The role of FAS to ezrin association in FAS-mediated apoptosis

The acquisition of a cell polarity is a crucial requirement for a number of cellular functions, including apoptosis. Cell polarization is an actin cytoskeleton-driven process, through a connection between actin and an increasing number of membrane proteins. The major actors in this connection are ezrin, radixin and moesin, a family of proteins with a high level of homology. Their structure includes an epitope that links to membrane proteins and the other that binds to the actin molecule. In this review we discuss recent data showing that the Fas linkage to the actin cytoskeleton is ezrin mediated and it is an essential requirement for susceptibility to the Fas-mediated apoptosis. The ezrin region responsible of Fas binding consists of 18 aminoacids mapped on the median lobe of the ezrin FERM domain. This binding is specific and of key importance in the control of cell homeostasis. Moreover, Fas-ezrin co-localization, ezrin phosphorylation and early acquisition of susceptibility to Fas-mediated apoptosis, may have a role in some human diseases in which programmed cell death seems to be a central pathogenetic mechanism, such as AIDS.Angelo De Milito was supported by a grant from the Swedish Research Council.     

Structure of the active N-terminal domain of Ezrin. Conformational and mobility changes identify keystone interactions

Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and also may function in signaling cascades that regulate the assembly of actin stress fibers. Here, we report a crystal structure for the free (activated) FERM domain (residues 2-297) of recombinant human ezrin at 2.3 A resolution. Structural comparison among the dormant moesin FERM domain structure and the three known active FERM domain structures (radixin, moesin, and now ezrin) allows the clear definition of regions that undergo structural changes during activation. The key regions affected are residues 135-150 and 155-180 in lobe F2 and residues 210-214 and 235-267 in lobe F3. Furthermore, we show that a large increase in the mobilities of lobes F2 and F3 accompanies activation, suggesting that their integrity is compromised. This leads us to propose a new concept that we refer to as keystone interactions. Keystone interactions occur when one protein (or protein part) contributes residues that allow another protein to complete folding, meaning that it becomes an integral part of the structure and would rarely dissociate. Such interactions are well suited for long-lived cytoskeletal protein interactions. The keystone interactions concept leads us to predict two specific docking sites within lobes F2 and F3 that are likely to bind target proteins.     

Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP₂) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP₂. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5 μM for PIP₂-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP₂-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30 nm²) binding to a single PIP₂ can block access to neighboring PIP₂ molecules and thus contributes to lower the accessible PIP₂ concentration. In addition, no evidence exists for a clustering of PIP₂ induced by ezrin addition.     

Structural basis for NHERF recognition by ERM proteins

The Na+/H+ exchanger regulatory factor (NHERF) is a key adaptor protein involved in the anchoring of ion channels and receptors to the actin cytoskeleton through binding to ERM (ezrin/radixin/moesin) proteins. NHERF binds the FERM domain of ERM proteins, although NHERF has no signature Motif-1 sequence for FERM binding found in adhesion molecules. The crystal structures of the radixin FERM domain complexed with the NHERF-1 and NHERF-2 C-terminal peptides revealed a peptide binding site of the FERM domain specific for the 13 residue motif MDWxxxxx(L/I)Fxx(L/F) (Motif-2), which is distinct from Motif-1. This Motif-2 forms an amphipathic alpha helix for hydrophobic docking to subdomain C of the FERM domain. This docking causes induced-fit conformational changes in subdomain C and affects binding to adhesion molecule peptides, while the two binding sites are not overlapped. Our studies provide structural paradigms for versatile ERM linkages between membrane proteins and the cytoskeleton.     

Calpain cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.     

Dual role of the Jak1 FERM and kinase domains in cytokine receptor binding and in stimulation-dependent Jak activation

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-gammaRalpha. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-gammaRalpha when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.     

The 2.7 A crystal structure of the activated FERM domain of moesin: an analysis of structural changes on activation

Moesin binds to a large range of proteins through its N terminal FERM (band 4.1, ezrin, radixin, moesin) domain. In full-length moesin isolated from cells, this binding is masked by binding to the C-terminal domain of moesin (C-ERMAD). Activation takes place by phosphorylation of Thr 558 in the C-ERMAD, which releases the C-ERMAD. A recently determined crystal structure of a noncovalent complex of the FERM and C-ERMAD domains showed for the first time that the structure of the FERM domain consists of three subdomains, each of which is similar to known structures. The structure reported here also contains a unique 47-residue helix pointing away from the FERM domain at the start of the alpha domain, in agreement with secondary structure predictions. Removal of the C-ERMAD does not result in a huge rearrangement of the FERM domain, but comparison with the activated radixin structure shows a consistent set of small changes. Not surprisingly, the exposed C-ERMAD binding area interacts in crystal contacts. More interestingly, a negatively charged peptide binds to the inositol site in a crystal contact and causes a greater conformational change in the structure than inositol.     

The FERM domain: organizing the structure and function of FAK

Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent structural work reveals that regulatory protein partners convert auto-inhibited FAK into its active state by binding to its FERM domain. Further, the identity of FAK FERM domain-interacting proteins yields clues as to how FAK coordinates diverse cellular responses, including cell adhesion, polarization, migration, survival and death, and suggests that FERM domains might mediate information transfer between the cell cortex and nucleus. Importantly, the FAK FERM domain might act as a paradigm for the actions of other FERM domain-containing proteins.     

Self-masking in an intact ERM-merlin protein: an active role for the central alpha-helical domain

Ezrin/radixin/moesin (ERM) family members provide a regulated link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and organization. Here, we report the crystal structure of intact insect moesin, revealing that its essential yet previously uncharacterized alpha-helical domain forms extensive interactions with conserved surfaces of the band four-point-one/ezrin/radixin/moesin (FERM) domain. These interdomain contacts provide a functional explanation for how PIP(2) binding and tyrosine phosphorylation of ezrin lead to activation, and provide an understanding of previously enigmatic loss-of-function missense mutations in the tumor suppressor merlin. Sequence conservation and biochemical results indicate that this structure represents a complete model for the closed state of all ERM-merlin proteins, wherein the central alpha-helical domain is an active participant in an extensive set of inhibitory interactions that can be unmasked, in a rheostat-like manner, by coincident regulatory factors that help determine cell polarity and membrane structure.     

Properties of an Ezrin Mutant Defective in F-actin Binding

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (αB, αC and αD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, αD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.     

Ezrin controls the macromolecular complexes formed between an adapter protein Na+/H+ exchanger regulatory factor and the cystic fibrosis transmembrane conductance regulator

Na(+)/H(+) exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)(2).NHERF.ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR.NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton.     

Unfurling of the band 4.1, ezrin, radixin, moesin (FERM) domain of the merlin tumor suppressor

The merlin-1 tumor suppressor is encoded by the Neurofibromatosis-2 (Nf2) gene and loss-of-function Nf2 mutations lead to nervous system tumors in man and to several tumor types in mice. Merlin is an ERM (ezrin, radixin, moesin) family cytoskeletal protein that interacts with other ERM proteins and with components of cell-cell adherens junctions (AJs). Merlin stabilizes the links of AJs to the actin cytoskeleton. Thus, its loss destabilizes AJs, promoting cell migration and invasion, which in Nf2(+/-) mice leads to highly metastatic tumors. Paradoxically, the "closed" conformation of merlin-1, where its N-terminal four-point-one, ezrin, radixin, moesin (FERM) domain binds to its C-terminal tail domain, directs its tumor suppressor functions. Here we report the crystal structure of the human merlin-1 head domain when crystallized in the presence of its tail domain. Remarkably, unlike other ERM head-tail interactions, this structure suggests that binding of the tail provokes dimerization and dynamic movement and unfurling of the F2 motif of the FERM domain. We conclude the "closed" tumor suppressor conformer of merlin-1 is in fact an "open" dimer whose functions are disabled by Nf2 mutations that disrupt this architecture.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

FERM domain phosphoinositide binding targets merlin to the membrane and is essential for its growth-suppressive function

The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP(2), via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.     

Structural determinants of integrin recognition by talin

The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior.     

CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.     

Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ~30 and ~100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH₂-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.     

Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells

Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho family. We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting of loss of stress fibers and formation of F-actin patches in membrane protrusions where the ERM protein moesin was recruited. Cell swelling increased the interaction between actin and moesin and induced the transition of moesin from an oligomeric to a monomeric functional conformation, characterized by both the COOH- and NH₂-terminal domains being exposed. In this conformation, which is stabilized by phosphorylation of a conserved threonine in the COOH-terminal domain by PKC or Rho kinase, moesin can bind interacting proteins. Interestingly, hypotonic stress increased the amount of threonine-phosphorylated moesin, which was prevented by the PKC- inhibitor Gö-6976 (50 nM). In contrast, the Rho kinase inhibitor Y-27632 (1 µM) did not affect the hypotonicity-induced increase in phosphorylated moesin. The present data represent the first evidence that hypotonicity-induced actin remodeling is associated with phosphorylated moesin recruitment at the cell border and interaction with actin. ezrin/radixin/moesin; protein kinase C; Rho     

ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.     

Ezrin mutants affecting dimerization and activation

ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane-associated proteins and the actin cytoskeleton. Previous work has shown that ezrin can exist in a dormant monomeric state in which the N-terminal FERM domain is tightly associated with the C-ERMAD (carboxyl-terminal ERM association domain), masking binding sites for at least some ligands, including F-actin and the scaffolding protein EBP50. Activation of ezrin requires relief of the intramolecular association, and this is believed to involve phosphorylation of threonine 567. Studies have therefore employed the T567D phosphomimetic mutant to explore the consequences of ezrin activation in vivo. Ezrin also exists as a stable dimer, in which the orientation of the two subunits is unknown, but might involve the central alpha-helical region predicted to form a coiled-coil. By characterization of ezrin mutants, we show that relief of the intramolecular association in the monomer results in unmasking of ligand binding sites and a significant conformational change, that the T567D mutation has a small effect on the biochemical activation of ezrin, and that the predicted coiled-coil region does not drive dimer formation. These results provide strong support for the conformational activation model of ezrin, elucidate the basis for dimer formation, and reveal that a mutant generally considered to be fully activated is not.