- Email: [email protected]
moesin) family: from cytoskeleton to signal transduction
70
ERM (ezrin/radixin/moesin) transduction Sachiko ERM
Tsukita*f,
family consists
Shigenobu
of three
and moesin,
family: from cytoskeleton
closely
Yonemura*
related
are thought
to work
as
and actin-based cytoskeletons.
Recent
of
proteins
involved
not
of the structure
and functions are
in cytoskeletal
organization
in
but also
and Shoichiro
to signal
Tsukita*$
in the band 4.1 superfamily [l,Z]. When the aminoand carboxy-terminal halves of ezrin were transfected into cultured fibroblasts, they were targeted to plasma membranes and actin-filament bundles, respectively [l]. This suggested that full-length ERM proteins work as plasma-membrane-actin-filament cross-linkers.
of the
neurofibromatosis to ERM interest
proteins,
has increased
Figure 1
in this family. Membrane-binding domain
Addresses of Cell Biology,
of Medicine,
Kyoto University,
Mouse Radixin
[email protected]
Mouse Moesin
Current
Actinbinding domain
in Cell Biology
Ltd ISSN 0955-0674
EGF
Mouse Ezrin
Kyoto
of Medical Technology,
a-helical domain
GDI
epidermal growth factor ezrin/radixin/moesin guanine
PIP
phosphatidylinositol
Drosophila Moesin Sea urchin Moesin C. elegans ERM
4-phosphate Human Merlin
Introduction cytoskeletons play a central role in cell motility and morphogenesis, and are believed to be involved in intracellular signal transduction. Compared with other cytoskeletal components such as intermediatesized filaments and microtubules, one of the characteristic aspects of actin filaments is their intimate interaction with plasma membranes. Detailed analyses of the molecular bases for this actin-filament-plasma-membrane interaction are required to increase understanding of the functions of the actin-based cytoskeleton.
Drosophila Merlin
Echinococcus
0 1997 Current Opinion in Cell Biolq ERM and related proteins in various species.
and homologues are found in various species (Fig. 1). The sequence of their amino-terminal halves is highly conserved (-80% identity for any pair in mouse or for any pair in human), and this sequence is also found in the amino-terminal ends of various submembranous proteins, including the erythrocyte membrane protein band 4.1 protein, indicating that the ERM family is included
ERM proteins consist of
three domains: a globular amino-terminal membrane-binding domain, followed by an extended a-helical domain and a positively charged carboxyterminal actin-binding domain. The percentage sequence identity with mouse moesin in each domain is indicated at the amino acid sequence
Three closely-related -80 kDa proteins, ezrin, radixin, and moesin, are thought to function as general cross-linkers between plasma membranes and actin-based cytoskeletons [l]. The encoding genes constitute a gene family, which is now called the ERM (ezrin/radixin/moesin) family,
multilocularis
EM10
level. P, polyproline stretch. These sequence
are available from the European
Molecular
data
Biology Laboratory
(URL:
http://www.ebi.ac.uk), GenBank (URL: http://www.ncbi.nlm.nih.gov), and DNA Data Bank of Japan (URL: http://www.ddbj.nig.ac.jp) databases under accession numbers X60671, X60672, S47577, L38909, the top.
U14180,
U10414,
L11353,
U49724
and M61186,
from
Independently of these lines of study, another ERMlike protein was identified as a tumour suppressor for hereditary neurofibromatosis type 2, and named merlin (moesin/ezrin/radixin-like protein) or schwannomin [l].
ERM family: from cytoskeleton
As shown in Figure 1, the merlin/schwannomin is strikingly
amino-terminal similar to that
half of of ERM
proteins. Here, we review current progress in studies of ERM proteins and merlin/schwannomin that were reported after the previous review of this subject in Current Opinion in Cell Biology [l]. Most of these studies consist of transfection experiments and in vitro binding experiments. To avoid confusion, merlin/schwannomin is not regarded as a member of the ERM family, as its carboxy-terminal half is not similar to that of ERM proteins [l,Z]. We initially take the position that ezrin, radixin, and moesin are essentially indistinguishable with respect to their properties, and then we discuss potential differences between these proteins. Furthermore, ERM proteins may interact with microtubule systems [3], and may be involved in measles virus infection [4,5] and rabies virus budding [6], but these aspects are not discussed in detail in this review.
Functional
analyses
Ezrin is directly involved in gastric acid secretion by parietal cells [l] and in the correct targeting of natural killer cells [7]. Takeuchi et al. [8] directly studied the in viva role of ERM proteins. They cultured mouse epithelial and thymoma cells, which coexpress all of the ERM proteins, in the presence of antisense phosphorothioate oligonucleotides complementary to ERM gene sequences. These antisense oligonucleotides selectively suppressed the expression of each ERM. Cells in which either one ERM protein or two different ERM proteins were suppressed showed no significant phenotypic changes, whereas the ezrin/radixin/moesin antisense oligonucleotide mixture suppressed the expression of all ERM proteins, inducing the destruction of cell-cell/cell-substrate adhesion in addition to the disappearance of microvilli. This is the most direct functions so far.
approach
to the
elucidation
of ERM
Considering that ERM proteins may work as cross-linkers through the respective interactions of their amino- and carboxy-terminal halves with plasma membranes and actin filaments [ 11, the amino- and carboxy-terminal-half constructs of ERM proteins should show some dominantnegative effects in viva. Several reports have indeed described dominant-negative effects. When the carboxyterminal half of ezrin is introduced into insect Sf9 cells or CHO cells, the actin-based cytoskeletal organization is remarkably affected, resulting in the formation of numerous cellular protrusions [9]. The introduction of the carboxy-terminal half of radixin into cultured fibroblasts appears to affect cytokinesis such that multinucleated cells form [lo’]. Furthermore, a Drosophila construct lacking the carboxy-terminal end of moesin reportedly interferes with the cytokinesis of fission yeast cells [ll]. These ERM
findings revealed proteins in cell-cell
the functional involvement of adhesion, cell-substrate adhesion,
to signal transduction
Tsukita, Yonemura and Tsukita
71
formation and maintenance of microvillar structures, and cytokinesis. As these cellular events are tightly coupled with the plasma-membrane-actin-filament interaction, these findings favored the notion that ERM proteins are general cross-linkers between plasma membranes and actin filaments.
Molecular bases for actin-filament plasma-membrane binding
and
As mentioned above, the carboxy-terminal half of ERM proteins is thought to be involved in their interaction with actin-based cytoskeletons [ 11. The recombinant carboxy-terminal half of ezrin and moesin bound directly to actin filaments with high affinity in vitro [l&13], and the actin-binding site of ezrin has been narrowed down to the carboxy-terminal-end 34 amino acids [la]. The carboxy-terminal domain of ezrin is highly conserved in radixin and moesin and shows sequence similarity to the potential actin-binding sites of other actin-binding proteins such as the myosin heavy chain and the B subunit of Cap2 [la]. Transfection studies showed that the remaining part of ERM proteins, namely the highly conserved aminoterminal half, is the membrane-binding domain [l]. This domain shows sequence similarity to the amino-terminal half of band 4.1 protein, which is reportedly responsible for binding to glycophorin C, a major glycosylated integral membrane protein in erythrocyte membranes [1,14]. This suggests that the amino-terminal region of ERM proteins is also directly associated with some integral membrane protein. Immunoprecipitation studies using BHK cells identified CD44, a polymorphic cell-surface glycoprotein expressed in a variety of cells, as one of the membrane-binding partners [14]. Direct binding between the amino-terminal halves of ERM proteins and the cytoplasmic domain of CD44 was also confirmed in vitro [15”]. Although CD44 is precisely colocalized with ERM proteins in cultured fibroblasts, it does not necessarily do so in tissue, suggesting that there may be other membrane-binding partners [ 1,16”]. Actually, CD44-deficient lymphoma cells appear normal [17], and CD43 and the H+/K+-ATPase pump precisely colocalize with ERM proteins in some cells [l]. Intercellular adhesion molecule-l may be directly associated with ezrin [7]. The presence of domains in ERM functions. Only about fibroblasts, however, membranes, where cross-linkers [8,15”].
both actinand proteins explains
membrane-binding their cross-linking
half of the ERM proteins in cultured are located just beneath the plasma they function as membrane-actin The other half of the ERM proteins
assume a soluble form in the cytoplasm and do not tightly associate with actin filaments. These findings suggest that a sophisticated regulation mechanism is involved in the cross-linking activity of ERM proteins.
72
Cytoskeleton
Regulation Model
of the cross-linking
for regulation
of the cross-linking
activity
Fiaure 2
activity
At least in vitro, both the actin-binding and the membranebinding sites of full-length native ERM proteins appear to be masked. As mentioned above, the carboxy-terminal half of ezrin binds to actin filaments with relatively high affinity [ 12,131. The interaction of denatured full-length ezrin and moesin with actin filaments has also been detected by gel overlay assays [ 131. The binding of native full-length ERM proteins to actin filaments has not, however, been directly established under physiological conditions. One recent paper describes that purified ezrin binds to nonmuscle fl-actin filaments with high affinity [18], whereas another reported the opposite [19]. At physiological ionic strength, full-length ERM proteins have very low affinity to the cytoplasmic domain of CD44 in vitro, whereas the amino-terminal halves of ERM proteins lacking carboxy-terminal halves bind to CD44 with high affinity [15”]. Considering that a significant amount of monomeric ezrin is detected as a soluble form in the cytoplasm [16”,20], these findings led to the proposal of an intramolecular head-to-tail association model for ERM protein activation and inactivation (Fig. 2). In native ERM proteins, the aminoand carboxy-terminal halves may mutually suppress each other’s functions, namely membraneand actin-binding, respectively, through an intramolecular head-to-tail association. Actually, the amino-terminal half of an ERM protein is directly associated with the carboxy-terminal half in vitro [‘Z-24]. The amino-terminal half of ezrin suppresses the dominant-negative effect of the ezrin in viva, that is, it suppresses protrusions [9].
carboxy-terminal half of the formation of cellular
This intramolecular head-to-tail association model is rather an oversimplification. Intermolecular head-to-tail association of ERM proteins is found in viva [16”,20], and the intermolecular and intramolecular interfaces between the amino- and carboxy-terminal halves appear to be distinct [ZO]. In the placenta, where ezrin and moesin are predominant, ezrin-ezrin, ezrin-moesin and moesin-moesin in addition
dimers and oligomers have been detected, to monomers. How the dimerization and
further oligomerization contribute to the activation inactivation of ERM proteins in viva remains unknown. Regulatory
or
signals
If the intramolecular mutual suppression mechanism is the cause of inactivation of ERM proteins, within cells some signals must release this suppression in order for ERM proteins to be able to function as cross-linkers just beneath the plasma membranes. Several types of signals can be considered. When people first began to study ezrin, they identified it as a good in viva substrate for tyrosine kinases such as epidermal growth factor (EGF) receptors [l]. Tyrosines 145 and 353 are phosphorylation sites in ezrin, and the former is also conserved in radixin and moesin
CD44 Lipid
’ bilayer
IIRM I
An intramolecular
head-to-tail
association
model for the activation
of ERM proteins. In native ERM proteins, their amino- and carboxyterminal halves (N and C, respectively) may mutually suppress their functions, namely membrane- and actin-binding respectively, through intramolecular head-to-tail association. Rho may release this suppression via some unidentified downstream signals to activate ERM proteins The carboxyl terminus
as membrane-actin-filament cross-linkers. of an ERM protein binds actin filaments,
whereas the amino terminus binds plasma membranes via a binding partner such as CD44, CD43, etc. This model is, however, rather an oversimplification,
as discussed
in the text.
[l]. Actually, radixin and moesin, are heavily tyrosine phosphorylated MDCK cells [25]. The physiological phosphorylation of ERM proteins
in addition to ezrin, in v-Src-transfected relevance of tyrosine remains unknown in
terms of ERM activation and inactivation, however, although it may be involved in their oligomerization [ 16”] and/or translocation to plasma membranes [26]. Another possible signal is serinelthreonine phosphorylation. Ezrin is also known to be serine phosphorylated, as well as tyrosine phosphorylated, in gastric parietal cells and cultured A431 cells [l]. In platelets, moesin is phosphorylated at a threonine residue in the carboxy-terminal actin-binding domain [27’]. This residue is also conserved in ezrin and radixin. Considering that, in kidney epithelial cells, the serine/threonine phosphorylated ezrin is reportedly concentrated in the insoluble fraction (probably the membrane fraction) [28], serine/threonine phosphorylation may be a key signal for the activation of ERM proteins. Two papers have described that ERM proteins specifically bind to phosphatidylinositol4-phosphate (PIP) and phosphatidylinositol 4,5_bisphosphate (PIP,) [15**,29]. As described above, native full-length ERM proteins have very low affinity for the cytoplasmic domain of CD44 at physiological ionic strength. PIP;! elevated this binding affinity up to a dissociation constant of -lOnM, suggesting that phosphoinositides are also key factors for the activation of ERM proteins [15”]. How are these signals controlled? In other words, what are the upstream factors required for activation of
ERM family: from cytoskeleton
phosphorylation and phosphoinositide synthesis? Rho, one of the small GTP-binding proteins, is now considered to be a general regulator of actin-based cytoskeletal organization, especially of actin-filament-plasma-membrane associations. Recent in vitro and in viva analyses suggest an intimate relationship between the Rho signalling pathway and activation of the actin-membrane cross-linking ability of ERM proteins [15”]. The in vitro binding of ERM proteins to the cytoplasmic domain of CD44 in the presence of crude cell homogenate is enhanced by GTPyS and completely suppressed by C3 toxin, a specific inhibitor of Rho. Furthermore, Rho guanine nucleotide dissociation inhibitor (GDI), an important regulator of Rho, is tightly associated with CD44-ERM complexes in viva [14]. In addition, Myc-tagged Rho introduced into MDCK cells colocalizes with ERM proteins [30]. Several intensive studies have been performed to identify the direct target of Rho, showing that Rho regulates phosphatidylinositol turnover [31]. For example, Rho activates PIP 5-kinase, which elevates the PIP, level in membranes. Rho also regulates the activity of some serine/threonine kinases [31]. It is possible that these kinases phosphorylate ERM proteins. It is thus fascinating to speculate that, through the activation and inactivation of ERM proteins, Rho can regulate the actin-based cytoskeletal organization in general.
Merlin/schwannomin Merlin/schwannomin was identified by two groups [l] as a tumour suppressor of neurofibromatosis type 2 that is associated with schwannomas (and sometimes meningiomas) [l]. This indicates that, at least in Schwann cells, the loss of merlin/schwannomin function results in the overgrowth of cells [32]. Current understanding of the physiological functions of this molecule remains frustratingly vague, however. Although its mRNA is detected in various tissues, histochemical analyses revealed that it is predominantly expressed in Schwann cells and smooth muscle cells [33,34]. The information about the subcellular distribution of endogenous merlin/schwannomin is still limited [35]. Some proteins are specifically bound to merlin/schwannomin in vitro, but they have not yet been well characterized [36]. Overexpression of merlin/schwannomin suppresses cell growth [37], and the suppression of its expression by antisense oligonucleotides results in reduced cell adhesion and increased growth of Schwann-like cells [38], but the molecular bases for these observations remain elusive. The lack of clear results from cell biological studies of merlin/schwannomin may be partly due to the difficulty of producing specific antibodies and partly due to its low level of expression. The fact that the amino-terminal half of merlin/schwannomin is highly homologous to that of ERM proteins suggests that merlin/schwannomin may compete with ERM proteins for their membrane-binding sites. If the expression level of merlin/schwannomin is much lower than that of
to signal transduction
Tsukita, Yonemura and Tsukita
73
ERM proteins, however, this notion is unlikely. Actually, Drosophila merlin/schwannomin and moesin have been identified, and their subcellular distributions are distinct from each other [39’]. Neurofibromatosis type 1 is also associated with overgrowth of Schwann cells, and its tumour suppressor, neurofibromin, functions as a GTPase-activating protein (GAP) of Ras. Merlin/schwannomin reportedly suppresses the Ras-induced transformation of fibroblasts [40]. As mentioned above, ERM proteins form a molecular complex with Rho-GDI, and ERM-CD44 complex formation is regulated by Rho [15”]. These findings suggest that merlin/schwannomin and ERM proteins are involved in distinct cellular events, and that their conserved amino-terminal-half domains are responsible for small GTP-binding protein dependent signaling pathways. This notion appears consistent with the fact that the carboxy-terminal end of merlin/schwannomin lacks the actin-binding sequence of ERM proteins [ lZ,Z?], although the actin-binding ability of merlin/schwannomin must be experimentally evaluated.
Similarities distribution moesin
and differences in the subcellular and functions of ezrin, radixin and
In this review, we have assumed the stance that ezrin, radixin, and moesin are essentially indistinguishable with respect to their properties. In cultured cells, all of them are concentrated at microvilli, ruffling membranes, cell-cell/cell-matrix adhesion sites (especially primordial forms of cell-cell/cell-matrix adherens junctions) and cleavage furrows, where actin filaments are densely associated with plasma membranes [ 1,8]. All of them have a similar binding affinity towards CD44 in vitro [ 15”], and bear putative actin-binding sites on their carboxy-terminal ends [la]. All of them bind similarly to phosphoinositides [15”], and appear to be tyrosine and/or serine/threonine phosphorylated. These findings favor the notion that the coexpression of ERM proteins in various types of cells is a safety measure. On the other hand, antisense-oligonucleotide experiments suggest that the physiological role of moesin is slightly different from those of ezrin and radixin [8]. In this context, among the ERM proteins only moesin lacks the polyproline stretch at the carboxy-terminal region (see Fig. 1). In tissues, the expression of ERM proteins varies depending on cell type [1,16”,41,42]. For example, in intestinal epithelial cells only ezrin and moesin are expressed, whereas hepatocytes express only radixin and moesin. Moesin is predominant in endothelial cells and platelets. This type of ERM protein expression in tissues suggests functional differences of ERM proteins. In this respect, it is notable that only moesin has been identified in Drosophila, despite an intensive search for ERM proteins [39’].
74
Cytoskeleton
Conclusions Although far more study is required to define the exact physiological functions of ERM proteins, there is no doubt that they are key players in plasma-membrane-actinfilament association. Experimental approaches to address the issues raised in this review will provide a more comprehensive picture of ERM-protein-dependent, as well as merlin/schwannomin-dependent, cellular events.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . l
*
of special interest of outstanding interest
1.
Arpin M, Algrain M, Louvard D: Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1. Curr Opin Cell Biol 1994, 6:136-l 41.
2.
Takeuchi K, Kawashima A, Nagafuchi A, Tsukita Sh: Structural diversity of band 4.1 superfamily members. J Cell SC; 1994, 107:1921-l 928.
3.
Winckler B, Gonzalez Agosti C, Magendantz M, Solomon F: Analysis of a cortical cytoskeletal structure: a role for ezrin-radixin-moesin (ERM proteins) in the marginal band of chicken erythrocytes. J Cell Sci 1994, 107:2523-2534.
4.
Dunster LM, Schneider-Schaulies J, Loffler S, Lankes W, SchwartzAlbiez R, Lottspeich F, Meulen V: Moesin: a cell membrane protein linked with susceptibility to measles virus infection. Virology 1994, 198:265-274.
5.
Schneider-Schaulies J, Dunster LM, Schwartz-Albiez R, Krohne G, Meulen V: Physical association of moesin and CD46 as a receptor complex for measles virus. J Krol 1995, 69:2248-2256.
6.
Sagara J, Tsukita Sa, Yonemura S, Tsukita Sh, Kawai A: Cellular actin-binding ezrin-radixin-moesin (ERM) family proteins are incorporated into the rabies virion and closely associated with viral envelope proteins in the cell. Virology 1995, 206:485-494.
7.
Helander TS, Carpen 0, Turunen 0, Kovanen PE, Vaheri A, Timonen T: ICAM- redistributed by ezrin as a target for killer cells. Nature 1996, 382:265-268.
8.
Takeuchi K, Sato N, Kasahara H, Funayama N, Nagafuchi A, Yonemura S, Tsukita Sa, Tsukita Sh: Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 1994, 125:1371-l 384.
9.
Martin M, Andreoli C, Sahuquet A, Montcourrier P, Algrain M, Manaeat P: Ezrin NH2-terminal domain inhibits the cell exteision activity of the COOH-terminal domain. J Cell Biol 1995, 128:1081-l 093.
10. .
Henry MD, Gonzalez Agosti C, Solomon F: Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains. J Cell Biol 1995, 129:1007-l 022. The authors of this paper suggest the distinct functions of the amino- and carboxy-terminal domains of radixin by means of transfection experiments. It is suggested that the functions of radixin in cells depend upon activities contributed by these two domains of the protein through modulating interactions between those domains. 11.
Edwards KA, Montague RA, Shepard S, Edgar BA, Erikson RL, Kiehart DP: Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. froc Nat/ Acad SC; USA 1994, 91:4589-4593.
12.
Turunen 0, Wahlstrom T, Vaheri A: Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol 1994, 126:1445-l 453.
13.
14.
Pestonjamasp K, Amieva MR, Strassel CP, Nauseef WM, Furthmayr H, Luna EJ: Moesin, ezrin, and ~205 are a&in-binding proteins associated with neutrophil plasma membranes. MO/ Biol Cell 1995, 6:247-259. Tsukita Sa, Oishi K, Sato N, Sagara J, Kawai A, Tsukita Sh: ERM family members as molecular linkers between the cell surface glycoprotein CD44 and a&in-based cytoskeletons. J Cell Biol 1994, 126:391-401.
15. ..
Hirao M, Sato N, Kondo T, Yonemura S, Monden M, Sasaki T, Takai Y, Tsukita Sh, Tsukita Sa: Regulation mechanism of ERM protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and rho-dependent signaling pathway. J Cell Biol 1996, 135~37-52. CD44 was identified as amembrane-binding partner of ERM proteins in BHK and L cells by immunoprecipitation using anti-moesin monoclonal antibody [141. This paper [15”1 shows that the binding of ERM proteins to the cytoplasmic domain of CD44 is regulated by phosphoinositides in vitro, and also provides evidence that Rho regulates the formation of the CD44-ERM complex. 16. ..
Berryman M, Gary R, Bretscher A: Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J Cell Biol 1995, 131 :1231-l 242. Shows that ezrin is a major component of isolated placental microvilli, where it exists primarily in oligomeric form, being cytoskeletally associated. This paper also describes the rapid formation of ezrin oligomers after tyrosine phospholylation of ezrin in EGF-stimulated A431 cells. 1 7.
Driessens MHE, Stroeken PJM, Rodriguez Erena NF, Van der Valk MA, Van Rijthoven EAM, Roos E: Targeted disruption of CD44 in MDAY-D2 lymphosarcoma cells has no effect on subcutaneous growth or metastatic capacity. J Cell Bioll995, 131 :1849-l 855.
18.
Yao X, Cheng L, Forte JG: Biochemical characterization of ezrin-actin interaction. J Biol Chem 1996, 271:7224-7229.
19.
Shuster CB, Herman IM: Indirect association of ezrin with Factin: isoform specificity and calcium sensitivity. J Cell Biol 1995, 128:837-848.
20.
Bretscher A, Gary R, Benyman M: Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains. Biochemistry 1995, 34:16830-l 6837.
21.
Gary R, Bretscher A: Heterotypic and homotypic associations between ezrin and moesin, two putative membranecytoskeletal linking proteins. froc Nat/ Acad Sci USA 1993, 90:10846-l 0850.
22.
Gary R, Bretscher A: Ezrin self-association involves binding of an N-terminal domain to a normallv masked C-terminal domain that includes the F-actin binding site. MO/ Biol Cell 1995, 6:1061-l 075.
23.
Andreoli C, Martin M, Le Borgne R, Reggio H, Mangeat P: Ezrin has properties to self-associate at the plasma membrane. J Cell Sci 1994, 107:2509-2521.
24.
Magendantz M, Henry MD, Lander A, Solomon F: Interdomain interactions of radixin in vitro. J Biol Chem 1995, 270:25324-25327.
25.
Takeda H, Nagafuchi A, Yonemura S, Tsukita Sa, Behrens J, Birchmeier W, Tsukita Sh: V-src kinase shifts the cadherinbased cell adhesion from the strong to the weak state and beta catenin is not required for the shift. J Cell Biol 1995, 131 :1839-l 847.
26.
Jiang WG, Hiscox S, Singhrao SK, Puntis MC, Nakamura T, Manse1 RE, Hallettt MB: Induction of tyrosine phosphorylation and translocation of ezrin by hepatocyte growth factor/scatter factor. Biocbem Siopbys Res Commun 1995, 217:1062-l 069.
27. .
Nakamura F, Amieva MR, Furthmayr H: Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J Biol Cbem 1995, 270:31377-31385. Reports the phospholylation and localization of moesin during early steps of platelet activation by thrombin. Upon exposure to thrombin, phospholylation of Thr558 of moesin remarkably increases, with drastic cytoskeletal rearrangement resulting, thus suggesting a model for regulated membrane-cytoskeleton interactions through moesin during platelet activation. 28.
Chen J, Cohn JA, Mandel U: Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. froc Nat/ Acad Sci USA 1995, 92:7495-7499.
29.
Niggli V, Andreoli C, Roy C, Mangeat P: Identification of a phosphatidylinositol-4,5-bisphosphate-binding domain in the N-terminal region of ezrin. FEES Leff 1995, 376:172-l 76.
30.
Takaishi K, Sasaki T, Kameyama T, Tsukita Sa, Tsukita Sh, Takai Y: Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 1995, 11:39-48.
ERM family: from cytoskeleton
31.
Nag& K, Hall A: The Rho GTPase regulates activity. Bioessays 1996, 18:529-531.
protein kinase
32.
Thomas G, Mere1 P, Sanson K, Hoang-Xuan K, Zucman J, Desmaze C, Melot T, Aurias A, Delattre 0: Neurofibromatosis type 2. Eur J Cancer 1994, 13:1981-l 987.
33.
Den Bakker MA, Riegman PH, Hekman RA, Boersma W, Janssen PJ, Van der Kwast TH, Zwarthoff EC: The product of the NF2 tumour suooressor aene localizes near the olasma membrane and is highly expressed in muscle cells. Oncogene 1995, 10:757-763.
34.
Sainz J, Huynh DP, Figueroa K, Ragge NK, Baser ME, Pulst SM: Mutations of the neurofibromatosis type 2 gene and lack of the gene product in vestibular schwannomas. Hum MO/ Genet 1994, 3:885-891.
35.
Gonzalez-Agosti C, XL. L, Pinney D, Beauchamp R, Hobbs W, Gusella J, Ramesh V: The merlin tumor suppressor localizes preferentially in membrane ruffles. Oncogene 1997, 13:in press.
36.
Takeshima H, lzawa I, Lee PS, Safdar N, Levin VA, Saya H: Detection of cellular proteins that interact with the NF2 tumor suppressor gene product. Oncogene 1994, 9:2135-2144.
37.
Lutchman M, Rouleau G: The neurofibromatosis type 2 gene product, schwannomin, suppresses growth of NIH3T3 cells. Cancer Res 1995, 55~2270-2274.
38.
to signal transduction
Tsukita, Yonemura and Tsukita
75
Huynh DP, Pulst SM: Neurofibromatosis 2 antisense oligonucleotides induce reversible inhibition of schwannomin synthesis and cell adhesion in STS26T and T98G cells. Oncogene 1996, 13:73-84.
39. .
McCartney BM, Fehon RG: Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J Cell Biol 1996, 133:843-852. Identifies and characterizes Drosophila homologues of moesin and of merlin, which show distinct subcellular localizationa It has been suggested that merlin has unique functions in the cell which differ from those of other ERM proteins. 40.
Tikoo A. Varaa M. Ramesh V. Gusella J. Maruta H: An antiRas function’ of neurofibromatosis type 2 gene product (NF2/Merlin). J Biol Chem 1994, 269:23387-23390.
41.
Amieva MR, Wilgenbus KK, Furthmayr H: Radixin is a component of hepatocyte microvili in situ. Exp Cell Res 1994, 210:140-l 44.
42.
Schwartz AR, Merling A, Spring H, Moller P, Koretz K: Differential expression of the microspike-associated protein moesin in human tissues. Eur J Cell Biol 1995, 67:189-l 98.
ERM (ezrin/radixin/moesin) transduction Sachiko ERM
Tsukita*f,
family consists
Shigenobu
of three
and moesin,
family: from cytoskeleton
closely
Yonemura*
related
are thought
to work
as
and actin-based cytoskeletons.
Recent
of
proteins
involved
not
of the structure
and functions are
in cytoskeletal
organization
in
but also
and Shoichiro
to signal
Tsukita*$
in the band 4.1 superfamily [l,Z]. When the aminoand carboxy-terminal halves of ezrin were transfected into cultured fibroblasts, they were targeted to plasma membranes and actin-filament bundles, respectively [l]. This suggested that full-length ERM proteins work as plasma-membrane-actin-filament cross-linkers.
of the
neurofibromatosis to ERM interest
proteins,
has increased
Figure 1
in this family. Membrane-binding domain
Addresses of Cell Biology,
of Medicine,
Kyoto University,
Mouse Radixin
[email protected]
Mouse Moesin
Current
Actinbinding domain
in Cell Biology
Ltd ISSN 0955-0674
EGF
Mouse Ezrin
Kyoto
of Medical Technology,
a-helical domain
GDI
epidermal growth factor ezrin/radixin/moesin guanine
PIP
phosphatidylinositol
Drosophila Moesin Sea urchin Moesin C. elegans ERM
4-phosphate Human Merlin
Introduction cytoskeletons play a central role in cell motility and morphogenesis, and are believed to be involved in intracellular signal transduction. Compared with other cytoskeletal components such as intermediatesized filaments and microtubules, one of the characteristic aspects of actin filaments is their intimate interaction with plasma membranes. Detailed analyses of the molecular bases for this actin-filament-plasma-membrane interaction are required to increase understanding of the functions of the actin-based cytoskeleton.
Drosophila Merlin
Echinococcus
0 1997 Current Opinion in Cell Biolq ERM and related proteins in various species.
and homologues are found in various species (Fig. 1). The sequence of their amino-terminal halves is highly conserved (-80% identity for any pair in mouse or for any pair in human), and this sequence is also found in the amino-terminal ends of various submembranous proteins, including the erythrocyte membrane protein band 4.1 protein, indicating that the ERM family is included
ERM proteins consist of
three domains: a globular amino-terminal membrane-binding domain, followed by an extended a-helical domain and a positively charged carboxyterminal actin-binding domain. The percentage sequence identity with mouse moesin in each domain is indicated at the amino acid sequence
Three closely-related -80 kDa proteins, ezrin, radixin, and moesin, are thought to function as general cross-linkers between plasma membranes and actin-based cytoskeletons [l]. The encoding genes constitute a gene family, which is now called the ERM (ezrin/radixin/moesin) family,
multilocularis
EM10
level. P, polyproline stretch. These sequence
are available from the European
Molecular
data
Biology Laboratory
(URL:
http://www.ebi.ac.uk), GenBank (URL: http://www.ncbi.nlm.nih.gov), and DNA Data Bank of Japan (URL: http://www.ddbj.nig.ac.jp) databases under accession numbers X60671, X60672, S47577, L38909, the top.
U14180,
U10414,
L11353,
U49724
and M61186,
from
Independently of these lines of study, another ERMlike protein was identified as a tumour suppressor for hereditary neurofibromatosis type 2, and named merlin (moesin/ezrin/radixin-like protein) or schwannomin [l].
ERM family: from cytoskeleton
As shown in Figure 1, the merlin/schwannomin is strikingly
amino-terminal similar to that
half of of ERM
proteins. Here, we review current progress in studies of ERM proteins and merlin/schwannomin that were reported after the previous review of this subject in Current Opinion in Cell Biology [l]. Most of these studies consist of transfection experiments and in vitro binding experiments. To avoid confusion, merlin/schwannomin is not regarded as a member of the ERM family, as its carboxy-terminal half is not similar to that of ERM proteins [l,Z]. We initially take the position that ezrin, radixin, and moesin are essentially indistinguishable with respect to their properties, and then we discuss potential differences between these proteins. Furthermore, ERM proteins may interact with microtubule systems [3], and may be involved in measles virus infection [4,5] and rabies virus budding [6], but these aspects are not discussed in detail in this review.
Functional
analyses
Ezrin is directly involved in gastric acid secretion by parietal cells [l] and in the correct targeting of natural killer cells [7]. Takeuchi et al. [8] directly studied the in viva role of ERM proteins. They cultured mouse epithelial and thymoma cells, which coexpress all of the ERM proteins, in the presence of antisense phosphorothioate oligonucleotides complementary to ERM gene sequences. These antisense oligonucleotides selectively suppressed the expression of each ERM. Cells in which either one ERM protein or two different ERM proteins were suppressed showed no significant phenotypic changes, whereas the ezrin/radixin/moesin antisense oligonucleotide mixture suppressed the expression of all ERM proteins, inducing the destruction of cell-cell/cell-substrate adhesion in addition to the disappearance of microvilli. This is the most direct functions so far.
approach
to the
elucidation
of ERM
Considering that ERM proteins may work as cross-linkers through the respective interactions of their amino- and carboxy-terminal halves with plasma membranes and actin filaments [ 11, the amino- and carboxy-terminal-half constructs of ERM proteins should show some dominantnegative effects in viva. Several reports have indeed described dominant-negative effects. When the carboxyterminal half of ezrin is introduced into insect Sf9 cells or CHO cells, the actin-based cytoskeletal organization is remarkably affected, resulting in the formation of numerous cellular protrusions [9]. The introduction of the carboxy-terminal half of radixin into cultured fibroblasts appears to affect cytokinesis such that multinucleated cells form [lo’]. Furthermore, a Drosophila construct lacking the carboxy-terminal end of moesin reportedly interferes with the cytokinesis of fission yeast cells [ll]. These ERM
findings revealed proteins in cell-cell
the functional involvement of adhesion, cell-substrate adhesion,
to signal transduction
Tsukita, Yonemura and Tsukita
71
formation and maintenance of microvillar structures, and cytokinesis. As these cellular events are tightly coupled with the plasma-membrane-actin-filament interaction, these findings favored the notion that ERM proteins are general cross-linkers between plasma membranes and actin filaments.
Molecular bases for actin-filament plasma-membrane binding
and
As mentioned above, the carboxy-terminal half of ERM proteins is thought to be involved in their interaction with actin-based cytoskeletons [ 11. The recombinant carboxy-terminal half of ezrin and moesin bound directly to actin filaments with high affinity in vitro [l&13], and the actin-binding site of ezrin has been narrowed down to the carboxy-terminal-end 34 amino acids [la]. The carboxy-terminal domain of ezrin is highly conserved in radixin and moesin and shows sequence similarity to the potential actin-binding sites of other actin-binding proteins such as the myosin heavy chain and the B subunit of Cap2 [la]. Transfection studies showed that the remaining part of ERM proteins, namely the highly conserved aminoterminal half, is the membrane-binding domain [l]. This domain shows sequence similarity to the amino-terminal half of band 4.1 protein, which is reportedly responsible for binding to glycophorin C, a major glycosylated integral membrane protein in erythrocyte membranes [1,14]. This suggests that the amino-terminal region of ERM proteins is also directly associated with some integral membrane protein. Immunoprecipitation studies using BHK cells identified CD44, a polymorphic cell-surface glycoprotein expressed in a variety of cells, as one of the membrane-binding partners [14]. Direct binding between the amino-terminal halves of ERM proteins and the cytoplasmic domain of CD44 was also confirmed in vitro [15”]. Although CD44 is precisely colocalized with ERM proteins in cultured fibroblasts, it does not necessarily do so in tissue, suggesting that there may be other membrane-binding partners [ 1,16”]. Actually, CD44-deficient lymphoma cells appear normal [17], and CD43 and the H+/K+-ATPase pump precisely colocalize with ERM proteins in some cells [l]. Intercellular adhesion molecule-l may be directly associated with ezrin [7]. The presence of domains in ERM functions. Only about fibroblasts, however, membranes, where cross-linkers [8,15”].
both actinand proteins explains
membrane-binding their cross-linking
half of the ERM proteins in cultured are located just beneath the plasma they function as membrane-actin The other half of the ERM proteins
assume a soluble form in the cytoplasm and do not tightly associate with actin filaments. These findings suggest that a sophisticated regulation mechanism is involved in the cross-linking activity of ERM proteins.
72
Cytoskeleton
Regulation Model
of the cross-linking
for regulation
of the cross-linking
activity
Fiaure 2
activity
At least in vitro, both the actin-binding and the membranebinding sites of full-length native ERM proteins appear to be masked. As mentioned above, the carboxy-terminal half of ezrin binds to actin filaments with relatively high affinity [ 12,131. The interaction of denatured full-length ezrin and moesin with actin filaments has also been detected by gel overlay assays [ 131. The binding of native full-length ERM proteins to actin filaments has not, however, been directly established under physiological conditions. One recent paper describes that purified ezrin binds to nonmuscle fl-actin filaments with high affinity [18], whereas another reported the opposite [19]. At physiological ionic strength, full-length ERM proteins have very low affinity to the cytoplasmic domain of CD44 in vitro, whereas the amino-terminal halves of ERM proteins lacking carboxy-terminal halves bind to CD44 with high affinity [15”]. Considering that a significant amount of monomeric ezrin is detected as a soluble form in the cytoplasm [16”,20], these findings led to the proposal of an intramolecular head-to-tail association model for ERM protein activation and inactivation (Fig. 2). In native ERM proteins, the aminoand carboxy-terminal halves may mutually suppress each other’s functions, namely membraneand actin-binding, respectively, through an intramolecular head-to-tail association. Actually, the amino-terminal half of an ERM protein is directly associated with the carboxy-terminal half in vitro [‘Z-24]. The amino-terminal half of ezrin suppresses the dominant-negative effect of the ezrin in viva, that is, it suppresses protrusions [9].
carboxy-terminal half of the formation of cellular
This intramolecular head-to-tail association model is rather an oversimplification. Intermolecular head-to-tail association of ERM proteins is found in viva [16”,20], and the intermolecular and intramolecular interfaces between the amino- and carboxy-terminal halves appear to be distinct [ZO]. In the placenta, where ezrin and moesin are predominant, ezrin-ezrin, ezrin-moesin and moesin-moesin in addition
dimers and oligomers have been detected, to monomers. How the dimerization and
further oligomerization contribute to the activation inactivation of ERM proteins in viva remains unknown. Regulatory
or
signals
If the intramolecular mutual suppression mechanism is the cause of inactivation of ERM proteins, within cells some signals must release this suppression in order for ERM proteins to be able to function as cross-linkers just beneath the plasma membranes. Several types of signals can be considered. When people first began to study ezrin, they identified it as a good in viva substrate for tyrosine kinases such as epidermal growth factor (EGF) receptors [l]. Tyrosines 145 and 353 are phosphorylation sites in ezrin, and the former is also conserved in radixin and moesin
CD44 Lipid
’ bilayer
IIRM I
An intramolecular
head-to-tail
association
model for the activation
of ERM proteins. In native ERM proteins, their amino- and carboxyterminal halves (N and C, respectively) may mutually suppress their functions, namely membrane- and actin-binding respectively, through intramolecular head-to-tail association. Rho may release this suppression via some unidentified downstream signals to activate ERM proteins The carboxyl terminus
as membrane-actin-filament cross-linkers. of an ERM protein binds actin filaments,
whereas the amino terminus binds plasma membranes via a binding partner such as CD44, CD43, etc. This model is, however, rather an oversimplification,
as discussed
in the text.
[l]. Actually, radixin and moesin, are heavily tyrosine phosphorylated MDCK cells [25]. The physiological phosphorylation of ERM proteins
in addition to ezrin, in v-Src-transfected relevance of tyrosine remains unknown in
terms of ERM activation and inactivation, however, although it may be involved in their oligomerization [ 16”] and/or translocation to plasma membranes [26]. Another possible signal is serinelthreonine phosphorylation. Ezrin is also known to be serine phosphorylated, as well as tyrosine phosphorylated, in gastric parietal cells and cultured A431 cells [l]. In platelets, moesin is phosphorylated at a threonine residue in the carboxy-terminal actin-binding domain [27’]. This residue is also conserved in ezrin and radixin. Considering that, in kidney epithelial cells, the serine/threonine phosphorylated ezrin is reportedly concentrated in the insoluble fraction (probably the membrane fraction) [28], serine/threonine phosphorylation may be a key signal for the activation of ERM proteins. Two papers have described that ERM proteins specifically bind to phosphatidylinositol4-phosphate (PIP) and phosphatidylinositol 4,5_bisphosphate (PIP,) [15**,29]. As described above, native full-length ERM proteins have very low affinity for the cytoplasmic domain of CD44 at physiological ionic strength. PIP;! elevated this binding affinity up to a dissociation constant of -lOnM, suggesting that phosphoinositides are also key factors for the activation of ERM proteins [15”]. How are these signals controlled? In other words, what are the upstream factors required for activation of
ERM family: from cytoskeleton
phosphorylation and phosphoinositide synthesis? Rho, one of the small GTP-binding proteins, is now considered to be a general regulator of actin-based cytoskeletal organization, especially of actin-filament-plasma-membrane associations. Recent in vitro and in viva analyses suggest an intimate relationship between the Rho signalling pathway and activation of the actin-membrane cross-linking ability of ERM proteins [15”]. The in vitro binding of ERM proteins to the cytoplasmic domain of CD44 in the presence of crude cell homogenate is enhanced by GTPyS and completely suppressed by C3 toxin, a specific inhibitor of Rho. Furthermore, Rho guanine nucleotide dissociation inhibitor (GDI), an important regulator of Rho, is tightly associated with CD44-ERM complexes in viva [14]. In addition, Myc-tagged Rho introduced into MDCK cells colocalizes with ERM proteins [30]. Several intensive studies have been performed to identify the direct target of Rho, showing that Rho regulates phosphatidylinositol turnover [31]. For example, Rho activates PIP 5-kinase, which elevates the PIP, level in membranes. Rho also regulates the activity of some serine/threonine kinases [31]. It is possible that these kinases phosphorylate ERM proteins. It is thus fascinating to speculate that, through the activation and inactivation of ERM proteins, Rho can regulate the actin-based cytoskeletal organization in general.
Merlin/schwannomin Merlin/schwannomin was identified by two groups [l] as a tumour suppressor of neurofibromatosis type 2 that is associated with schwannomas (and sometimes meningiomas) [l]. This indicates that, at least in Schwann cells, the loss of merlin/schwannomin function results in the overgrowth of cells [32]. Current understanding of the physiological functions of this molecule remains frustratingly vague, however. Although its mRNA is detected in various tissues, histochemical analyses revealed that it is predominantly expressed in Schwann cells and smooth muscle cells [33,34]. The information about the subcellular distribution of endogenous merlin/schwannomin is still limited [35]. Some proteins are specifically bound to merlin/schwannomin in vitro, but they have not yet been well characterized [36]. Overexpression of merlin/schwannomin suppresses cell growth [37], and the suppression of its expression by antisense oligonucleotides results in reduced cell adhesion and increased growth of Schwann-like cells [38], but the molecular bases for these observations remain elusive. The lack of clear results from cell biological studies of merlin/schwannomin may be partly due to the difficulty of producing specific antibodies and partly due to its low level of expression. The fact that the amino-terminal half of merlin/schwannomin is highly homologous to that of ERM proteins suggests that merlin/schwannomin may compete with ERM proteins for their membrane-binding sites. If the expression level of merlin/schwannomin is much lower than that of
to signal transduction
Tsukita, Yonemura and Tsukita
73
ERM proteins, however, this notion is unlikely. Actually, Drosophila merlin/schwannomin and moesin have been identified, and their subcellular distributions are distinct from each other [39’]. Neurofibromatosis type 1 is also associated with overgrowth of Schwann cells, and its tumour suppressor, neurofibromin, functions as a GTPase-activating protein (GAP) of Ras. Merlin/schwannomin reportedly suppresses the Ras-induced transformation of fibroblasts [40]. As mentioned above, ERM proteins form a molecular complex with Rho-GDI, and ERM-CD44 complex formation is regulated by Rho [15”]. These findings suggest that merlin/schwannomin and ERM proteins are involved in distinct cellular events, and that their conserved amino-terminal-half domains are responsible for small GTP-binding protein dependent signaling pathways. This notion appears consistent with the fact that the carboxy-terminal end of merlin/schwannomin lacks the actin-binding sequence of ERM proteins [ lZ,Z?], although the actin-binding ability of merlin/schwannomin must be experimentally evaluated.
Similarities distribution moesin
and differences in the subcellular and functions of ezrin, radixin and
In this review, we have assumed the stance that ezrin, radixin, and moesin are essentially indistinguishable with respect to their properties. In cultured cells, all of them are concentrated at microvilli, ruffling membranes, cell-cell/cell-matrix adhesion sites (especially primordial forms of cell-cell/cell-matrix adherens junctions) and cleavage furrows, where actin filaments are densely associated with plasma membranes [ 1,8]. All of them have a similar binding affinity towards CD44 in vitro [ 15”], and bear putative actin-binding sites on their carboxy-terminal ends [la]. All of them bind similarly to phosphoinositides [15”], and appear to be tyrosine and/or serine/threonine phosphorylated. These findings favor the notion that the coexpression of ERM proteins in various types of cells is a safety measure. On the other hand, antisense-oligonucleotide experiments suggest that the physiological role of moesin is slightly different from those of ezrin and radixin [8]. In this context, among the ERM proteins only moesin lacks the polyproline stretch at the carboxy-terminal region (see Fig. 1). In tissues, the expression of ERM proteins varies depending on cell type [1,16”,41,42]. For example, in intestinal epithelial cells only ezrin and moesin are expressed, whereas hepatocytes express only radixin and moesin. Moesin is predominant in endothelial cells and platelets. This type of ERM protein expression in tissues suggests functional differences of ERM proteins. In this respect, it is notable that only moesin has been identified in Drosophila, despite an intensive search for ERM proteins [39’].
74
Cytoskeleton
Conclusions Although far more study is required to define the exact physiological functions of ERM proteins, there is no doubt that they are key players in plasma-membrane-actinfilament association. Experimental approaches to address the issues raised in this review will provide a more comprehensive picture of ERM-protein-dependent, as well as merlin/schwannomin-dependent, cellular events.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . l
*
of special interest of outstanding interest
1.
Arpin M, Algrain M, Louvard D: Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1. Curr Opin Cell Biol 1994, 6:136-l 41.
2.
Takeuchi K, Kawashima A, Nagafuchi A, Tsukita Sh: Structural diversity of band 4.1 superfamily members. J Cell SC; 1994, 107:1921-l 928.
3.
Winckler B, Gonzalez Agosti C, Magendantz M, Solomon F: Analysis of a cortical cytoskeletal structure: a role for ezrin-radixin-moesin (ERM proteins) in the marginal band of chicken erythrocytes. J Cell Sci 1994, 107:2523-2534.
4.
Dunster LM, Schneider-Schaulies J, Loffler S, Lankes W, SchwartzAlbiez R, Lottspeich F, Meulen V: Moesin: a cell membrane protein linked with susceptibility to measles virus infection. Virology 1994, 198:265-274.
5.
Schneider-Schaulies J, Dunster LM, Schwartz-Albiez R, Krohne G, Meulen V: Physical association of moesin and CD46 as a receptor complex for measles virus. J Krol 1995, 69:2248-2256.
6.
Sagara J, Tsukita Sa, Yonemura S, Tsukita Sh, Kawai A: Cellular actin-binding ezrin-radixin-moesin (ERM) family proteins are incorporated into the rabies virion and closely associated with viral envelope proteins in the cell. Virology 1995, 206:485-494.
7.
Helander TS, Carpen 0, Turunen 0, Kovanen PE, Vaheri A, Timonen T: ICAM- redistributed by ezrin as a target for killer cells. Nature 1996, 382:265-268.
8.
Takeuchi K, Sato N, Kasahara H, Funayama N, Nagafuchi A, Yonemura S, Tsukita Sa, Tsukita Sh: Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 1994, 125:1371-l 384.
9.
Martin M, Andreoli C, Sahuquet A, Montcourrier P, Algrain M, Manaeat P: Ezrin NH2-terminal domain inhibits the cell exteision activity of the COOH-terminal domain. J Cell Biol 1995, 128:1081-l 093.
10. .
Henry MD, Gonzalez Agosti C, Solomon F: Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains. J Cell Biol 1995, 129:1007-l 022. The authors of this paper suggest the distinct functions of the amino- and carboxy-terminal domains of radixin by means of transfection experiments. It is suggested that the functions of radixin in cells depend upon activities contributed by these two domains of the protein through modulating interactions between those domains. 11.
Edwards KA, Montague RA, Shepard S, Edgar BA, Erikson RL, Kiehart DP: Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. froc Nat/ Acad SC; USA 1994, 91:4589-4593.
12.
Turunen 0, Wahlstrom T, Vaheri A: Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol 1994, 126:1445-l 453.
13.
14.
Pestonjamasp K, Amieva MR, Strassel CP, Nauseef WM, Furthmayr H, Luna EJ: Moesin, ezrin, and ~205 are a&in-binding proteins associated with neutrophil plasma membranes. MO/ Biol Cell 1995, 6:247-259. Tsukita Sa, Oishi K, Sato N, Sagara J, Kawai A, Tsukita Sh: ERM family members as molecular linkers between the cell surface glycoprotein CD44 and a&in-based cytoskeletons. J Cell Biol 1994, 126:391-401.
15. ..
Hirao M, Sato N, Kondo T, Yonemura S, Monden M, Sasaki T, Takai Y, Tsukita Sh, Tsukita Sa: Regulation mechanism of ERM protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and rho-dependent signaling pathway. J Cell Biol 1996, 135~37-52. CD44 was identified as amembrane-binding partner of ERM proteins in BHK and L cells by immunoprecipitation using anti-moesin monoclonal antibody [141. This paper [15”1 shows that the binding of ERM proteins to the cytoplasmic domain of CD44 is regulated by phosphoinositides in vitro, and also provides evidence that Rho regulates the formation of the CD44-ERM complex. 16. ..
Berryman M, Gary R, Bretscher A: Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J Cell Biol 1995, 131 :1231-l 242. Shows that ezrin is a major component of isolated placental microvilli, where it exists primarily in oligomeric form, being cytoskeletally associated. This paper also describes the rapid formation of ezrin oligomers after tyrosine phospholylation of ezrin in EGF-stimulated A431 cells. 1 7.
Driessens MHE, Stroeken PJM, Rodriguez Erena NF, Van der Valk MA, Van Rijthoven EAM, Roos E: Targeted disruption of CD44 in MDAY-D2 lymphosarcoma cells has no effect on subcutaneous growth or metastatic capacity. J Cell Bioll995, 131 :1849-l 855.
18.
Yao X, Cheng L, Forte JG: Biochemical characterization of ezrin-actin interaction. J Biol Chem 1996, 271:7224-7229.
19.
Shuster CB, Herman IM: Indirect association of ezrin with Factin: isoform specificity and calcium sensitivity. J Cell Biol 1995, 128:837-848.
20.
Bretscher A, Gary R, Benyman M: Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains. Biochemistry 1995, 34:16830-l 6837.
21.
Gary R, Bretscher A: Heterotypic and homotypic associations between ezrin and moesin, two putative membranecytoskeletal linking proteins. froc Nat/ Acad Sci USA 1993, 90:10846-l 0850.
22.
Gary R, Bretscher A: Ezrin self-association involves binding of an N-terminal domain to a normallv masked C-terminal domain that includes the F-actin binding site. MO/ Biol Cell 1995, 6:1061-l 075.
23.
Andreoli C, Martin M, Le Borgne R, Reggio H, Mangeat P: Ezrin has properties to self-associate at the plasma membrane. J Cell Sci 1994, 107:2509-2521.
24.
Magendantz M, Henry MD, Lander A, Solomon F: Interdomain interactions of radixin in vitro. J Biol Chem 1995, 270:25324-25327.
25.
Takeda H, Nagafuchi A, Yonemura S, Tsukita Sa, Behrens J, Birchmeier W, Tsukita Sh: V-src kinase shifts the cadherinbased cell adhesion from the strong to the weak state and beta catenin is not required for the shift. J Cell Biol 1995, 131 :1839-l 847.
26.
Jiang WG, Hiscox S, Singhrao SK, Puntis MC, Nakamura T, Manse1 RE, Hallettt MB: Induction of tyrosine phosphorylation and translocation of ezrin by hepatocyte growth factor/scatter factor. Biocbem Siopbys Res Commun 1995, 217:1062-l 069.
27. .
Nakamura F, Amieva MR, Furthmayr H: Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J Biol Cbem 1995, 270:31377-31385. Reports the phospholylation and localization of moesin during early steps of platelet activation by thrombin. Upon exposure to thrombin, phospholylation of Thr558 of moesin remarkably increases, with drastic cytoskeletal rearrangement resulting, thus suggesting a model for regulated membrane-cytoskeleton interactions through moesin during platelet activation. 28.
Chen J, Cohn JA, Mandel U: Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. froc Nat/ Acad Sci USA 1995, 92:7495-7499.
29.
Niggli V, Andreoli C, Roy C, Mangeat P: Identification of a phosphatidylinositol-4,5-bisphosphate-binding domain in the N-terminal region of ezrin. FEES Leff 1995, 376:172-l 76.
30.
Takaishi K, Sasaki T, Kameyama T, Tsukita Sa, Tsukita Sh, Takai Y: Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 1995, 11:39-48.
ERM family: from cytoskeleton
31.
Nag& K, Hall A: The Rho GTPase regulates activity. Bioessays 1996, 18:529-531.
protein kinase
32.
Thomas G, Mere1 P, Sanson K, Hoang-Xuan K, Zucman J, Desmaze C, Melot T, Aurias A, Delattre 0: Neurofibromatosis type 2. Eur J Cancer 1994, 13:1981-l 987.
33.
Den Bakker MA, Riegman PH, Hekman RA, Boersma W, Janssen PJ, Van der Kwast TH, Zwarthoff EC: The product of the NF2 tumour suooressor aene localizes near the olasma membrane and is highly expressed in muscle cells. Oncogene 1995, 10:757-763.
34.
Sainz J, Huynh DP, Figueroa K, Ragge NK, Baser ME, Pulst SM: Mutations of the neurofibromatosis type 2 gene and lack of the gene product in vestibular schwannomas. Hum MO/ Genet 1994, 3:885-891.
35.
Gonzalez-Agosti C, XL. L, Pinney D, Beauchamp R, Hobbs W, Gusella J, Ramesh V: The merlin tumor suppressor localizes preferentially in membrane ruffles. Oncogene 1997, 13:in press.
36.
Takeshima H, lzawa I, Lee PS, Safdar N, Levin VA, Saya H: Detection of cellular proteins that interact with the NF2 tumor suppressor gene product. Oncogene 1994, 9:2135-2144.
37.
Lutchman M, Rouleau G: The neurofibromatosis type 2 gene product, schwannomin, suppresses growth of NIH3T3 cells. Cancer Res 1995, 55~2270-2274.
38.
to signal transduction
Tsukita, Yonemura and Tsukita
75
Huynh DP, Pulst SM: Neurofibromatosis 2 antisense oligonucleotides induce reversible inhibition of schwannomin synthesis and cell adhesion in STS26T and T98G cells. Oncogene 1996, 13:73-84.
39. .
McCartney BM, Fehon RG: Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J Cell Biol 1996, 133:843-852. Identifies and characterizes Drosophila homologues of moesin and of merlin, which show distinct subcellular localizationa It has been suggested that merlin has unique functions in the cell which differ from those of other ERM proteins. 40.
Tikoo A. Varaa M. Ramesh V. Gusella J. Maruta H: An antiRas function’ of neurofibromatosis type 2 gene product (NF2/Merlin). J Biol Chem 1994, 269:23387-23390.
41.
Amieva MR, Wilgenbus KK, Furthmayr H: Radixin is a component of hepatocyte microvili in situ. Exp Cell Res 1994, 210:140-l 44.
42.
Schwartz AR, Merling A, Spring H, Moller P, Koretz K: Differential expression of the microspike-associated protein moesin in human tissues. Eur J Cell Biol 1995, 67:189-l 98.