Ezrin/Radixin/Moesin (ERM) Proteins Bind to a Positively Charged Amino Acid Cluster in the Juxta-Membrane Cytoplasmic Domain of CD44, CD43, and ICAM-2

Abstract. CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20–30–amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.     

ERM (Ezrin/Radixin/Moesin)-based Molecular Mechanism of Microvillar Breakdown at an Early Stage of Apoptosis

Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)–induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1β–converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein–based microvillar breakdown in apoptosis will be discussed.     

Emerging role for ERM proteins in cell adhesion and migration

The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration.Key words: epithelial cells, membrane-cytoskeleton interface, morphogenesis, ERM proteins, cell adhesion     

Characterization of a novel interaction between ELMO1 and ERM proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.     

ERM proteins: from cellular architecture to cell signaling

ERM (ezrin/radixin/moesin) proteins, concentrated in actin rich cell-surface structures, cross-link actin filaments with the plasma membrane. They are involved in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell motility and membrane trafficking. Recent analyses reveal that they are not only involved in cytoskeleton organization but also in signaling pathway. They play an important role in the activation of members of the Rho family by recruiting their regulators. The functions of ERM proteins are regulated by their conformational charges: the intramolecular interaction between the N- and C-terminal domains of ERM proteins charges masks several binding sites, leading to a dormant protein. Different activation signals regulate ERM proteins functions by modulating these intramolecular interactions. The involvement of ERM proteins in many signaling pathways has led to study their role during development of different species.     

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.     

Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins

The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.     

Differential expression of ezrin/radixin/moesin (ERM) and ERM-associated adhesion molecules in the blastocyst and uterus suggests their functions during implantation

Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.     

Actin microdomains on endothelial cells: association with CD44, ERM proteins,and signaling molecules during quiescence and wound healing

During studies of the actin cytoskeleton in cultured endothelial cells we have observed that the luminal side of many cells contains F-actin microdomains that are rich in the hyaluronan receptor CD44 and in ezrin-radixin-moesin (ERM) proteins. A small subpopulation of the domains are also enriched in tyrosine phosphorylated proteins and signaling molecules. Confocal microscopy of rat aortic endothelial cells in situ demonstrated that similar microdomains occur in vivo. During healing of endothelial wounds, characteristic alterations of the actin cytoskeleton occurred. Thus, in many cells close to the wound, focal F-actin branching points appeared. The branching points were similar to the microdomains in that they colocalized with CD44 and ERM proteins, but, in addition, they formed centers for actin filament branching and were associated with phosphorylated protein kinase C /_II. These colocalization data are consonant with the view that activated PKC is responsible for activating ERM-mediated crosslinking between CD44 and the actin cytoskeleton. Importantly, inhibition of PKC activity decreased staining for phosphorylated ERM proteins, decreased the frequency of F-actin branching points, and inhibited monolayer wound healing. Together, our data show that endothelial cells contain a novel actin cytoskeletal structure, the F-actin microdomain, and suggest that during wound healing such structures become associated with activated signaling molecules and thereby enhance actin cytoskeletal remodeling.     

Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.     

Formation of microvilli and phosphorylation of ERM family proteins by CD43, a potent inhibitor for cell adhesion: Cell detachment is a potential cue for ERM phosphorylation and organization of cell morphology

CD43/sialophorin/leukosialin, a common leukocyte antigen, is known as an inhibitor for cell adhesion. The ectodomain of CD43 is considered as a molecular barrier for cell adhesion, while the cytoplasmic domain has a binding site for Ezrin/Radixin/Moesin (ERM). We found expression of CD43 induced cell rounding, inhibition of cell re-attachment, augmentation of microvilli and phosphorylation of ERM in HE K293T cells. Mutant studies revealed the ectodomain of CD43, but not the intracellular domain, essential and sufficient for all these phenomena. We also found that forced cell detachment by itself induced phosphorylation of ERM in HE K293T cells. Taken together, these findings indicate that inhibition of cell adhesion by the ectodomain of CD43 induces phosphorylation of ERM, microvilli formation and eventual cell rounding. Furthermore, our study suggests a novel possibility that cell detachment itself induces activation of ERM and modification of cell shape.Key words: cell adhesion, CD43, microvilli, ERM, integrin, cell rounding, phosphorylation, mucin     

Direct binding of the Na--H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation

The association of actin filaments with the plasma membrane maintains cell shape and adhesion. Here, we show that the plasma membrane ion exchanger NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton. This occurs through a previously unrecognized structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM). NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, have impaired organization of focal adhesions and actin stress fibers, and an irregular cell shape. We propose a structural role for NHE1 in regulating the cortical cytoskeleton that is independent of its function as an ion exchanger.     

Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons

In leukocytes such as thymocytes and basophilic leukemia cells, a glycosilated integral membrane protein called CD43 (leukosialin or sialophorin), which is defective in patients with Wiskott-Aldrich syndrome, was highly concentrated in the cleavage furrow during cytokinesis. Not only at the mitotic phase but also at interphase, CD43 was precisely colocalized with ezrin-radixin-moesin family members. (ERM), which were previously reported to play an important role in the plasma membrane-actin filament association in general. At the electron microscopic level, throughout the cell cycle, both CD43 and ERM were tightly associated with microvilli, providing membrane attachment sites for actin filaments. We constructed a cDNA encoding a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and the transmembrane/cytoplasmic domain of rat CD43, and introduced it into mouse L fibroblasts lacking both endogenous CD43 and E-cadherin. In dividing transfectants, the chimeric molecules were concentrated in the cleavage furrow together with ERM, and both proteins were precisely colocalized throughout the cell cycle. Furthermore, using this transfection system, we narrowed down the domain responsible for the CD43-concentration in the cleavage furrow. Based on these findings, we conclude that CD43 is concentrated in the cleavage furrow through the direct or indirect interaction of its cytoplasmic domain with ERM and actin filaments.     

Formation of microvilli and phosphorylation of ERM family proteins by CD43, a potent inhibitor for cell adhesion

CD43/sialophorin/leukosialin, a common leukocyte antigen, is known as an inhibitor for cell adhesion. The ectodomain of CD43 is considered as a molecular barrier for cell adhesion, while the cytoplasmic domain has a binding site for Ezrin/Radixin/Moesin (ERM). We found expression of CD43 induced cell rounding, inhibition of cell re-attachment, augmentation of microvilli, and phosphorylation of ERM in HEK293T cells. Mutant studies revealed the ectodomain of CD43, but not the intracellular domain, essential and sufficient for all these phenomena. We also found that forced cell detachment by itself induced phosphorylation of ERM in HEK293T cells. Taken together, these findings indicate that inhibition of cell adhesion by the ectodomain of CD43 induces phosphorylation of ERM, microvilli formation, and eventual cell rounding. Furthermore, our study suggests a novel possibility that cell detachment itself induces activation of ERM and modification of cell shape.     

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.     

Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases.

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1] [2] [3] [4] [5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6] [7] increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) [8] [9] [10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase [11]. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha [12] [13] [14], but not a kinase-inactive mutant [15], increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro [16], our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.     

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.     

CD43 interaction with ezrin-radixin-moesin (ERM) proteins regulates T-cell trafficking and CD43 phosphorylation

Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase CΘ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation-dependent mechanism.     

Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane-cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.