scholar), 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 (15Kissil J.L. Wilker E.W. Johnson K.C. Eckman M.S. Yaffe M.B. Jacks T. Mol. Cell. 2003; 12: 841-849Google Scholar). 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 (16Brandon E.P. Idzerda R.L. McKnight G.S. Curr. Opin. Neurobiol. 1997; 7: 397-403Google Scholar, 17Albright T.D. Kandel E.R. Posner M.I. Curr. Opin. Neurobiol. 2000; 10: 612-624Google Scholar). In Schwann cells activation of the cAMP/PKA pathway promotes cell growth and cell cycle progression (18Kim H.A. DeClue J.E. Ratner N. J. Neurosci. Res. 1997; 49: 236-247Google Scholar), and cAMP/PKA is also required for myelin formation (19Howe D.G. McCarthy K.D. J. Neurosci. 2000; 20: 3513-3521Google Scholar). PKA has a number of physiological substrates, and, recently, ezrin also has been reported to be phosphorylated by PKA (20Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Google Scholar). 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) (21Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Google Scholar, 22Edwards A.S. Scott J.D. Curr. Opin. Cell Biol. 2000; 12: 217-221Google Scholar, 23Feliciello A. Gottesman M.E. Avvedimento E.V. J. Mol. Biol. 2001; 308: 99-114Google Scholar). We have recently shown that merlin binds to a regulatory subunit (RIβ) of PKA and may function as an AKAP for RIβ-containing PKA both in the central nervous system and in cultured neuronal cells (24Grönholm M. Vossebein L. Carlson C.R. Kuja-Panula J. Teesalu T. Alfthan K. Vaheri A. Rauvala H. Herberg F.W. Taskén K. Carpén O. J. Biol. Chem. 2003; 278: 41167-41172Google Scholar). 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. Plasmids—The human merlin isoform 1 cDNA in pcDNA3 vector (Invitrogen) has been described earlier (25Sainio M. Zhao F. Heiska L. Turunen O. den Bakker M. Zwarthoff E. Lutchman M. Rouleau G.A. Jääskeläinen J. Vaheri A. Carpén O. J. Cell Sci. 1997; 110: 2249-2260Google Scholar). The plasmid encoding the catalytic subunit (C) of human PKA (PKAc) (26Taskén K. Andersson K.B. Erikstein B.K. Hansson V. Jahnsen T. Blomhoff H.K. Endocrinology. 1994; 135: 2109-2119Google Scholar) 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 (27Mizushima S. Nagata S. Nucleic Acids Res. 1990; 185322Google Scholar) 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), α-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 (28Turunen O. Wahlström T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Google Scholar), and the plasmid encoding the vesicular stomatitis glycoprotein (VSVG)-tagged N-domain of ezrin (aa 1–309) (29Algrain M. Turunen O. Vaheri A. Louvard D. Arpin M. J. Cell Biol. 1993; 120: 129-139Google Scholar) 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) (30den Bakker M.A. Tascilar M. Riegman P.H. Hekman A.C. Boersma W. Janssen P.J. de Jong T.A. Hendriks W. van der Kwast T.H. Zwarthoff E.C. Am. J. Pathol. 1995; 147: 1339-1349Google Scholar) and sc-331 rabbit antiserum (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used to detect merlin. HM2175 antiserum (13Kissil J.L. Johnson K.C. Eckman M.S. Jacks T. J. Biol. Chem. 2002; 277: 10394-10399Google Scholar), 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 (31Böhling T. Turunen O. Jääskeläinen J. Carpén O. Sainio M. Wahlström T. Vaheri A. Haltia M. Am. J. Pathol. 1996; 148: 367-373Google Scholar) 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 × 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 5× 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 [γ-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 1× 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 × 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 × 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 (13Kissil J.L. Johnson K.C. Eckman M.S. Jacks T. J. Biol. Chem. 2002; 277: 10394-10399Google Scholar). 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 × 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. In Vivo Phosphorylation of Merlin by PKA—We previously characterized merlin as an AKAP for the RIβ subunit containing PKA (24Grönholm M. Vossebein L. Carlson C.R. Kuja-Panula J. Teesalu T. Alfthan K. Vaheri A. Rauvala H. Herberg F.W. Taskén K. Carpén O. J. Biol. Chem. 2003; 278: 41167-41172Google Scholar). 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 (32Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Google Scholar) and is a selective although not a specific PKA inhibitor (33Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Google Scholar). 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 (12Shaw R.J. Paez J.G. Curto M. Yaktine A. Pruitt W.M. Saotome I. O′Bryan J.P. Gupta V. Ratner N. Der C.J. Jacks T. McClatchey A.I. Dev. Cell. 2001; 1: 63-72Google Scholar, 13Kissil J.L. Johnson K.C. Eckman M.S. Jacks T. J. Biol. Chem. 2002; 277: 10394-10399Google Scholar, 14Xiao G.-H. Beeser A. Chernoff J. Testa J.R. J. Biol. Chem. 2002; 277: 883-886Google Scholar). 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 α-helical domain (aa 314–477), and the C terminus (aa 492–595). Incubation of these fusion proteins with [γ-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 α-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). 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 (20Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Google Scholar). 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) (34Shabb J.B. Chem. Rev. 2001; 101: 2381-2411Google Scholar). 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 (8Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Google Scholar, 9Pietromonaco S.F. Simons P.C. Altman A. Elias L. J. Biol. Chem. 1998; 273: 7594-7603Google Scholar, 10Ng T. Parsons M. Hughes W.E. Monypenny J. Zicha D. Gautreau A. Arpin M. Gschmeissner S. Verveer P.J. Bastiaens P.I.H. Parker P.J. EMBO J. 2001; 20: 2723-2741Google Scholar). 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 (13Kissil J.L. Johnson K.C. Eckman M.S. Jacks T. J. Biol. Chem. 2002; 277: 10394-10399Google Scholar, 14Xiao G.-H. Beeser A. Chernoff J. Testa J.R. J. Biol. Chem. 2002; 277: 883-886Google Scholar). 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. 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 (35Howe A.K. Juliano R.L. Nat. Cell Biol. 2000; 2: 593-600Google Scholar). 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 (36Zenke F.T. King C.C. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 32565-32573Google Scholar). 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). 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 stimulatio" @default.
• W2080708346 created "2016-06-24" @default.
• W2080708346 creator A5030148031 @default.
• W2080708346 creator A5043081202 @default.
• W2080708346 creator A5054627640 @default.
• W2080708346 creator A5069434219 @default.
• W2080708346 creator A5083030451 @default.
• W2080708346 date "2004-04-01" @default.
• W2080708346 modified "2023-10-05" @default.
• W2080708346 title "Cyclic AMP-dependent Protein Kinase Phosphorylates Merlin at Serine 518 Independently of p21-activated Kinase and Promotes Merlin-Ezrin Heterodimerization" @default.
• W2080708346 cites W1481438484 @default.
• W2080708346 cites W1487918137 @default.
• W2080708346 cites W1590118434 @default.
• W2080708346 cites W1964377967 @default.
• W2080708346 cites W1964484264 @default.
• W2080708346 cites W1967571967 @default.
• W2080708346 cites W1977675485 @default.
• W2080708346 cites W1981327350 @default.
• W2080708346 cites W1996275548 @default.
• W2080708346 cites W1998183313 @default.
• W2080708346 cites W2000253969 @default.
• W2080708346 cites W2000990440 @default.
• W2080708346 cites W2005252392 @default.
• W2080708346 cites W2005487999 @default.
• W2080708346 cites W2012819315 @default.
• W2080708346 cites W2016569169 @default.
• W2080708346 cites W2027310689 @default.
• W2080708346 cites W2029888446 @default.
• W2080708346 cites W2029976610 @default.
• W2080708346 cites W2037632318 @default.
• W2080708346 cites W2041421563 @default.
• W2080708346 cites W2060655307 @default.
• W2080708346 cites W2062483095 @default.
• W2080708346 cites W2065839189 @default.
• W2080708346 cites W2089444196 @default.
• W2080708346 cites W2094565395 @default.
• W2080708346 cites W2097930500 @default.
• W2080708346 cites W2109200753 @default.
• W2080708346 cites W2111622605 @default.
• W2080708346 cites W2112387151 @default.
• W2080708346 cites W2112630320 @default.
• W2080708346 cites W2117524604 @default.
• W2080708346 cites W2117822148 @default.
• W2080708346 cites W2119785069 @default.
• W2080708346 cites W2122666046 @default.
• W2080708346 cites W2127890262 @default.
• W2080708346 cites W2134360487 @default.
• W2080708346 cites W2150172003 @default.
• W2080708346 cites W2161807488 @default.
• W2080708346 cites W2185731407 @default.
• W2080708346 cites W2315552227 @default.
• W2080708346 cites W2953375613 @default.
• W2080708346 cites W4247904703 @default.
• W2080708346 cites W4250421628 @default.
• W2080708346 doi "https://doi.org/10.1074/jbc.m313916200" @default.
• W2080708346 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14981079" @default.
• W2080708346 hasPublicationYear "2004" @default.
• W2080708346 type Work @default.
• W2080708346 sameAs 2080708346 @default.
• W2080708346 citedByCount "127" @default.
• W2080708346 countsByYear W20807083462012 @default.
• W2080708346 countsByYear W20807083462013 @default.
• W2080708346 countsByYear W20807083462014 @default.
• W2080708346 countsByYear W20807083462015 @default.
• W2080708346 countsByYear W20807083462016 @default.
• W2080708346 countsByYear W20807083462017 @default.
• W2080708346 countsByYear W20807083462018 @default.
• W2080708346 countsByYear W20807083462019 @default.
• W2080708346 countsByYear W20807083462020 @default.
• W2080708346 countsByYear W20807083462021 @default.
• W2080708346 countsByYear W20807083462022 @default.
• W2080708346 countsByYear W20807083462023 @default.
• W2080708346 crossrefType "journal-article" @default.
• W2080708346 hasAuthorship W2080708346A5030148031 @default.
• W2080708346 hasAuthorship W2080708346A5043081202 @default.
• W2080708346 hasAuthorship W2080708346A5054627640 @default.
• W2080708346 hasAuthorship W2080708346A5069434219 @default.
• W2080708346 hasAuthorship W2080708346A5083030451 @default.
• W2080708346 hasBestOaLocation W20807083461 @default.
• W2080708346 hasConcept C104317684 @default.
• W2080708346 hasConcept C11960822 @default.
• W2080708346 hasConcept C142669718 @default.
• W2080708346 hasConcept C1491633281 @default.
• W2080708346 hasConcept C178628643 @default.
• W2080708346 hasConcept C179185449 @default.
• W2080708346 hasConcept C184235292 @default.
• W2080708346 hasConcept C185592680 @default.
• W2080708346 hasConcept C2776414213 @default.
• W2080708346 hasConcept C2780246058 @default.
• W2080708346 hasConcept C55493867 @default.
• W2080708346 hasConcept C59143045 @default.
• W2080708346 hasConcept C86803240 @default.
• W2080708346 hasConcept C90934575 @default.
• W2080708346 hasConcept C95444343 @default.
• W2080708346 hasConcept C97029542 @default.
• W2080708346 hasConceptScore W2080708346C104317684 @default.
• W2080708346 hasConceptScore W2080708346C11960822 @default.
• W2080708346 hasConceptScore W2080708346C142669718 @default.

• next