The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Anthony Bretscher - One of the best experts on this subject based on the ideXlab platform.
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Ezrin activation by lok phosphorylation involves a pip2 dependent wedge mechanism
eLife, 2017Co-Authors: Thaher Pelaseyed, Raghuvir Viswanatha, Cecile Sauvanet, Joshua J Filter, Michael L Goldberg, Anthony BretscherAbstract:How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein Ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates Ezrin to activate it. Binding of PIP2 to Ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of Ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that Ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.
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local phosphocycling mediated by lok slk restricts Ezrin function to the apical aspect of epithelial cells
Journal of Cell Biology, 2012Co-Authors: Raghuvir Viswanatha, Patrice Y Ohouo, Marcus B Smolka, Anthony BretscherAbstract:In this paper, we describe how a dynamic regulatory process is necessary to restrict microvilli to the apical aspect of polarized epithelial cells. We found that local phosphocycling regulation of Ezrin, a critical plasma membrane–cytoskeletal linker of microvilli, was required to restrict its function to the apical membrane. Proteomic approaches and ribonucleic acid interference knockdown identified lymphocyte-oriented kinase (LOK) and SLK as the relevant kinases. Using drug-resistant LOK and SLK variants showed that these kinases were sufficient to restrict Ezrin function to the apical domain. Both kinases were enriched in microvilli and locally activated there. Unregulated kinase activity caused Ezrin mislocalization toward the basolateral domain, whereas expression of the kinase regulatory regions of LOK or SLK resulted in local inhibition of Ezrin phosphorylation by the endogenous kinases. Thus, the domain-specific presence of microvilli is a dynamic process requiring a localized kinase driving the phosphocycling of Ezrin to continually bias its function to the apical membrane.
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immune synapse formation requires zap 70 recruitment by Ezrin and cd43 removal by moesin
Journal of Cell Biology, 2007Co-Authors: Tal Ilani, Chand Khanna, Ming Zhou, Timothy D Veenstra, Anthony BretscherAbstract:Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of Ezrin and moesin, the two Ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that Ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that Ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of Ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, Ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.
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the carboxyl terminal region of ebp50 binds to a site in the amino terminal domain of Ezrin that is masked in the dormant molecule
Journal of Biological Chemistry, 1998Co-Authors: David Reczek, Anthony BretscherAbstract:Abstract EBP50 (Ezrin-radixin-moesin-bindingphosphoprotein 50) was recently identified by affinity chromatography on the immobilized NH2-terminal domain of Ezrin. Here we map and characterize the regions in EBP50 and Ezrin necessary for this association. Using blot overlays and in solution binding assays, the COOH-terminal 30 residues of EBP50 were found to be sufficient for an association with residues 1–286 of Ezrin. EBP50 did not bind to full-length (1–585) Ezrin, indicating that the EBP50 binding site is masked in the full-length molecule. Ezrin contains two complementary self-association domains known as N- and C-ERMADs (Ezrin-radixin-moesin-associationdomains), encompassing residues 1–296 and 479–585, respectively. An Ezrin 1–583 construct lacking the two terminal residues necessary for this association was found to have an unmasked EBP50 binding site. Moreover, binding of EBP50 and the C-ERMAD to Ezrin residues 1–296 was found to be mutually exclusive, with the C-ERMAD having a higher affinity. These results suggest that in full-length Ezrin, the binding site for EBP50 is masked through an intramolecular N/C-ERMAD association. Based on these and additional results, we propose a model whereby dormant Ezrin can be activated to bind EBP50 on its NH2-terminal end and F-actin on its COOH-terminal end. Since EBP50 is proposed to bind membrane proteins through its PDZ domains, this provides a molecular description of the regulated linkage of microfilaments to membranes in cell surface microvilli.
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Ezrin a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures
Journal of Cell Science, 1997Co-Authors: Anthony Bretscher, David Reczek, Mark BerrymanAbstract:The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (Ezrin-radixin-moesin) family of proteins, of which Ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. The present knowledge about Ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that Ezrin can exist in a dormant conformation that requires activation to expose otherwise masked association sites. Current results indicate that activated Ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of Ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that Ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of Ezrin and its association with different binding partners is regulated.
Monique Arpin - One of the best experts on this subject based on the ideXlab platform.
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Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells
BMC Cancer, 2012Co-Authors: Hannah Mak, Sandip Sengupta, Monique Arpin, Alexandra Naba, Sonal Varma, Colleen Schick, Andrew G Day, Bruce E ElliottAbstract:The membrane cytoskeletal crosslinker, Ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that Ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, Ezrin phosphorylation on Y477 by Src is specific to Ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in Ezrin on tumor progression. Using a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of Ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model. AC2M2 cells over-expressing Y477F Ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F Ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F Ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F Ezrin reduces the number of lung metastatic lesions. Our study implicates a role of Y477 Ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/Ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.
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the membrane cytoskeletal crosslinker Ezrin is required for metastasis of breast carcinoma cells
Breast Cancer Research, 2005Co-Authors: Bruce E Elliott, Jalna Meens, Sandip Sengupta, Daniel Louvard, Monique ArpinAbstract:The membrane cytoskeletal crosslinker Ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by Ezrin in breast cancer metastasis has not been clearly delineated. We examined the role of Ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type Ezrin or a dominant-negative amino-terminal domain of Ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model. Parental AC2M2 cells and cells overexpressing wild-type Ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal Ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal Ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type Ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type Ezrin. We further found that overexpression of amino-terminal Ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type Ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal Ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system. Our findings indicate that Ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of Ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking Ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis.
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phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of Ezrin
Journal of Cell Biology, 2004Co-Authors: Bruno Fievet, Christian Roy, Paul Mangeat, Daniel Louvard, Alexis Gautreau, Laurence Del Maestro, Monique ArpinAbstract:Ezrin, a membrane–actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that Ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of Ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of Ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow Ezrin to exert its cellular functions.
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Ezrin regulates e cadherin dependent adherens junction assembly through rac1 activation
Molecular Biology of the Cell, 2003Co-Authors: Philippe Pujuguet, Daniel Louvard, Alexis Gautreau, Laurence Del Maestro, Monique ArpinAbstract:Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of Ezrin, Ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in Ezrin T567D-expressing cells, compared with cells expressing wild-type Ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in Ezrin T567D-producing cells. In confluent cells, Ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for Ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane.
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morphogenic effects of Ezrin require a phosphorylation induced transition from oligomers to monomers at the plasma membrane
Journal of Cell Biology, 2000Co-Authors: Alexis Gautreau, Daniel Louvard, Monique ArpinAbstract:ERM (Ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in Ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D Ezrin from LLC-PK1 epithelial cells. We found that T567A Ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D Ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D Ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D Ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.
Chand Khanna - One of the best experts on this subject based on the ideXlab platform.
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role of Ezrin in osteosarcoma metastasis
Advances in Experimental Medicine and Biology, 2014Co-Authors: Ling Ren, Chand KhannaAbstract:The cause of death for the vast majority of cancer patients is the development of metastases at sites distant from that of the primary tumor. For most pediatric sarcoma patients such as those with osteosarcoma (OS), despite successful management of the primary tumor through multimodality approaches, the development of metastases, commonly to the lungs, is the cause of death. Significant improvements in long-term outcome for these patients have not been seen in more than 30 years. Furthermore, the long-term outcome for patients who present with metastatic disease is grave [1-5]. New treatment options are needed.Opportunities to improve outcomes for patients who present with metastases and those at-risk for progression and metastasis require an improved understanding of cancer progression and metastasis. With this goal in mind we and others have identified Ezrin as a metastasis-associated protein that associated with OS and other cancers. Ezrin is the prototypical ERM (Ezrin/Radixin/Moesin) protein family member. ERMs function as linker proteins connecting the actin cytoskeleton and the plasma membrane. Since our initial identification of Ezrin in pediatric sarcoma, an increasing understanding the role of Ezrin in metastasis has emerged. Briefly, Ezrin appears to allow metastatic cells to overcome a number of stresses experienced during the metastatic cascade, most notably the stress experienced as cells interact with the microenvironment of the secondary site. Cells must rapidly adapt to this environment in order to survive. Evidence now suggests a connection between Ezrin expression and a variety of mechanisms linked to this important cellular adaptation including the ability of metastatic cells to initiate the translation of new proteins and to allow the efficient generation of ATP through a variety of sources. This understanding of the role of Ezrin in the biology of metastasis is now sufficient to consider Ezrin as an important therapeutic target in osteosarcoma patients. This chapter reviews our understanding of Ezrin and the related ERM proteins in normal tissues and physiology, summarizes the expression of Ezrin in human cancers and associations with clinical parameters of disease progression, reviews reports that detail a biological understanding of Ezrin's role in metastatic progression, and concludes with a rationale that may be considered to target Ezrin and Ezrin biology in osteosarcoma.
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protein kinase c regulates Ezrin radixin moesin phosphorylation in canine osteosarcoma cells
Veterinary and Comparative Oncology, 2011Co-Authors: Sunghyeok Hong, Ling Ren, Tanasa S Osborne, Joseph Briggs, Christina Mazcko, Sandra Burkett, Chand KhannaAbstract:The development of metastasis is the most significant cause of death for both canine and human patients with osteosarcoma (OS). Ezrin has been associated with tumour progression and metastasis in human, canine and murine OS. Ezrin activation is dynamically regulated by protein kinase C (PKC) during metastatic progression in human and murine OS. To include the dog in the development of therapeutics that target Ezrin biology, we characterized four new canine OS cell lines and confirmed the relationship between PKC and Ezrin in these cells. Three of four cell lines formed tumours in mice that were histologically consistent with OS. All cell lines were markedly aneuploid and expressed Ezrin and PKC. Finally, both Ezrin phosphorylation and cell migration were inhibited using a PKC inhibitor. These data suggest that an association between PKC-mediated activation of Ezrin and the metastatic phenotype in canine OS cells.
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the actin cytoskeleton linker protein Ezrin is regulated during osteosarcoma metastasis by pkc
Oncogene, 2009Co-Authors: Ling Ren, Sunghyeok Hong, Jessica Cassavaugh, Tanasa S Osborne, Alexander J Chou, Su Young Kim, Richard Gorlick, Stephen M Hewitt, Chand KhannaAbstract:Ezrin is a member of the ERM (Ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that Ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of Ezrin, and that Ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of Ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to Ezrin phosphorylation. These data are the first to suggest a dynamic regulation of Ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.
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immune synapse formation requires zap 70 recruitment by Ezrin and cd43 removal by moesin
Journal of Cell Biology, 2007Co-Authors: Tal Ilani, Chand Khanna, Ming Zhou, Timothy D Veenstra, Anthony BretscherAbstract:Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of Ezrin and moesin, the two Ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that Ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that Ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of Ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, Ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.
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rapamycin inhibits Ezrin mediated metastatic behavior in a murine model of osteosarcoma
Cancer Research, 2005Co-Authors: Xiaolin Wan, Chand Khanna, Arnulfo Mendoza, Lee J HelmanAbstract:Osteosarcoma is the most frequent primary malignant tumor of bone with a high propensity for metastasis. We have previously showed that Ezrin expression is necessary for metastatic behavior in a murine model of osteosarcoma (K7M2). In this study, we found that a mechanism of Ezrin-related metastatic behavior is linked to an Akt-dependent mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (S6K1)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) pathway. Suppression of Ezrin expression either by antisense transfection or by small interfering RNAs or disruption of Ezrin function by transfection of a dominant-negative Ezrin-T567A mutant led to decreased expression and decreased phosphorylation of both S6K1 and 4E-BP1. Proteosomal inhibition by MG132 reversed antisense-mediated decrease of S6K1 and 4E-BP1 protein expression, but failed to affect the effect of Ezrin on phosphorylation of S6K1 and 4E-BP1. Blockade of the mTOR pathway with rapamycin or its analog, cell cycle inhibitor-779 led to significant inhibition of experimental lung metastasis in vivo. These results suggest that blocking the mTOR/S6K1/4E-BP1 pathway may be an appropriate target for strategies to reduce tumor cell metastasis.
Matthew J Mahon - One of the best experts on this subject based on the ideXlab platform.
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fluorescent ligand directed co localization of the parathyroid hormone 1 receptor with the brush border scaffold complex of the proximal tubule reveals hormone dependent changes in Ezrin immunoreactivity consistent with inactivation
Biochimica et Biophysica Acta, 2012Co-Authors: Jun Guo, Lige Song, Minlin Liu, Matthew J MahonAbstract:Through binding to parathyroid hormone (PTH), PTH1R interacts with kidney-specific scaffold proteins, including the sodium hydrogen exchanger regulatory factors 1 and 2 (NHERFs), and Ezrin. To facilitate in vivo localization, tetramethylrhodamine-labeled PTH (PTH-TMR) was used as a fluorescent probe. In mice, PTH-TMR localizes to luminal surfaces of tubular S1 segments that overlap PTH1R immunostaining, but does not directly overlap with megalin-specific antibodies. PTH-TMR staining directly overlaps with Npt2a in nascent, endocytic vesicles, marking the location of transporter regulation. PKA substrate antibodies display marked staining increases in segments labeled with PTH-TMR, demonstrating a functional effect. In the presence of secondary hyperparathyroidism, PTH-TMR staining is markedly reduced and shifts to co-localizing with megalin. At 15min post-injection, PTH-TMR-labeled vesicles do not co-localize with either NHERF or Ezrin, suggesting PTH1R dissociation from the scaffold complex. At the 5min time point, PTH-TMR stains the base of microvilli where it localizes with both NHERF2 and Ezrin, and only partially with NHERF1. Strikingly, the bulk of Ezrin protein becomes undetectable with the polyclonal, CS3145 antibody, revealing a PTH-induced conformational change in the scaffold. A second Ezrin antibody (3C12) is capable of detecting the altered Ezrin protein. The CS3145 antibody only binds to the active form of Ezrin and fails to recognize the inactive form, while the 3C12 reagent can detect either active or inactive Ezrin. Here we show that the PTH1R is part of the Ezrin scaffold complex and that acute actions of PTH suggest a rapid inactivation of Ezrin in a spatially defined manner.
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apical membrane segregation of phosphatidylinositol 4 5 bisphosphate influences parathyroid hormone 1 receptor compartmental signaling and localization via direct regulation of Ezrin in llc pk1 cells
Cellular Signalling, 2011Co-Authors: Matthew J MahonAbstract:The parathyroid hormone 1 receptor (PTH1R), a primary regulator of mineral ion homeostasis, is expressed on both the apical and basolateral membranes of kidney proximal tubules and in the LLC-PK1 kidney cell line. In LLC-PK1 cells, apical PTH1R subpopulations are far more effective at signaling via phospholipase (PLC) than basolateral counterparts, revealing the presence of compartmental signaling. Apical PTH1R localization is dependent upon direct interactions with Ezrin, an actin-membrane cross-linking scaffold protein. Ezrin undergoes an activation process that is dependent upon phosphorylation and binding to phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that is selectively concentrated to apical surfaces of polarized epithelia. Consistently, the intracellular probe for PIP2, GFP-PLCδ1-PH, localizes to the apical membranes of LLC-PK1 cells, directly overlapping Ezrin and PTH1R expression. Activation of the apical PTH1R shifts the GFP-PLCδ1-PH probe from the apical membrane to the cytosol and basolateral membranes, reflecting domain-specific activation of PLC and hydrolysis of PIP2. This compartmental signaling is likely due to the polarized localization of PIP2, the substrate for PLC. PIP2 degradation using a membrane-directed phosphatase shifts Ezrin localization to the cytosol and induces Ezrin de-phosphorylation, processes consistent with inactivation. PIP2 degradation also shifts PTH1R expression from brush border microvilli to basolateral membranes and markedly blunts PTH-elicited activation of the MAPK pathway. Transient expression of Ezrin in HEK293 cells shifts PTH1R expression from the plasma membrane to microvilli-like surface projections that also contain PIP2. As a result, Ezrin enhances PTH mediated activation of the PLC pathway in this cell model with increasing total receptor surface expression. Collectively, these findings demonstrate that the apical segregation of PIP2 to the apical domains not only promotes the activation of Ezrin and the subsequent formation of the PTH1R containing scaffold, but also ensures the presence of ample substrate for propagating the PLC pathway.
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the parathyroid hormone 1 receptor directly binds to the ferm domain of Ezrin an interaction that supports apical receptor localization and signaling in llc pk1 cells
Molecular Endocrinology, 2009Co-Authors: Matthew J MahonAbstract:PTH 1 receptor (PTH1R) regulates mineral ion homeostasis. Both apical and basolateral PTH1R subpopulations exist within the renal proximal tubule. The purpose of this research was to examine determinants within the PTH1R that direct apical localization. When expressed in LLC-PK1 cells, a proximal tubule cell model, the PTH1R localizes to both apical and basolateral membranes. The C terminus of the PTH1R contains a psd-95, discs large, ZO-1 domain interaction motif that binds the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1), a renal tubule scaffold protein. Receptors lacking the psd-95, discs large, ZO-1 domain interaction motif (PTH1R-CDelta4) partly localize to apical membranes, suggesting that additional factors may be involved. Ezrin, a membrane-cytoskeleton linking protein, directly binds NHERF-1 and thus links assembled complexes to actin. In vitro, subdomain C of the Ezrin band 4.1, Ezrin, radixin, domain interacts with the C-terminal tail of the PTH1R on a site that is mutually exclusive from the NHERF-1 interaction domain, suggesting the presence of a ternary complex. Mutating the lysine-arginine-lysine motif within the juxtamembrane region of the PTH1R C-terminal tail to alanines markedly disrupts interactions with the band 4.1, Ezrin, radixin, domain of Ezrin both in vitro and within cells. Inclusion of these mutations in the context of the full-length PTH1R disrupts apical localization with no effect on basolateral expression. Expression of a dominant-negative Ezrin selectively disrupts apical expression and signaling of the PTH1R. However, dominant-negative Ezrin does not affect expression or signaling of the basolateral PTH1R subpopulation. These findings reveal that direct Ezrin interactions promote PTH1R apical localization and signaling in LLC-PK1 cells.
Olli Carpen - One of the best experts on this subject based on the ideXlab platform.
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Ezrin is key regulator of Src-induced malignant phenotype in three-dimensional environment.
Oncogene, 2011Co-Authors: Leena Heiska, Michaela Melikova, Andrea I. Mcclatchey, Ichiko Saotome, Fang Zhao, Olli CarpenAbstract:The oncogenic tyrosine kinase Src has a role in cancer development, especially by promoting invasive and metastatic behavior. It is, however, unclear which of the Src-regulated signaling cascades induce malignant phenotype in three-dimensional environment. One of Src substrates is Ezrin, a cytoskeletal organiser and regulator of signal transduction. Ezrin expression correlates with poor outcome of diverse cancers and is essential in experimental metastatic osteosarcoma. We reconstituted genetically Ezrin-deficient cells with wild-type (WT) or phosphorylation-deficient Y477F Ezrin together with constitutively active Src. In two-dimensional cultures, Src induced malignant features regardless of the presence or absence of Ezrin. In contrast, only WT Ezrin-expressing cells grew efficiently in soft agar or in suspension. In Matrigel, only WT Ezrin significantly promoted growth and invasion, and was targeted to specific regions on the plasma membrane. WT and Y477F Ezrin-expressing cells showed marked differences only when growing or scattering in three-dimensional matrix. Additional experiments showed that Y477-phosphorylated Ezrin is also needed for the growth of Src-transformed epithelial cells in three-dimensional matrix. Cells lacking functional Ezrin had reduced cyclin D levels and fewer cells in G2+S phase, possibly as a consequence of abnormal mTOR signaling, as Ezrin Y477F cells showed lower expression of phosphorylated intermediates downstream of mTOR than WT cells. We conclude that the pathways activated by Src depend on the type of environment and that Ezrin is a crucial element of Src-induced malignant features in cells growing inside three-dimensional environment.
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intense cytoplasmic Ezrin immunoreactivity predicts poor survival in colorectal cancer
Human Pathology, 2008Co-Authors: Adam Elzagheid, Antti Vaheri, Eija Korkeila, Riyad Bendardaf, Abdelbaset Buhmeida, Suvi Heikkila, Kari Syrjanen, Seppo Pyrhonen, Olli CarpenAbstract:Ezrin is a membrane-cytoskeleton anchor, which, in experimental models, regulates tumor cell invasion and metastatic ability. We carried out immunohistochemical analysis of Ezrin in 74 advanced colorectal cancer patients and correlated it to clinicopathologic variables and disease outcome. In contrast to the predominantly membraneous immunoreactivity of normal colorectal epithelium, Ezrin expression in the colorectal cells was typically cytoplasmic. Altogether, 16.2% (12/74) of the tumors showed negative/weak Ezrin staining, 35.1% (26/74) had moderate staining, and 48.6% (36/74) had intense staining. The expression was more intense in colon than in rectal carcinomas (P = .003). Increased Ezrin expression was associated with adverse outcome, that is, shorter disease-specific survival; 48.3 months and 36.6 months for negative-weak versus intense expression (P = .041) as well as shorter survival with metastases at 36 months (P = .030); the metastases(36) rates in Ezrin(neg/weak), Ezrin(moderate), Ezrin(intense) are 58.3%, 25.0%, and 18.4%, respectively. In univariate survival analysis, dichotomized (negative/weak versus moderate/strong) Ezrin expression significantly predicted both the 5-year disease specific survival (P = .035) and 5-year metastases (P = .018) but lost this predictive power in multivariate (Cox) analysis. High Ezrin expression was also related to high E-cadherin (cytoplasmic) expression, DNA aneuploidy, and high thymidylate synthase expression (P = .046, P = .042, P = .046, respectively). These results suggest that Ezrin may play a role in colorectal cancer progression and that Ezrin expression might provide clinically valuable information in predicting the biological behavior of colorectal cancer.
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inhibition of t cell activation by cyclic adenosine 5 monophosphate requires lipid raft targeting of protein kinase a type i by the a kinase anchoring protein Ezrin
Journal of Immunology, 2007Co-Authors: Anja Ruppelt, Olli Carpen, Mikaela Gronholm, Randi Mosenden, Einar Martin Aandahl, Derek Tobin, Cathrine R Carlson, Hilde Abrahamsen, Friedrich W HerbergAbstract:cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.
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Ezrin in primary cutaneous melanoma
Modern Pathology, 2005Co-Authors: Suvi Ilmonen, Antti Vaheri, Sirpa Asko-seljavaara, Olli CarpenAbstract:Ezrin is a member of the Ezrin–radixin–moesin family of proteins that link the actin-containing cytoskeleton to the plasma membrane. Ezrin is also connected to signaling molecules involved in the regulation of cell survival, proliferation and migration. Here, we examined the expression of Ezrin in 95 primary cutaneous melanomas and correlated Ezrin expression with conventional prognostic factors and biomarkers. From 12 patients metastatic tissue samples were also examined. In addition to Ezrin staining, Mib-1 proliferation antigen, p53 and Bcl-2 were evaluated. Ezrin immunoreactivity was seen in most tumors; only 19 (20%) melanomas were negative. A total of 48 (51%) tumors had weak immunoreactivity and 28 (29%) strong immunoreactivity. The intensity of Ezrin immunoreactivity was associated with tumor thickness (Breslow, P =0.0008) and with tumor invasion level (Clark, P =0.004), thicker tumors having stronger immunoreactivity. Also, there was a correlation between higher Mib-1 index in tumors and strong Ezrin expression. All metastatic samples ( n =12) showed positive Ezrin immunoreactivity. In univariate analysis of survival, patients ( n =76) with positive Ezrin immunoreactivity had worse clinical disease behavior than those ( n =19) without Ezrin immunoreactivity, but the difference was not significant ( P =0.19). In multivariate analysis of survival, the Ezrin immunoreactivity was not a significant marker. The results indicate that Ezrin is expressed in most primary melanomas of the skin and in all metastatic tumors. Ezrin expression correlates with tumor thickness and level of invasion suggesting an association between Ezrin expression and tumor progression.
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Src phosphorylates Ezrin at tyrosine 477 and induces a phosphospecific association between Ezrin and a kelch-repeat protein family member.
Journal of Biological Chemistry, 2004Co-Authors: Leena Heiska, Olli CarpenAbstract:Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of Ezrin in adherent cells. We show that Ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, Ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect Ezrin head-to-tail association or phosphorylation of Ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of Ezrin. A modified yeast two-hybrid screen in which Ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of Ezrin.
