Title: Cortical Actin Organization: Lessons from ERM (Ezrin/Radixin/Moesin) Proteins
Abstract: ezrin/radixin/moesin ERM-binding phosphoprotein of 50 kDa protein-tyrosine phosphatase Na+/H+ exchanger regulatory factor protein kinase A ERM-association domain phosphatidylinositol 4,5-bisphosphate guanosine 5′-O-(3-thio)triphosphate phosphatidylinositol 4-phosphate 5-kinase GDP dissociation inhibitor In recent years to clarify the molecular mechanism of dynamic organization of the cortical actin filaments, which is important not only for the determination of cell-surface structures but also for the functions of integral membrane proteins themselves, various types of submembrane proteins involved in cortical actin filament/plasma membrane interaction have been intensively studied. In this minireview, we focus on ezrin/radixin/moesin (ERM)1 proteins, which are general cross-linkers between cortical actin filaments and plasma membranes and are involved in the formation of microvilli, cell adhesion sites, ruffling membranes, and cleavage furrows. ERM proteins have attracted a great deal of interest because their functions have been shown to be regulated by the Rho signaling pathway (for recent reviews, see Refs. 1Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 2Tsukita Sa Yonemura S. Tsukita Sh Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (313) Google Scholar, 3Tsukita Sa Yonemura S. Tsukita Sh Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (275) Google Scholar, 4Vaheri A. Carpén O. Heiska L. Helander T.S. Jääskeläinen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (165) Google Scholar, 5del Pozo M.A. Nieto M. Serrador J.M. Sancho D. Vicente-Manzanares M. Martinez C. Sanchez-Madrid F. Cell Adhes. Commun. 1998; 6: 125-133Crossref PubMed Scopus (68) Google Scholar, 6Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (334) Google Scholar, 7Mangeat P. Roy C. Martin M. Trends Cell Biol. 1999; 9: 187-192Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). The ERM family consists of three closely related proteins, ezrin, radixin, and moesin (ERM proteins) (8Sato N. Funayama N. Nagafuchi A. Yonemura S. Tsukita Sa Tsukita Sh J. Cell Sci. 1992; 103: 131-143PubMed Google Scholar) (Fig.1). Ezrin (∼82 kDa) was first isolated from chicken intestinal brush borders as a component of microvilli (9Bretcher A. J. Cell Biol. 1983; 97: 425-432Crossref PubMed Scopus (256) Google Scholar). Molecular cloning revealed that ezrin was identical to cytovillin, which was enriched in microvilli of human placental syncytiotrophoblasts (10Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (244) Google Scholar, 11Turunen O. Winqvist R. Pakkanen R. Grzeschik K.-H. Wahlström T. Vaheri A. J. Biol. Chem. 1989; 264: 16727-16732Abstract Full Text PDF PubMed Google Scholar). Radixin (∼80 kDa) was isolated from rat liver as a component of adherens junctions (12Tsukita Sa Hieda Y. Tsukita Sh J. Cell Biol. 1989; 108: 2369-2382Crossref PubMed Scopus (187) Google Scholar). Moesin (∼75 kDa) was isolated from bovine uterus abundant in smooth muscle cells as a heparin-binding protein (13Lankes W. Griesmacher A. Grünwald J. Schwartz-Albiez R. Keller R. Biochem. J. 1988; 251: 831-842Crossref PubMed Scopus (117) Google Scholar). Homologues for ERM proteins have been found from Caenorhabditis elegans to human, although the number of family members appears to vary from one to three depending on species (2Tsukita Sa Yonemura S. Tsukita Sh Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (313) Google Scholar). The sequences of their N-terminal halves are highly conserved (∼85% identity) and similar to the N-terminal half of human erythroid band 4.1 protein (∼78 kDa), indicating that the ERM family is included in the band 4.1 superfamily that contains merlin/schwannomin (a tumor suppressor molecule for neurofibromatosis type II), talin, PTP-H1, and PTP-MEG. Among these, merlin (isoforms I–III)(∼70 kDa) is fairly similar to ERM proteins (∼60% identity). The sequence, which is conserved among the members of the band 4.1 superfamily and referred to as the FERM (4.1 and ERM) domain, is a membrane-binding site in band 4.1 protein, and similar sequences have recently been found in the central portion of PTP-BAS and the C-terminal domain of myosin VIIA. In ERM proteins, the N-terminal FERM domain is followed by an extended α-helical domain and a charged C-terminal domain, which includes a consensus sequence motif for actin binding. Thus, from their structure, ERM proteins have been suggested to function as cross-linkers between actin filaments and plasma membranes (1Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 2Tsukita Sa Yonemura S. Tsukita Sh Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (313) Google Scholar, 3Tsukita Sa Yonemura S. Tsukita Sh Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (275) Google Scholar, 4Vaheri A. Carpén O. Heiska L. Helander T.S. Jääskeläinen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (165) Google Scholar, 5del Pozo M.A. Nieto M. Serrador J.M. Sancho D. Vicente-Manzanares M. Martinez C. Sanchez-Madrid F. Cell Adhes. Commun. 1998; 6: 125-133Crossref PubMed Scopus (68) Google Scholar, 6Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (334) Google Scholar, 7Mangeat P. Roy C. Martin M. Trends Cell Biol. 1999; 9: 187-192Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Immunoblotting analysis and immunofluorescence microscopy revealed that in most cultured cells all ERM proteins are co-expressed and co-localized but that in organs their expression and distribution pattern appear to be regulated in a cell type-specific manner (1Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 2Tsukita Sa Yonemura S. Tsukita Sh Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (313) Google Scholar, 3Tsukita Sa Yonemura S. Tsukita Sh Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (275) Google Scholar, 4Vaheri A. Carpén O. Heiska L. Helander T.S. Jääskeläinen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (165) Google Scholar, 5del Pozo M.A. Nieto M. Serrador J.M. Sancho D. Vicente-Manzanares M. Martinez C. Sanchez-Madrid F. Cell Adhes. Commun. 1998; 6: 125-133Crossref PubMed Scopus (68) Google Scholar, 6Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (334) Google Scholar, 7Mangeat P. Roy C. Martin M. Trends Cell Biol. 1999; 9: 187-192Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Immunofluorescence studies of cultured fibroblasts and epithelial cells have revealed that ERM proteins are co-expressed and co-concentrated at cell-surface structures such as microvilli, filopodia, uropods, ruffling membranes, retraction fibers, and cell adhesion sites where actin filaments are associated with plasma membranes (8Sato N. Funayama N. Nagafuchi A. Yonemura S. Tsukita Sa Tsukita Sh J. Cell Sci. 1992; 103: 131-143PubMed Google Scholar, 14Sato N. Yonemura S. Obinata T. Tsukita Sa Tsukita Sh J. Cell Biol. 1991; 113: 321-330Crossref PubMed Scopus (115) Google Scholar, 15Franck Z. Gary R. Bretscher A. J. Cell Sci. 1993; 105: 219-231Crossref PubMed Google Scholar, 16Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar, 17Serrador J.M. Alonso-Lebrero J.L. del Pozo M.A. Furthmayr H. Schwartz-Albiez R. Calvo J. Lozano F. Sánchez-Madrid F. J. Cell Biol. 1997; 138: 1409-1423Crossref PubMed Scopus (205) Google Scholar) (Fig.2). ERM proteins are also concentrated specifically at cleavage furrows in dividing cells (14Sato N. Yonemura S. Obinata T. Tsukita Sa Tsukita Sh J. Cell Biol. 1991; 113: 321-330Crossref PubMed Scopus (115) Google Scholar) but not along cytoplasmic actin filaments such as stress fibers, in contrast to filamin and α-actinin, which are concentrated in both sites (18Nunnally M.H. D'Angelo J.M. Graig S.W. J. Cell Biol. 1980; 87: 219-226Crossref PubMed Scopus (66) Google Scholar). Suppression of the expression of all ERM proteins with antisense oligonucleotides in cultured fibroblasts/epithelial cells destroyed microvillus formation as well as cell-to-cell/cell-to-substrate adhesion (19Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita Sa Tsukita Sh J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (326) Google Scholar). Similarly, in cultured neurons that contain mainly radixin and moesin, antisense oligonucleotides of radixin and moesin severely affected the morphology, motility, and process formation of growth cones (20Paglini G. Kunda P. Quiroga S. Kosik K. Cáceres A. J. Cell Biol. 1998; 143: 443-455Crossref PubMed Scopus (142) Google Scholar). Specific ezrin ablation by MicroCALI (chromatophore-assisted laser irradiation) blocked membrane ruffling and motility (21Lamb R.F. Ozanne B.W. Roy C. McGarry L. Stipp C. Mangeat P. Jay D.G. Curr. Biol. 1997; 7: 682-688Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Furthermore, overproduction of full-length ERM proteins appeared to enhance cell adhesion, whereas that of their C-terminal halves perturbed the cell-surface morphology and inhibited cytokinesis (22Martin M. Andréoli C. Sahuquet A. Montcourrier P. Algrain M. Mangeat P. J. Cell Biol. 1995; 128: 1081-1093Crossref PubMed Scopus (122) Google Scholar, 23Henry M.D. Agosti C.G. Solomon F. J. Cell Biol. 1995; 129: 1007-1022Crossref PubMed Scopus (99) Google Scholar). These findings suggested that ERM proteins were involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and plasma membranes. Extensive functional analyses suggested the possible functional redundancy of ERM proteins at least at the cellular level. Recently, moesin-deficient mice were generated by gene targeting, and they appeared normal without any compensatory up-regulation of ezrin or radixin (24Doi Y. Itoh M. Yonemura S. Ishihara S. Takano H. Noda T. Tsukita Sh Tsukita Sa J. Biol. Chem. 1999; 274: 2315-2321Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Therefore, also at the whole body level ERM proteins appear to be functionally redundant, although ERM proteins are not necessarily co-localized and co-expressed at the organ level. Targeted disruption of ezrin and radixin genes will allow clarification of this redundancy problem in the near future. The C-terminal halves of ERM proteins bind to F-actin through their major actin-binding sites, the C-terminal 34 amino acids, which are highly conserved among these proteins (25Turunen O. Wahlström T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (356) Google Scholar). In addition to this domain, two more actin-binding domains have recently been identified in their N-terminal and middle regions, which bind to F-actin and both F- and G-actin, respectively (26Roy C. Martin M. Mangeat P. J. Biol. Chem. 1997; 272: 20088-20095Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although the physiological relevance of these newly identified actin-binding sites in ERM proteins is not clear at present, the mode of association of actin filaments with ERM proteins does not appear to be simple. G-actin binding affinity in their middle regions would explain the actin barbed end-capping activity of ERM proteins, which was detected in radixin at low ionic strength (12Tsukita Sa Hieda Y. Tsukita Sh J. Cell Biol. 1989; 108: 2369-2382Crossref PubMed Scopus (187) Google Scholar). On the other hand, the N-terminal halves of ERM proteins were reported to directly bind to the cytoplasmic domains of CD44 (27Tsukita Sa Oishi K. Sato N. Sagara J. Kawai A. 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Nature. 1996; 382: 265-268Crossref PubMed Scopus (201) Google Scholar, 30Heiska L. Alfthan K. Grönholm M. Vilja P. Vaheri A. Carpén O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 31Yonemura S. Nagafuchi A. Sato N. Tsukita Sh J. Cell Biol. 1993; 120: 437-449Crossref PubMed Scopus (140) Google Scholar, 32Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita Sa Tsukita Sh J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (514) Google Scholar). Although the cytoplasmic domains of these integral membrane proteins have no shared sequences, their juxtamembrane positively charged amino acid clusters are thought to be responsible for their binding to ERM proteins (32Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita Sa Tsukita Sh J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (514) Google Scholar, 33Legg J.W. Isacke C.M. Curr. Biol. 1998; 8: 705-708Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). This direct binding of ERM proteins with integral membrane proteins was shown to be essential for cell-surface morphogenesis such as microvillus formation (34Yonemura S. Tsukita Sa Tsukita Sh J. Cell Biol. 1999; 145: 1497-1509Crossref PubMed Scopus (182) Google Scholar, 35Kenney D. Cairns L. Remold-O'Donnell E. Peterson J. Rosen F.S. Parkman R. Blood. 1986; 68: 1329-1332Crossref PubMed Google Scholar). Ezrin effected the function of ICAM-2 in thymoma cells for being targeted by natural killer cells (29Helander T.S. Carpén O. Turunen O. Kovanen P.E. Vaheri A. Timmonen T. Nature. 1996; 382: 265-268Crossref PubMed Scopus (201) Google Scholar). The mechanism of indirect binding of ERM proteins to integral membrane proteins has also been reported. EBP-50 (ERM-binding phosphoprotein of 50 kDa) was identified as a cytoplasmic protein, the C-terminal region of which binds to the N-terminal half of ezrin (36Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (519) Google Scholar). Sequence analyses revealed that EBP-50 is identical to a Na+/H+exchanger regulatory factor (NHE-RF). This NHE-RF and its isoform, E3KARP, bear two PDZ domains, which were shown to directly bind to the C terminus of NHE3 (36Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (519) Google Scholar, 37Reczek D. Bretscher A. J. Biol. Chem. 1998; 273: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Thus, NHE-RF and E3KARP can function as adapters between NHE3 and ezrin (38Dransfield D.T. Bradford A.J. Smith J. Martin M. Roy C. Mangeat P.H. Goldenring J.R. EMBO J. 1997; 16: 35-43Crossref PubMed Scopus (270) Google Scholar). Interestingly, because NHE-RF regulates the NHE3 function in a protein kinase A (PKA)-dependent manner and because ezrin specifically binds to the RII subunit of PKA (38Dransfield D.T. Bradford A.J. Smith J. Martin M. Roy C. Mangeat P.H. Goldenring J.R. EMBO J. 1997; 16: 35-43Crossref PubMed Scopus (270) Google Scholar), ezrin appears to play an important role in recruiting PKA to the NHE3·NHE-RF complex to regulate the function of NHE3. Furthermore, NHE-RF and E3KARP were found to also be associated with other integral membrane proteins such as the β2-adrenergic receptor and the cystic fibrosis transmembrane conductance regulator (39Hall R.A. Premont R.T. Chow C.-W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (524) Google Scholar, 40Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), which are not always co-localized with ERM proteins (41Moyer B.D. Loffing J. Schweibert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The physiological relevance of the existence of two mechanisms of binding of ERM proteins to integral membrane proteins, direct and indirect, is an interesting subject for future study. Given that the cortical actin filaments are dynamically organized in response to various signals, the cross-linking activity of ERM proteins between actin filaments and plasma membranes is expected to be dynamically regulated. Indeed, the pioneering work by Bretscher (42Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (376) Google Scholar) or Hanzel et al. (43Hanzel D.K. Urushidani T. Usinger W.R. Smolka A. Forte J.G. Am. J. Physiol. 1989; 256: G1082-G1089Crossref PubMed Google Scholar) showed that EGF treatment of A431 cells or the secretion-stimulation of parietal cells rapidly recruited substantial amounts of ERM proteins to the cortical actin layer with concomitant phosphorylation of ERM proteins. When conventionally cultured cells were homogenized and centrifuged in physiological saline, ERM proteins were partitioned almost equally into the soluble and insoluble fractions (44Kondo T. Takeuchi K. Doi Y. Yonemura S. Nagata S. Tsukita Sh Tsukita Sa J. Cell Biol. 1997; 139: 749-758Crossref PubMed Scopus (139) Google Scholar). These findings suggested that there are active (insoluble) and inactive (soluble) forms of ERM proteins in terms of their cross-linking activity inside cells. Evidence has accumulated in vitro and in vivothat the N- and C-terminal halves of ERM proteins mutually interact intramolecularly and suppress their actin filament and membrane binding activities, respectively (45Andréoli C. Martin M. Le Borgne R. Reggio H. Mangeat P. J. Cell Sci. 1994; 107: 2509-2521PubMed Google Scholar, 46Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (380) Google Scholar, 47Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Recently, it was shown that when two amino acid residues were deleted from the C-terminal end of ezrin, which do not correspond to the EBP-50-binding domain, the interdomain interaction was affected, allowing ezrin to directly interact with EBP-50 (37Reczek D. Bretscher A. J. Biol. Chem. 1998; 273: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). These findings indicated that conformational masking by intramolecular interdomain interaction is the molecular mechanism behind the inactivation of ERM proteins. The region involved in the interdomain interaction was narrowed down to residues 1–297 and 480–586 (1–296 and 479–585 when methionine 1 is posttranslationally removed) as exemplified in ezrin, and these regions are called N- and C-ERMADs (ERM-association domains), respectively (46Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (380) Google Scholar). Initially, these N- and C-ERMADs were thought to be responsible for oligomerization of ERM proteins, i.e. intermolecular interaction of ERM proteins, but it has been suggested that they are also important for intramolecular interaction (6Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (334) Google Scholar, 7Mangeat P. Roy C. Martin M. Trends Cell Biol. 1999; 9: 187-192Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Furthermore, yeast two-hybrid analyses identified several middle regions between N- and C-ERMADs that interact with N- and C-ERMADs (48Bhartur S.G. Goldenring J.R. Biochem. Biophys. Res. Commun. 1998; 243: 874-877Crossref PubMed Scopus (8) Google Scholar). Although the molecular mechanism and physiological relevance of dimerization and oligomerization of ERM proteins remain elusive, it is now accepted that the intramolecular mutual suppression mechanism keeps ERM proteins in an inactive state and that some activation signal may release this suppression to activate ERM proteins inside cells. To date, two molecular events have been shown to generate and/or maintain the active form of ERM proteins in vitro: phosphorylation of their C-terminal threonine residue and PIP2 binding to their N-terminal domains. The phosphorylation of ERM proteins, especially ezrin, has been examined in detail. EGF stimulation induced tyrosine and serine phosphorylation of ezrin in A431 cells with concomitant translocation from the cytoplasm to the cortical actin layer (42Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (376) Google Scholar). The tyrosine residues that were phosphorylated in A431 cells were shown to be Tyr-146 and Tyr-354 (Tyr-145 and Tyr-353 when methionine 1 is posttranslationally removed) in ezrin, the former of which was conserved in radixin and moesin (49Krieg J. Hunter T. . 1992; 267: 19258-19265Google Scholar). ERM proteins were also reported to be heavily tyrosine-phosphorylated by v-Src and hepatocyte growth factor (50Takeda H. Nagafuchi A. Yonemura S. Tsukita Sa Behrens J. Birchmeier W. Tsukita Sh J. Cell Biol. 1995; 131: 1839-1847Crossref PubMed Scopus (199) Google Scholar, 51Crepaldi T. Gautreau A. Comoglio P.M. Louvard D. Arpin M. J. Cell Biol. 1997; 138: 423-434Crossref PubMed Scopus (287) Google Scholar). However, the substitution of Tyr-146 with phenylalanine in ezrin showed some effects on cell motility but did not affect the cortical localization of ezrin induced by hepatocyte growth factor. Thus, the direct activation of ERM proteins by tyrosine phosphorylation is unlikely. Secretion/stimulation in gastric parietal cells was reported to induce serine/threonine phosphorylation of ezrin (43Hanzel D.K. Urushidani T. Usinger W.R. Smolka A. Forte J.G. Am. J. Physiol. 1989; 256: G1082-G1089Crossref PubMed Google Scholar). In platelets thrombin activation induced the phosphorylation of moesin at a specific C-terminal threonine residue (Thr-558) (52Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1995; 270: 31377-31385Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), causing filopodia formation. This site was effectively phosphorylated in vitro by ROKα/ROCK-II/Rho-kinase in radixin (Thr-564) and by PKC-θ (53Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita Sa Tsukita Sh J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (733) Google Scholar, 54Pietromonaco S.F. Simons P.C. Altman A. Elias L. J. Biol. Chem. 1998; 273: 7594-7603Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, it is likely that ROCK kinases do not phosphorylate ERM proteins in vivo (55Matsui T. Yonemura S. Tsukita Sh Tsukita Sa Curr. Biol. 1999; 9: 1259-1262Abstract Full Text Full Text PDF PubMed Google Scholar). In vitrofunctional analyses suggested that the C-terminal threonine phosphorylation maintains ERM proteins in the active state by suppressing the intramolecular interaction (53Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita Sa Tsukita Sh J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (733) Google Scholar). Immunofluorescence microscopy with monoclonal antibodies specific for C-terminal threonine-phosphorylated ERM proteins revealed that ERM proteins localized beneath plasma membranes were actually phosphorylated at the C-terminal threonine in vivo (53Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita Sa Tsukita Sh J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (733) Google Scholar, 56Hayashi K. Yonemura S. Matsui T. Tsukita Sa Tsukita Sh J. Cell Sci. 1999; 112: 1149-1158Crossref PubMed Google Scholar). Taken all together, it seems that the threonine phosphorylation just maintains the activated ERM proteins. Another candidate for the activation signal for ERM proteins is PIP2, which has been shown to directly bind to the N-terminal halves of ERM proteins in vitro (28Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh Tsukita Sa J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (513) Google Scholar, 57Niggli V. Andréoli C. Roy C. Mangeat P. FEBS Lett. 1995; 376: 172-176Crossref PubMed Scopus (166) Google Scholar). Recently, it has been shown that PIP2 is a key factor for the activation of ERM proteins in vivo (55Matsui T. Yonemura S. Tsukita Sh Tsukita Sa Curr. Biol. 1999; 9: 1259-1262Abstract Full Text Full Text PDF PubMed Google Scholar). The question has thus arisen as to the identities of the upstream factors required for activation of ERM proteins. Rho, one of the small GTP-binding proteins, is now considered to be a general regulator of actin-based cytoskeletal organization. To date, in vitro as well as in vivo analyses have suggested an intimate relationship between the Rho signaling pathway and activation of ERM proteins. First, the binding ability of ERM proteins to the cytoplasmic domain of CD44 in crude cell homogenate was reported to be enhanced by activation of Rho (28Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh Tsukita Sa J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (513) Google Scholar). In semi-permeabilized Swiss 3T3 cells, at least one of the ERM proteins was shown to be required for Rho-dependent formation of stress fibers and focal contacts (58Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20686Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Furthermore, transfection of the constitutively active mutant of RhoA (V14RhoA), but not that of Rac1 or Cdc42, induced microvillus formation to which ERM proteins were recruited (55Matsui T. Yonemura S. Tsukita Sh Tsukita Sa Curr. Biol. 1999; 9: 1259-1262Abstract Full Text Full Text PDF PubMed Google Scholar, 59Shaw R.J. Henry M. Solomon F. Jacks T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar). Thus, it is now accepted that when Rho is activated in vivo, ERM proteins in the cytoplasm are activated and recruited to plasma membranes to form microvilli. Although ERM proteins are also suggested to be located downstream of Rac (58Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20686Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar), the activation of ERM proteins to form microvilli specifically depends on Rho but not on Rac. Rho has been reported to activate several serine/threonine kinases such as ROKα/ROCK-II/Rho-kinase, ROKβ/ROCK-I, citron kinase, protein kinase N, and protein kinase C1 (58Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20686Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Immunoblotting with a monoclonal antibody specific for C-terminal threonine-phosphorylated ERM proteins revealed that in serum-starved Swiss 3T3 cells Rho activation by lysophosphatidic acid stimulation increased the levels of the C-terminal threonine phosphorylation of ERM proteins (53Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita Sa Tsukita Sh J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (733) Google Scholar). Rho-kinase, which effectively phosphorylates the C-terminal threonines of ERM proteins in vitro, is not responsible for this Rho-dependent threonine phosphorylation of ERM proteins in vivo. PIP2-producing phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) has also been reported to be a direct Rho effector (for a review, see Ref. 60Ren X.-D. Schwartz M.A. Curr. Opin. Genet. Dev. 1998; 8: 63-67Crossref PubMed Scopus (78) Google Scholar). Because activation of dormant ERM proteins was induced by PIP2 in vivo as well asin vitro (28Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh Tsukita Sa J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (513) Google Scholar, 55Matsui T. Yonemura S. Tsukita Sh Tsukita Sa Curr. Biol. 1999; 9: 1259-1262Abstract Full Text Full Text PDF PubMed Google Scholar), one possible pathway for the activation of ERM proteins is as follows. Rho may activate PI4P5K, which in turn increases the amount of PIP2. PIP2 then activates ERM proteins by inhibiting their interdomain interaction, which allows phosphorylation of their C-terminal threonine residue by unidentified kinases. The C-terminally threonine-phosphorylated ERM proteins are stabilized as activated forms, which function as actin filament/plasma membrane cross-linkers to form microvilli (Fig.3). On the other hand, immunoprecipitation experiments identified Rho-GDI (GDP dissociation inhibitor) in the CD44-ERM protein complex (28Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh Tsukita Sa J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (513) Google Scholar). Anin vitro binding study then revealed that active but not inactive forms of ERM proteins directly bound to Rho-GDI at their N-terminal halves (61Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita Sa Tsukita Sh Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Interestingly, this binding of ERM proteins suppressed GDI activity of Rho-GDI, i.e. GDP-Rho was released from Rho-GDI, followed by activation as GTP-Rho possibly through Dbl (GDP/GTP exchange protein) (61Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita Sa Tsukita Sh Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 62Takahashi K. Sasaki T. Mammoto A. Hotta I. Takaishi K. Imamura H. Nakano K. Kodama A. Takai Y. Oncogene. 1998; 16: 3279-3284Crossref PubMed Scopus (104) Google Scholar). These findings suggested that ERM proteins, once activated, can activate Rho, which again activates ERM proteins as a positive feedback system. In this sense, ERM proteins are located not only downstream but also upstream of Rho. Because native monomers of ERM proteins have a tendency to inactivate themselves by the interdomain interaction, the down-regulation of activation signals may inactivate ERM proteins inside cells. For example, at the initial phase of apoptosis, ERM proteins were dephosphorylated and inactivated, resulting in their cytoplasmic translocation with concomitant microvillar breakdown (44Kondo T. Takeuchi K. Doi Y. Yonemura S. Nagata S. Tsukita Sh Tsukita Sa J. Cell Biol. 1997; 139: 749-758Crossref PubMed Scopus (139) Google Scholar). In this process, the C-terminal threonine was confirmed to be dephosphorylated by the monoclonal antibody specific for C-terminal threonine-phosphorylated ERM proteins. 2Sa. Tsukita, unpublished data. Recently, myosin light chain phosphatase was shown to bind to moesin through its myosin-binding subunit in vitro, although its physiological relevance remains unclear (63Fukata Y. Kimura K. Oshiro N. Saya H. Matsuura Y. Kaibuchi K. J. Cell Biol. 1998; 141: 409-418Crossref PubMed Scopus (183) Google Scholar). On the other hand, proteolysis could be another method of inactivation of ERM proteins because ezrin is a good substrate for calpain in vitro (6Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (334) Google Scholar, 7Mangeat P. Roy C. Martin M. Trends Cell Biol. 1999; 9: 187-192Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Although evidence is still lacking, on and off activation and/or inactivation signals and their balance may regulate the function of ERM proteins. Some, but not all, of the characteristics of ERM proteins appear to be shared by merlin. The subcellular localization of ERM proteins was similar to that of merlin in fibroblasts (microvilli and ruffling membranes) but different in epithelial cells; merlin, but not ERM proteins, was concentrated at lateral membranes together with E-cadherin (64Maeda M. Matsui T. Imamura M. Tsukita Sh Tsukita Sa Oncogene. 1999; 18: 4788-4797Crossref PubMed Scopus (67) Google Scholar). The N-terminal half of merlin bound to the cytoplasmic domains of CD44 (65Sainio 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: 2249-2260Crossref PubMed Google Scholar) and NHE-RF (66Murthy A. Gonzalez-Agosti C. Cordero E. Pinney D. Candia C. Solomon F. Gusella J. Ramesh V. J. Biol. Chem. 1998; 273: 1273-1276Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) as well as Rho-GDI in vitro (64Maeda M. Matsui T. Imamura M. Tsukita Sh Tsukita Sa Oncogene. 1999; 18: 4788-4797Crossref PubMed Scopus (67) Google Scholar). Merlin has two major alternatively spliced isoforms, I and II, which differ at their C-terminal ends (67Hara T. Bianchi A.B. Seizinger B.R. Kley N. Cancer Res. 1994; 54: 330-335PubMed Google Scholar). Although neither of these C-terminal ends shows any similarity to those of ERM proteins, the major actin-binding domains, merlin has been reported to bind to actin filaments at its middle region (68Xu H.-M. Gutmann D.H. J. Neurosci. Res. 1998; 51: 403-415Crossref PubMed Scopus (142) Google Scholar). Interdomain interaction between the N- and C-terminal halves has been suggested in isoform-I, but the interaction does not appear to affect its binding affinity to actin filaments or to NHE-RF (66Murthy A. Gonzalez-Agosti C. Cordero E. Pinney D. Candia C. Solomon F. Gusella J. Ramesh V. J. Biol. Chem. 1998; 273: 1273-1276Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 68Xu H.-M. Gutmann D.H. J. Neurosci. Res. 1998; 51: 403-415Crossref PubMed Scopus (142) Google Scholar). No interdomain interaction has been detected in isoform-II (69Sherman L. Xu H.-M. Geist R.T. Saporito-Irwin S. Howells N. Ponta H. Herrlich P. Gutmann D.H. Oncogene. 1997; 15: 2505-2509Crossref PubMed Scopus (205) Google Scholar). At present, there are three distinct possible explanations for the sequence similarity between ERM proteins and merlin. First, it is possible that merlin functions in cells by competing for shared binding partners such as CD44, NHE-RF, and Rho-GDI. However, because the molar ratio of endogenous merlin/ERM was calculated to be 0.05–0.15 in cultured fibroblasts and epithelial cells, the ERM-merlin competition is not likely in vivo. Second, merlin would be functionally redundant for ERM proteins. Data from the suppression experiments of ERM proteins by antisense oligonucleotides discussed above are inconsistent with this explanation. Furthermore, this explanation was not supported by the observation that merlin-deficient mice showed an embryonic lethal phenotype (70McClatchey A.I. Saotome I. Mercer K. Crowley D. Gusella J.F. Bronson R.T. Jacks T. Genes Dev. 1998; 12: 1121-1133Crossref PubMed Scopus (325) Google Scholar). Third, it is also possible that merlin shows sequence similarity to ERM proteins because both are involved in Rho signaling pathways; for example, both bind to Rho-GDI. If this is the case, loss of function mutations of merlin may induce tumorigenesis through some disturbance in the Rho signaling pathway. In this connection, it should be pointed out that neurofibromin, which is responsible for neurofibromatosis type I, shows GTPase-activating protein activity for Ras (71Xu G.F. O'Connell P. Viskochil D. Cawthon R. Robertson M. Culver M. Dunn D. Stevens J. Gesteland R. White R. Weiss R. Cell. 1990; 62: 599-608Abstract Full Text PDF PubMed Scopus (986) Google Scholar). It is reasonable to speculate that both neurofibromatosis types I and II are caused by some dysregulation of small GTP-binding protein-dependent signaling pathways. In the past decade, it has been established that ERM proteins function as general cross-linkers in the cortical layer, coupled with signal transduction pathways such as Rho signaling. Because ERM proteins are expressed almost ubiquitously, this cross-linking system would be involved in various cellular events in various types of cells. Thus, in the coming decade, ERM proteins will attract increasing interest in many fields from not only biological but also medical researchers. For example, in the immune system ERM proteins are thought to play an important role in cell recognition of T lymphocytes by producing uropods (17Serrador J.M. Alonso-Lebrero J.L. del Pozo M.A. Furthmayr H. Schwartz-Albiez R. Calvo J. Lozano F. Sánchez-Madrid F. J. Cell Biol. 1997; 138: 1409-1423Crossref PubMed Scopus (205) Google Scholar, 29Helander T.S. Carpén O. Turunen O. Kovanen P.E. Vaheri A. Timmonen T. Nature. 1996; 382: 265-268Crossref PubMed Scopus (201) Google Scholar). It will also be interesting to study the interactions between ERM proteins and microtubules. Radixin was characterized as a marginal microtubule band-associated protein in nucleated erythrocytes (72Birgbauer E. Solomon F. J. Cell Biol. 1989; 109: 1609-1620Crossref PubMed Scopus (52) Google Scholar), and in activated T-lymphocytes tubulin was co-concentrated at the uropods together with ERM proteins (17Serrador J.M. Alonso-Lebrero J.L. del Pozo M.A. Furthmayr H. Schwartz-Albiez R. Calvo J. Lozano F. Sánchez-Madrid F. J. Cell Biol. 1997; 138: 1409-1423Crossref PubMed Scopus (205) Google Scholar). Furthermore, it was also shown that ERM proteins have some homology with Tea 1, which is localized on the ends of microtubules and is critical for polarization in yeast (73Vega L.R. Solomon F. Cell. 1997; 89: 825-828Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Further studies of the ERM proteins-microtubule interaction will provide new insight into the physiological functions of ERM proteins as well as the cortical actin layer.