Available online at www.sciencedirect.com

The International Journal of Biochemistry & Cell Biology 40 (2008) 344–349

Molecules in focus //moesin: Versatile controllers of signaling molecules and of the cortical Verena Niggli ∗,Jer´ emie´ Rossy Department of Pathology, University of Bern, Murtenstr. 31, CH-3010 Bern, Switzerland Received 1 February 2007; received in revised form 15 February 2007; accepted 15 February 2007 Available online 22 February 2007

Abstract Ezrin, radixin and moesin (ERM) are widely distributed proteins located in the cellular cortex, in microvilli and adherens junctions. They feature an N-terminal membrane binding domain linked by an ␣-helical domain to the C-terminal -binding domain. In the dormant state, binding sites in the N-terminal domain are masked by interactions with the C-terminal region. The ␣-helical domain also contributes to masking of binding sites. A specific sequence of signaling events results in dissociation of these intramolecular interactions resulting in ERM activation. ERM molecules have been implicated in mediating actin–membrane linkage and in regulating signaling molecules. They are involved in cell membrane organization, cell migration, phagocytosis and apoptosis, and may also play cell-specific roles in tumor progression. Their precise involvement in these processes has yet to be elucidated. © 2007 Elsevier Ltd. All rights reserved.

Keywords: ERM; Actin–membrane linkage; Membrane organization; Cell migration

1. Introduction membrane proteins. ERMs were originally characterized 20 years ago as structural components of the cell cortex, Reversible actin–membrane linkage is essential for localized in microvilli and adherens junctions. Recent maintenance of cell shape, for cell adhesion, migra- studies in mice suggest redundant functions of the three tion and division. Ezrin (cytovillin), radixin and moesin proteins (Bretscher, Edwards, & Fehon, 2002; Fievet,´ (ERM) proteins are closely related proteins linking actin Louvard, & Arpin, 2007). For lack of space this review filaments to the membrane either (i) directly via bind- will not discuss the putative tumor suppressor , an ing to cytoplasmic tails of transmembrane proteins or ERM-related . (ii) indirectly via scaffolding proteins attached to trans- 2. Structure Abbreviations: C-ERMAD, C-terminal ERM association domain; ERM, ezrin, radixin, moesin; FERM, four-point one ERM; GDI, The for ezrin (13 exons) has been mapped to guanine nucleotide dissociation inhibitor; MRP2, multidrug resis- 6, that of moesin to chromosome X and that tance protein 2; N-ERMAD, N-terminal ERM association domain; of radixin to chromosome 11. The closely related ERMs NIK, Nck-interacting kinase; PI(4,5)P2, phosphatidylinositol 4,5- arose by gene duplication in vertebrates. ERMs feature bisphosphate; PKC, protein kinase C ∗ Corresponding author. Tel.: +41 31 632 87 44; a conserved N-terminal lipid and membrane binding fax: +41 31 381 24 12. domain of approximately 300 amino acids called the E-mail address: [email protected] (V. Niggli). FERM (4.1-ezrin–radixin–moesin) domain, that adopts

1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.02.012 V. Niggli, J. Rossy / The International Journal of Biochemistry & Cell Biology 40 (2008) 344–349 345

Fig. 1. Domain structure of ERM proteins. (A) Comparison of the domain organization and sequence identity between human ERM proteins (NCBI accession numbers: ezrin, NP 003370; radixin, NP 002897; moesin, NP 002435). CTD, C-terminal domain; P, polyproline domain. The F-actin-binding site is located within the last 30 residues at the C-terminus of the ERM proteins (Bretscher et al., 2002). (B) Ribbon structure of dormant moesin from Spodoptera frugiperda (3.0 A˚ structure; accession number 2I1K; Li et al., 2007). Lobes F1, F2 and F3 of the FERM domain are colored yellow and the C-terminal domain in red. The ␣-helical domain (blue) folds into three extended helices (␣A, ␣B and ␣C), containing elements that contact the FERM domain. The linker between the ␣-helical domain and the C-terminal domain (amino acids 461–472) colored in violet interacts mainly with lobe F1 of the FERM domain. The C-terminal domain associates with lobes F2 and F3 of the FERM domain. Backbones of conserved amino acids implicated in interaction with PI(4,5)P2 are colored black (from top to bottom: lysines 64, 63, 60, 278, arginine 253, lysines 254, 262, 263). The following parts of the molecule are not observed in the crystal (dashed black lines): the ␣B-␣C loop of the ␣-helical domain and the connection between the linker (violet), and the beginning of the C-terminal domain. a tri-lobed structure. This domain is connected via a far. The three isoforms are widely distributed in verte- central ␣-helix-rich domain to the C-terminal domain brates, whereas other species only express one isoform. which contains a major F-actin-binding site, enabling In mice, most tissues co-express all three isoforms in these proteins to link actin filaments to the plasma variable expression ratios, but the liver and intestine membrane. In the absence of activating signals, ERM lack ezrin and radixin expression, respectively. Ezrin is proteins are maintained in an inactive conformation enriched at the apical side of epithelial cells, moesin through intramolecular interactions between the N- is a major isoform in endothelial cells and leukocytes, terminal ERM association domain (N-ERMAD) and and radixin is especially abundant in liver (Doi et al., the C-terminal ERM association domain (C-ERMAD), 1999). masking membrane and F-actin-binding sites (Fig. 1A The activation state of ERM proteins is tightly reg- and B). Recent data by Li et al. (2007), based on the first ulated by signaling. Binding of the protein to the crystal structure of intact insect moesin in the closed membrane lipid phosphatidylinositol 4,5-bisphosphate state, show that interactions of the central ␣-helix-rich (PI(4,5)P2) and subsequent phosphorylation of a con- domain and linker regions with lobes F1 and F2 of the served C-terminal threonine are thought to disrupt FERM domain also importantly contribute to masking the intramolecular associations, thus unmasking sites of binding sites, especially those for phosphoinositides involved in interaction with other proteins (Fig. 2A). (Fig. 1B) (Bretscher et al., 2002; Fievet´ et al., 2007; Li Based on crystallization studies and site-directed muta- et al., 2007). genesis, the binding site for PI(4,5)P2 has been located in a basic groove between subdomains F1 and F3 involving 3. Expression and activation conserved, positively charged amino acids contributed from both lobes (Barret, Roy, Montcourrier, Mangeat, encoding for ERM proteins have been identi- & Niggli, 2000; Hamada, Shimizu, Matsui, Tsukita, & fied in all multicellular metazoan organisms analyzed so Hakoshima, 2000)(Fig. 1B). According to the crystal 346 V. Niggli, J. Rossy / The International Journal of Biochemistry & Cell Biology 40 (2008) 344–349

Fig. 2. Activation and cell-type dependent functions of ERM proteins. (A) For stable activation, ERM proteins in a first step interact via their N- terminal FERM domain with the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), followed by C-terminal phosphorylation on a conserved threonine residue. The stably activated protein can then link actin filaments (F-actin) to single pass or multiple pass membrane proteins, or interact for example with GDI proteins that regulate functions of small GTP-binding proteins. F1, F2 and F3 correspond to the three lobes of the FERM domain. ROCK, Rho-kinase; ␤2AR, ␤2-adrenergic receptor; CFTR, cystic fibrosis transmembrane conductance regulator; ICAM-1, intercellular adhesion molecule 1. For the other abbreviations see text. (B) Localization and putative functional roles of ERM proteins in different cell types. IS, immunological synapse; APC, antigen-presenting cell. For details see text.

structure obtained for intact insect moesin, association Interaction with PI(4,5)P2 is a prerequisite for phos- of the ␣-helical domain and the linker region reduces the phorylation of ezrin on a C-terminally located, conserved positive charge of the protein surface in addition to mask- threonine residue, but is not required for its subse- ing the lipid binding domain on lobe F1 (Fig. 1B). Thus, quent membrane interaction (Fievet´ et al., 2007). ERM large conformational changes are required to expose var- phosphorylation on this C-terminal threonine requires ious binding sites including that for PI(4,5)P2 (Li et al., activity of the small GTP-binding protein Rho in 2007). However, dormant intact ezrin interacts at least most cell types. Rho may induce localized production as well with PI(4,5)P2 as the isolated FERM domain, in of PI(4,5)P2. Kinases implicated in ERM activation vitro (Niggli, Andreoli,´ Roy, & Mangeat, 1995). It is pos- include protein kinase C (PKC) ␣, Rho-kinase and sible that interaction of the inactive intact molecule with Nck-interacting kinase (NIK) (Fig. 2A). In addition, the lipid involves residues identified in lobe F3 (Barret phosphorylation of ezrin on other sites by for example et al., 2000), which may still be available for binding in tyrosine kinases may also be required for specific func- the closed form (Fig. 1B). tions (Baumgartner et al., 2006; Fievet´ et al., 2007; Ivetic V. Niggli, J. Rossy / The International Journal of Biochemistry & Cell Biology 40 (2008) 344–349 347

& Ridley, 2004). Based on the findings of Li et al. (2007), tions in human radixin also correlate with neurosensory phosphorylation of Tyr353 in ezrin would be expected to hearing loss (Khan et al., 2007). Interestingly, radixin induce release of the ␣-helical domain from the FERM appears to specifically mediate anchoring of the GABAA domain. receptor ␣5 subunit to the actin cytoskeleton, depen- dent on radixin-phosphoinositide binding and activation. 4. Biological functions GABAA receptor clustering is not observed in hippocam- pal slices of mice lacking radixin. Ezrin and moesin ERM proteins interact via their FERM domains with in contrast do not appear to interact with this receptor three types of proteins: transmembrane proteins, scaf- (Loebrich et al., 2006). This is one of the first stud- folding proteins that bind to transmembrane receptors ies suggesting ERM proteins have an isoform-specific and signaling molecules such as phosphatidylinositol 3- function. kinase or a Rho-specific guanine nucleotide dissociation Moesin knockout mice show no apparent defects. inhibitor (GDI) (Fig. 2A; Bretscher et al., 2002; Ivetic This is surprising as moesin shows some structural dif- & Ridley, 2004; Loebrich, Bahring,¨ Katsuno, Tsukita, ferences from ezrin and radixin, lacking a polyproline & Kneussel, 2006). These findings predict a role for stretch present in the two other proteins, and as moesin ERMs in organizing transmembrane receptors and in is not phosphorylated on tyrosine (Doi et al., 1999). signal transduction. Data obtained using knockout mice Functions of ERM proteins appear to differ depend- lacking individual ERM isoforms especially support a ing on the cell type (Fig. 2B). In leukocytes, ERMs membrane-organizing role of ERM proteins. For each may control signaling molecules such as the small of the ERM proteins, knockout mice have been gener- GTP-binding protein Rho and the enzyme phosphatidyli- ated. The data obtained with these mice mostly support nositol 3-kinase. ERMs are required for formation and the functional redundancy of the three proteins. Defects maintenance of microvilli and modulation of important were only observed for those organs where only one of functions such as clustering of cell adhesion molecules the ERM proteins was expressed. in lymphocyte uropods, tail retraction of migrating cells, A lack of ezrin, the only isoform expressed in part phagocytosis, T cell apoptosis and so forth (Fig. 2B) of the polarized epithelia of the intestine, results in (Charrin & Alcover, 2006; Ivetic & Ridley, 2004). abnormal villus morphogenesis and a disrupted temi- However, the molecular role of ERM proteins in these nal web in these cells. As a consequence, neonate mice functions is unclear, particularly their involvement in lacking ezrin fail to thrive and do not survive past wean- cell migration, for which conflicting data exists. Obser- ing (Saotome, Curto, & McClatchey, 2004). Using a vations in neutrophils and T lymphocytes suggest that knockdown approach in mice, a role of ezrin has been ERM inactivation may be required for tail retraction and demonstrated in formation/expansion of canicular api- cell polarization. In contrast to these findings, express- cal membranes in gastric parietal cells, which express ing activated phosphomimetic ezrin in a spontaneously only ezrin, resulting in achlorhydria. This work suggests polarized T lymphoma cell line, which exhibits enrich- ERM proteins play a role in vesicle trafficking (Tamura ment of P-ERM in the uropod, enhances uropod size et al., 2005). and chemotaxis (Charrin & Alcover, 2006; Ivetic & Radixin is the dominant ERM protein in hepatocytes Ridley, 2004; Lee et al., 2004). In a rat tumor cell line and the only detectable isoform present in stereocilia (most likely monocytoid in origin), attenuation of ERM of the cochlea of adult mice. Mice lacking radixin expression does not affect cell polarization, but signif- develop normally but show mild liver injury after 8 icantly reduces the fraction of spontaneously migrating weeks past birth. Radixin appears to mediate localiza- cells. This finding cannot be explained by requirement tion of the multidrug resistance protein 2 (MRP2) in bile of ERM proteins for Rho activation, as Rho activity canicular membranes via a direct interaction. MRP2 is was not impaired by ERM depletion (Rossy, Gutjahr, involved in secretion of conjugated bilirubin into bile Blaser, Schlicht, & Niggli, 2007). ERM proteins may (Kikuchi et al., 2002). The lack of radixin also results thus have cell-type specific functions in polarization and in deafness in mice. This is due to defective stere- migration. ocilia in the inner and outer hair cells, which normally Concerning the role of ERM isoforms in signaling, exclusively express radixin. No defects are observed in they may act as scaffolding proteins, recruiting for exam- vestibular stereocilia, where the small amounts of ezrin ple Ras into a complex with its activator at the membrane expressed appear to compensate for the lack of radixin upon receptor occupancy (Orian-Rousseau et al., 2007). (Kitajiri et al., 2004). This is a clear-cut example for the ERM proteins could also regulate signaling by modu- functional redundancy of radixin and ezrin in vivo. Muta- lating organization of lipid rafts which are thought to 348 V. Niggli, J. Rossy / The International Journal of Biochemistry & Cell Biology 40 (2008) 344–349 be platforms for organization of signaling molecules. Acknowledgements In resting B lymphocytes, ezrin appears to link lipid rafts to the cortical actin network. Transient activation- We thank E. Sigel for careful reading of the dependent dissociation of ezrin from lipid rafts appears manuscript and Y. Fidalgo for help with preparing the to be a trigger for the release of lipid rafts from the corti- figures. Our work is supported by the Swiss National cal actin network, allowing stimulus-dependent lipid raft Science Foundation (grant no. 3100A0-103708/1). coalescence (Gupta et al., 2006). References 5. Possible medical application Baumgartner, M., Sillman, A. L., Blackwood, E. M., Srivastava, J., In two pediatric tumors, ezrin overexpression Madson, N., Schilling, J. W., et al. (2006). The Nck-interacting kinase phosphorylates ERM proteins for formation of lamel- has been shown to correlate with clinical stage. lipodium by growth factors. Proc. Natl. Acad. Sci. U.S.A., 103, Concomitantly, reduction of ezrin expression in rhab- 13391–13396. domyosarcoma or osteosarcoma cell lines results in a Barret, C., Roy, C., Montcourrier, P., Mangeat, P., & Niggli, V. (2000). decrease in pulmonary metastases in mice. In these Mutagenesis of the PIP2 binding site in the N-terminal domain of cell lines, ezrin may contribute to metastasis by sup- ezrin correlates with its altered cellular distribution. J. Cell Biol., 151, 1067–1079. pressing apoptosis, disturbing cell–cell adhesion and Bretscher, A., Edwards, K., & Fehon, R. G. (2002). ERM proteins and activating Rho. Depending on the type of adult human merlin: Integrators at the cell cortex. Nat. Rev. Mol. Cell Biol., 3, tumor, loss or upregulation of ezrin correlates with poor 586–599. prognosis indicating cell-specific functions of ezrin in Charrin, S., & Alcover, A. (2006). Role of ERM (ezrin-radixin-moesin) tumor progression (Hunter, 2004). As ERM proteins proteins in T lymphocyte polarization, immune synapse forma- tion and in T cell receptor-mediated signalling. Front. Biosci., 11, have been implicated in Ras signaling (Orian-Rousseau 1987–1997. et al., 2007), uncontrolled Ras activation via ERM pro- Doi, Y., Itoh, M., Yonemura, S., Ishihara, S., Takano, H., Noda, T., et teins could for example promote tumor progression. al. (1999). Normal development of mice and unimpaired cell adhe- sion/cell motility/actin-based cytoskeleton without compensatory up-regulation of ezrin or radixin in moesin gene knockout. J. Biol. 6. Open questions Chem., 274, 2315–2321. Fievet,´ B., Louvard, D., & Arpin, M. (2007). ERM proteins in epithelial Concerning structure–function relationship, crys- cell organization and functions. Biochim. Biophys. Acta., 1773, 653–660. tal structures of intact dormant and activated ERM Gupta, N., Wollscheid, B., Watts, J. D., Scheer, B., Aebersold, R., & molecules are required for the full understanding of the DeFranco, A. L. (2006). Quantitative proteomic analysis of B cell molecular events during activation. The binding site for lipid rafts reveals that ezrin regulates antigen receptor-mediated phosphoinositides should be defined more precisely, as lipid raft dynamics. Nat. Immunol., 7, 625–633. well as the conformational changes resulting from its Hamada, K., Shimizu, T., Matsui, T., Tsukita, S., & Hakoshima, T. (2000). Structural basis of the membrane-targeting and unmask- occupancy and from phosphorylation of the C-terminal ing mechanisms of the radixin FERM domain. EMBO J., 19, threonine. Information on specific protein binding sites 4449–4462. (except for C-ERMAD-N-ERMAD interactions) is yet Hunter, K. W. (2004). Ezrin, a key component in tumor metastasis. lacking. It is also not clear whether ERM proteins can Trends Mol. Med., 10, 201–204. interact simultaneously with several interaction partners. Ivetic, A., & Ridley, A. J. (2004). Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes. Immunology, 112, 165–176. Concerning biological functions, the precise func- Khan, S. Y., Ahmed, Z. M., Shabbir, M. I., Kitajiri, S. I., Kalsoom, tions of ERM proteins in cell polarization, tail retraction S., Tasneem, S., et al. (2007). Mutations of the RDX gene cause and migration will have to be elucidated using downreg- nonsyndromic hearing loss at the DFNB24 locus. Hum. Mut., 28, ulation of specific isoforms. Targeted disruption of all 417–423. three ERM proteins in tissues is required for elucidation Kikuchi, M., Hata, K., Fukumoto, Y., Yamane, Y., Matsui, T., Tamura, A., et al. (2002). Radixin deficiency causes conjugated hyperbiliru- of their in vivo functions. binemia with loss of Mrp2 from bile canalicular membranes. Nat. More data on the role of ezrin and the other ERM Genet., 31, 320–325. proteins in human tumor development and metastasis are Kitajiri, S., Fukumoto, K., Hata, M., Sasaki, H., Katsuno, T., Nak- required to decide, whether the role of ERM proteins in agawa, T., et al. (2004). Radixin deficiency causes deafness these processes varies depending on the tissue, whether associated with progressive degeneration of cochlear stereocilia. J. Cell Biol., 166, 559–570. different isoforms play different roles and whether their Lee, J. H., Katakai, T., Hara, T., Gonda, H., Sugai, M., & Shimizu, A. role in tumor progression can for example be explained (2004). Roles of p-ERM and Rho-ROCK signaling in lymphocyte by their impact on Ras signaling. polarity and uropod formation. J. Cell Biol., 167, 327–337. V. Niggli, J. Rossy / The International Journal of Biochemistry & Cell Biology 40 (2008) 344–349 349

Li, Q., Nance, M. R., Kulikauskas, R., Nyberg, K., Fehon, R., Karplus, Ras activation requires ERM proteins linked to both CD44␯6 and P. A., et al. (2007). Self-masking in an intact ERM-merlin protein: F-actin. Mol. Biol. Cell, 18, 76–83. An active role for the central ␣-helical domain. J. Mol. Biol., 365, Rossy, J., Gutjahr, M. C., Blaser, N., Schlicht, D., & Niggli, V. (2007). 1446–1459. Ezrin/moesin in motile Walker 256 carcinosarcoma cells: Signal- Loebrich, S., Bahring,¨ R., Katsuno, T., Tsukita, S., & Kneussel, M. dependent relocalization and role in cell migration. Exp. Cell Res., (2006). Activated radixin is essential for GABAA receptor ␣5 313, 1106–1120. subunit anchoring at the actin cytoskeleton. EMBO J., 25, 987– Saotome, I., Curto, M., & McClatchey, A. I. (2004). Ezrin is essen- 999. tial for epithelial organization and villus morphogenesis in the Niggli, V., Andreoli,´ C., Roy, C., & Mangeat, P. (1995). Identification developing intestine. Dev. Cell., 6, 855–864. of a phosphatidylinositol-4,5-bisphosphate-binding domain in the Tamura, A., Kikuchi, S., Hata, M., Katsuno, T., Matsui, T., Hayashi, N-terminal region of ezrin. FEBS Lett., 376, 172–176. H., et al. (2005). Achlorhydria by ezrin knockdown: Defects in the Orian-Rousseau, V., Morrisson, H., Matzke, A., Kastilan, T., Pace, formation/expansion of apical canaliculi in gastric parietal cells. J. G., Herrlich, P., et al. (2007). Hepatocyte growth factor-induced Cell Biol., 169, 21–28.