Moesin Function Research Articles

FBXW2 suppresses breast tumorigenesis by targeting AKT-Moesin-SKP2 axis

Oncogene Moesin plays critical role in initiation, progression, and metastasis of multiple cancers. It exerts oncogenic activity due to its high-level expression as well as posttranslational modification in cancer. However, factors responsible for its high-level expression remain elusive. In this study, we identified positive as well as negative regulators of Moesin. Our study reveals that Moesin is a cellular target of F-box protein FBXW2. We showed that FBXW2 suppresses breast cancer progression through directing proteasomal degradation of Moesin. In contrast, AKT kinase plays an important role in oncogenic function of Moesin by protecting it from FBXW2-mediated proteasomal degradation. Mechanistically, AKT phosphorylates Moesin at Thr-558 and thereby prevents its degradation by FBXW2 via weakening the association between FBXW2 and Moesin. Further, accumulated Moesin prevents FBXW2-mediated degradation of oncogene SKP2, showing that Moesin functions as an upstream regulator of oncogene SKP2. In turn, SKP2 stabilizes Moesin by directing its non-degradable form of polyubiquitination and therefore AKT-Moesin-SKP2 oncogenic axis plays crucial role in breast cancer progression. Collectively, our study reveals that FBXW2 functions as a tumor suppressor in breast cancer by restricting AKT-Moesin-SKP2 axis. Thus, AKT-Moesin-SKP2 axis may be explored for the development of therapeutics for cancer treatment.

Discovery of a Potent Dual SLK/STK10 Inhibitor Based on a Maleimide Scaffold.

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila.

Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the invivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression ofprotein chaperones requires the nuclear functions of Moesin. DATABASE: GEO accession number: GSE155778.

Internalization of NKCC2 is impaired in thick ascending limb of Henle in moesin knockout mice.

Moesin is expressed in several types of cells including epithelial and endothelial cells. Several groups reported that moesin plays important roles in the regulation of the cellular motility, and the process of internalization of membrane proteins. However, the physiological roles of moesin in the kidney still remain unclear. Herein, we examined the physiological function of moesin in the kidney using moesin knockout (Msn -/y ) mice. There was no obvious abnormality in the renal morphology of Msn -/y mice. However, we found that Msn -/y mice exhibited mild hyperchloremia, and reduced glomerular filtration rate compared to wild type (WT) mice. Absolute electrolytes excretions of NaCl in Msn -/y mice were not significantly changed compared to WT mice. In the renal medulla, moesin was detected in thick ascending limb of Henle (TALH) as previously reported. To determine the physiological function of moesin in TALH, we examined the expression and subcellular localization of NKCC2 in Msn -/y mice. Interestingly, apical surface expression level, but not total expression of NKCC2 was increased in Msn -/y mice. Subcellular fractionation of renal medulla lysate and internalization assay using tubular suspension showed that the process of NKCC2 endocytosis is impaired. Since the distribution of NKCC2 in lipid raft fractions was decreased in Msn -/y mice, moesin may regulate the NKCC2 distribution to microdomain. These results suggest that moesin regulates the internalization of NKCC2. Furthermore, euhydration by water loading caused hyponatremina in Msn -/y mice, suggesting that dysfunction of moesin is associated with the nephrogenic syndrome of inappropriate antidiuresis (NSIAD).

Crumbs, Moesin and Yurt regulate junctional stability and dynamics for a proper morphogenesis of the Drosophila pupal wing epithelium

The Crumbs (Crb) complex is a key epithelial determinant. To understand its role in morphogenesis, we examined its function in the Drosophila pupal wing, an epithelium undergoing hexagonal packing and formation of planar-oriented hairs. Crb distribution is dynamic, being stabilized to the subapical region just before hair formation. Lack of crb or stardust, but not DPatj, affects hexagonal packing and delays hair formation, without impairing epithelial polarities but with increased fluctuations in cell junctions and perimeter length, fragmentation of adherens junctions and the actomyosin cytoskeleton. Crb interacts with Moesin and Yurt, FERM proteins regulating the actomyosin network. We found that Moesin and Yurt distribution at the subapical region depends on Crb. In contrast to previous reports, yurt, but not moesin, mutants phenocopy crb junctional defects. Moreover, while unaffected in crb mutants, cell perimeter increases in yurt mutant cells and decreases in the absence of moesin function. Our data suggest that Crb coordinates proper hexagonal packing and hair formation, by modulating junction integrity via Yurt and stabilizing cell perimeter via both Yurt and Moesin. The Drosophila pupal wing thus appears as a useful system to investigate the functional diversification of the Crb complex during morphogenesis, independently of its role in polarity.

Moesin-deficient mice reveal a non-redundant role for moesin in lymphocyte homeostasis

Moesin is a member of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins. These proteins organize membrane domains by interacting with plasma membrane proteins and the actin cytoskeleton. Because of their high sequence similarity, ERM proteins are usually thought to be functionally redundant. Lymphocytes express two ERM proteins, ezrin and moesin. Whether each ERM plays a specialized role in lymphocytes, particularly in vivo, remains unknown. Here, we show that moesin has a crucial, non-redundant role in lymphocyte homeostasis. Moesin-deficient mice exhibited decreases in both T and B cells in the peripheral blood and lymph nodes, but not in the spleen. This phenotype was recapitulated in bone marrow (BM) chimeras with a hematopoietic moesin deficiency. Although the T and B cells apparently developed without major defects in the moesin-deficient mice, T cell egress from the thymus and immature B cell egress from the BM were impaired. In the periphery, both T and B cells showed delayed egress from lymphoid organs. We showed that moesin is the primary phosphorylated ERM subject to dynamic regulation during cell shape changes and migration. Our findings identify a previously unknown, non-redundant function of moesin in lymphocyte homeostasis in regulating lymphocyte egress from lymphoid organs.

Moesin-induced signaling in response to lipopolysaccharide in macrophages

Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane-organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)-mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS-induced moesin signaling pathways in macrophages. Macrophages were stimulated with 500 ng/mL LPS in macrophage serum-free medium. For blocking experiments, cells were pre-incubated with anti-moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real-time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD-2 and Toll-like receptor 4 (TLR4) and co-immunoprecipitation of MyD88-interleukin-1 receptor-associated kinase (IRAK) and IRAK-tumor necrosis factor receptor-activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor kappaB (NF-kappaB) and IkappaBalpha were studied. Tumor necrosis factor alpha, interleukin-1beta and interferon beta were measured by ELISA. Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl-terminus. Moesin is temporally associated with TLR4 and MD-2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide-induced signaling is transferred downstream to p38, p44/42 MAPK and NF-kappaB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF-kappaB activation and translocation to the nucleus. These results suggest an important role for moesin in the innate immune response and TLR4-mediated pattern recognition in periodontal disease.

Moesin/ezrin: a specific role in cell metastasis?

Barsh, G.S. (2006). Regulation of pigment type switching by agouti, melanocortin signaling, attractin and mahoganoid. In The Pigmentary System, 2nd edn. J.J. Nordlund, R.E. Boissy, V.J. Hearing, R.A. King, W.S. Oetting, and J.P. Ortonne, eds (Massachusetts: Blackwell Publishing), pp. 395–409. Candille, S.I., Kaelin, C.B., Cattanach, B.M., Yu, B., Thompson, D.A., Nix, M.A., Kerns, J.A., Schmutz, S.M., Millhauser, G.L., and Barsh, G.S. (2007). A b-defensin mutation causes black coat color in domestic dogs. Science 318, 1418–1423. Chakrabarti, O., and Hegde, R.S. (2009). Functional depletion of mahogunin by cytosolically exposed prion protein contributes to neurodegeneration. Cell 137, 1136–1147. Hida, T., Wakamatsu, K., Sviderskaya, E.V. et al. (2009). Agouti protein, mahogunin, and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes: a cAMP-independent pathway. Pigment Cell Melanoma Res. 22, 623–634. Jiao, J., Kim, H.Y., Liu, R.R., Hogan, C.A., Sun, K., Tam, L.M., and Gunn, T.M. (2009). Transgenic analysis of the physiological functions of Mahogunin Ring Finger-1 isoforms. Genesis 47, 524–534.

Ezrin and Moesin Function Together to Promote T Cell Activation

The highly homologous proteins ezrin, radixin, and moesin link proteins to the actin cytoskeleton. The two family members expressed in T cells, ezrin and moesin, are implicated in promoting T cell activation and polarity. To elucidate the contributions of ezrin and moesin, we conducted a systematic analysis of their function during T cell activation. In response to TCR engagement, ezrin and moesin were phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localized to the distal pole complex (DPC). However, ezrin exhibited unique behaviors, including tyrosine phosphorylation and transient localization to the immunological synapse before movement to the DPC. To ask whether these differences reflect unique requirements for ezrin vs moesin in T cell signaling, we generated mice with conditional deletion of ezrin in mature T cells. Ezrin-/- T cells exhibited normal immunological synapse organization based upon localization of protein kinase C-theta, talin, and phospho-ZAP70. DPC localization of CD43 and RhoGDP dissociation inhibitor, as well as the novel DPC protein Src homology region 2 domain-containing phosphatase-1, was also unaffected. However, recruitment of three novel DPC proteins, ezrin binding protein of 50 kDa, Csk binding protein, and the p85 subunit of PI3K was partially perturbed. Biochemical analysis of ezrin-/- T cells or T cells suppressed for moesin using small interfering RNA showed intact early TCR signaling, but diminished levels of IL-2. The defects in IL-2 production were more pronounced in T cells deficient for both ezrin and moesin. These cells also exhibited diminished phospholipase C-gamma1 phosphorylation and calcium flux. We conclude that despite their unique movement and phosphorylation patterns, ezrin and moesin function together to promote T cell activation.

Role for moesin in lipopolysaccharide-stimulated signal transduction.

Moesin is a 78-kDa protein with diverse functions in linking the cytoskeleton to the membrane while controlling cell shape, adhesion, locomotion, and signaling. The aim of this study was to characterize the expression and localization of moesin in mononuclear phagocytes by using confocal microscopy, flow cytometry, immunoprecipitation, and Western blotting and to analyze the function of moesin as a lipopolysaccharide receptor, utilizing an antisense oligonucleotide approach to knock down the moesin gene. Results revealed that moesin is expressed on the surface of monocytes/macrophages and surface expression is increased after lipopolysaccharide stimulation. The total protein mass of moesin is increased in monocytes after lipopolysaccharide stimulation. Immunoprecipitation showed that moesin coprecipitates with TLR4, a well-known lipopolysaccharide receptor, suggesting an early role of moesin in the formation of the initiation complex for lipopolysaccharide signaling. Two antisense and two control sense oligonucleotides were synthesized and introduced every 4 h for 48 h in adherent macrophage-like cells. Cells were then stimulated with lipopolysaccharide for 4 h, and the supernatants were assayed for tumor necrosis factor alpha (TNF-alpha) production. Cell lysates were assayed for moesin expression by Western blotting immediately after the 48-h treatment period and also after 116 h of recovery to assess the return of moesin expression and function. Moesin gene expression was completely suppressed after 48 h of incubation with antisense oligonucleotides. The antisense elimination of moesin gene expression led to a significant reduction of lipopolysaccharide-induced TNF-alpha secretion. Restoration of moesin gene expression led to restoration of TNF-alpha production. These data suggest an important role for moesin in lipopolysaccharide-induced TNF-alpha production, highlighting its importance in lipopolysaccharide-mediated signal transduction.

A role for moesin in polarity

Three groups have recently characterized defects arising in development owing to mutations in the gene encoding Dmoesin, which is the sole ezrin–radixin–moesin (ERM) protein in Drosophila. Previously, studies in cultured mammalian cells suggested that ERM proteins are important for actin–membrane associations. However, mutations in moesin and radixin in mice do not cause severe defects, indicating functional overlap among vertebrate ERM paralogs. In Drosophila, however, mutations in Dmoesin result in lethality. Actin organization in imaginal disc epithelia is abnormal and apical–basal polarity is lost. When moesin function is reduced in the female germ-line, defects in cortical actin organization are also observed. Localization of informational molecules at the oocyte posterior is strongly affected, thus indicating a role for moesin in anchoring these determinants.

Moesin as Rho Inhibitor

Proteins of the ERM (for ezrin, radixin, and moesin) family are thought to function in linking proteins in the plasma membrane to the actin cytoskeleton. Speck et al. examined the function of ERM proteins in Drosophila , which have only one family member, moesin. Cells in the epithelium of imaginal discs that lacked moesin had defects in actin assembly and apical-basal polarity and showed inappropriate migration and invasive properties. Reducing expression of the small guanosine triphosphatase (GTPase) Rho by one half suppressed the effects of loss of moesin, and overexpression of Rho in epithelial cells produced a phenotype similar to that caused by lack of moesin. Thus, moesin's effects on epithelial cells appear to result from inhibition of Rho signaling. ERM proteins have been proposed to serve a structural role in linkage of the cytoskeletion to the plasma membrane, but the rescue of cells lacking moesin by modulation of Rho signaling indicates that inhibition of Rho activity may be a more critical function of moesin. ERM proteins are also themselves regulated by Rho: ERM proteins exist in inactive and active conformations and Rho promotes formation of the active form. The authors propose that the negative feedback loop produced by Rho's activation of ERM may be an important mechanism that prevents the excessive migratory and invasive properties characteristic of metastatic cancer cells. O. Speck, S. C. Hughes, N. K. Noren, R. M. Kulikauskas, R. G. Fehon, Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 421 , 83-87 (2003). [Online Journal]

Moesin as Rho Inhibitor

Proteins of the ERM (for ezrin, radixin, and moesin) family are thought to function in linking proteins in the plasma membrane to the actin cytoskeleton. Speck et al. examined the function of ERM proteins in Drosophila, which have only one family member, moesin. Cells in the epithelium of imaginal discs that lacked moesin had defects in actin assembly and apical-basal polarity and showed inappropriate migration and invasive properties. Reducing expression of the small guanosine triphosphatase (GTPase) Rho by one half suppressed the effects of loss of moesin, and overexpression of Rho in epithelial cells produced a phenotype similar to that caused by lack of moesin. Thus, moesin's effects on epithelial cells appear to result from inhibition of Rho signaling. ERM proteins have been proposed to serve a structural role in linkage of the cytoskeletion to the plasma membrane, but the rescue of cells lacking moesin by modulation of Rho signaling indicates that inhibition of Rho activity may be a more critical function of moesin. ERM proteins are also themselves regulated by Rho: ERM proteins exist in inactive and active conformations and Rho promotes formation of the active form. The authors propose that the negative feedback loop produced by Rho's activation of ERM may be an important mechanism that prevents the excessive migratory and invasive properties characteristic of metastatic cancer cells.O. Speck, S. C. Hughes, N. K. Noren, R. M. Kulikauskas, R. G. Fehon, Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 421, 83-87 (2003). [Online Journal]

Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

Moesin, a member of the ezrin-radixin-moesin (ERM) family of membrane/cytoskeletal linkage proteins, is known to be threonine-phosphorylated at Thr558 in activated platelets within its conserved putative actin-binding domain. The pathway leading to this phosphorylation step and its control have not been previously elucidated. We have detected and characterized reactions leading to moesin phosphorylation in human leukocyte extracts. In vitro phosphorylation of endogenous moesin, which was identified by peptide microsequencing, was dependent on phosphatidylglycerol (PG) or to a lesser extent, phosphatidylinositol (PI), but not phosphatidylserine (PS) and diacylglycerol (DAG). Analysis of charge shifts, phosphoamino acid analysis, and stoichiometry was consistent with a single phosphorylation site. By using mass spectroscopy and direct microsequencing of CNBr fragments of phospho-moesin, the phosphorylation site was identified as KYKT*LRQIR (where * indicates the phosphorylation site) (Thr558), which is conserved in the ERM family. Recombinant moesin demonstrated similar in vitro phospholipid-dependent phosphorylation compared with the endogenous protein. The phosphorylation site sequence of moesin displays a high degree of conservation with the pseudosubstrate sequences of the protein kinase C (PKC) family. We identified the kinase activity as PKC-theta on the basis of immunodepletion of the moesin kinase activity and copurification of PKC-theta with the enzymic activity. We further demonstrate that PKC-theta displays a preference for PG vesicles over PI or PS/DAG, with minimal activation by DAG, as well as specificity for moesin compared with myelin basic protein, histone H1, or other cellular proteins. Expression of a human His6-tagged PKC-theta in Jurkat cells and purification by Ni2+ chelate chromatography yield an active enzyme that phosphorylates moesin. PG vesicle binding experiments with expressed PKC-theta and moesin demonstrate that both bind to vesicles independently of one another. Thus, PKC-theta is identified as a major kinase within cells with specificity for moesin and with activation under non-classical PKC conditions. It appears likely that this activity corresponds to a specific intracellular pathway controlling the function of moesin as well as other ERM proteins.