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Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues
K. Hayashi, S. Yonemura, T. Matsui, S. Tsukita
Journal of Cell Science 1999 112: 1149-1158;

Summary

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

REFERENCES

    1. Algrain M.,
    2. Turunen O.,
    3. Vaheri A.,
    4. Louvard D. and
    5. Arpin M.
    (1993). Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J. CellBiol 120, 129–139
    OpenUrlAbstract/FREE Full Text
    1. Arpin M.,
    2. Algrain M. and
    3. Louvard D.
    (1994). Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1. Curr. Opin. Cell Biol 6, 136–141
    OpenUrlCrossRefPubMedWeb of Science
    1. Berryman M.,
    2. Franck Z. and
    3. Bretscher A.
    (1993). Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J. Cell Sci 105, 1025–1043
    OpenUrlAbstract/FREE Full Text
    1. Berryman M.,
    2. Gary R. and
    3. Bretscher A.
    (1995). Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J. Cell Biol 131, 1231–1242
    OpenUrlAbstract/FREE Full Text
    1. Bretscher A.
    (1983). Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J. Cell Biol 97, 425–432
    OpenUrlAbstract/FREE Full Text
    1. Bretscher A.
    (1989). Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J. Cell Biol 108, 921–930
    OpenUrlAbstract/FREE Full Text
    1. Bretscher A.,
    2. Gary R. and
    3. Berryman M.
    (1995). Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains. Biochemistry 34, 16830–16837
    OpenUrlCrossRefPubMed
    1. Bretscher A.,
    2. Reczek D. and
    3. Berryman M.
    (1997). Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J. Cell Sci 110, 3011–3018
    OpenUrlAbstract/FREE Full Text
    1. Chen J.,
    2. Cohn J. A. and
    3. Mandel L. J.
    (1995). Dephosphorylation of ezrin as anearly event in renal microvillar breakdown and anoxic injury. Proc. Nat. Acad. Sci. USA 92, 7495–7499
    OpenUrlAbstract/FREE Full Text
    1. Chen J. and
    2. Mandel L. J.
    (1997). Unopposed phosphatase action initiates ezrin dysfunction: a potential mechanism for anoxic injury. Am. J.Physiol 273, 710–.
    OpenUrl
    1. Clark R. K. and
    2. Damjanov I.
    (1986). Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives. J. Histochem Cytochem 34, 679–682
    OpenUrlAbstract/FREE Full Text
    1. Earl W. R.
    (1943). The mouse fibroblast cultures and changes seen in the living cells. J. Nat. Cancer Inst 4, 165–212
    OpenUrlFREE Full Text
    1. Edwards K. A.,
    2. Montague R. A.,
    3. Shepard S.,
    4. Edgar B. A.,
    5. Erikson R. L. and
    6. Kiehart D. P.
    (1994). Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. Proc. Nat. Acad. Sci. USA 91, 4589–4593
    OpenUrlAbstract/FREE Full Text
    1. Enami J.,
    2. Enami S. and
    3. Koga M.
    (1984). Isolation of an insulin-responsive preadipose cell line and a mammary tumor-producing, dome-forming epithelial cell line from a mouse mammary tumor. Dev. GrowthDiffer 26, 223–234
    OpenUrlCrossRef
    1. Fabricant R. N.,
    2. De Larco J. E. and
    3. Todaro G. J.
    (1977). Nerve growth factor receptors on human melanoma cells in culture. Proc. Nat.Acad. Sci. USA 74, 565–569
    OpenUrlAbstract/FREE Full Text
    1. Gary R. and
    2. Bretscher A.
    (1995). Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061–1075
    OpenUrlAbstract/FREE Full Text
    1. Gaush C. R.,
    2. Hard W. L. and
    3. Smith T. F.
    (1966). Characterization of an established line of canine kidney cells (MDCK). Proc. Soc. Exp. Biol.Med 122, 931–935
    OpenUrlAbstract/FREE Full Text
    1. Henry M. D.,
    2. Gonzalez Agosti C. and
    3. Solomon F.
    (1995). Molecular dissection of radixin: distinct and interdependent functions of the amino-and carboxy-terminal domains. J. Cell Biol 129, 1007–1022
    OpenUrlAbstract/FREE Full Text
    1. Herman I. M.,
    2. Crisona N. J. and
    3. Pollard T. D.
    (1981). Relation between cell activity and the distribution of cytoplasmic actin and myosin. J. Cell Biol 90, 84–91
    OpenUrlAbstract/FREE Full Text
    1. Hirano S.,
    2. Nose A.,
    3. Hatta K.,
    4. Kawakami A. and
    5. Takeichi M.
    (1987). Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles. J. CellBiol 105, 2501–2510
    OpenUrlAbstract/FREE Full Text
    1. Hirao M.,
    2. Sato N.,
    3. Kondo T.,
    4. Yonemura S.,
    5. Monden M.,
    6. Sasaki T.,
    7. Takai Y.,
    8. Tsukita Sh. and
    9. Tsukita Sa
    (1996). Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol 135, 37–51
    OpenUrlAbstract/FREE Full Text
    1. Ikeda K.,
    2. Monden T.,
    3. Kanoh T.,
    4. Tsujie M.,
    5. Izawa H.,
    6. Haba A.,
    7. Ohnishi T.,
    8. Sekimoto M.,
    9. Tomita N.,
    10. Shiozaki H. and
    11. Monden M.
    (1998). Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J. Histochem.Cytochem 46, 397–403
    OpenUrlAbstract/FREE Full Text
    1. Ishizaki T.,
    2. Maekawa M.,
    3. Fujisawa K.,
    4. Okawa K.,
    5. Iwamatsu A.,
    6. Fujita A.,
    7. Watanabe N.,
    8. Saito Y.,
    9. Kakizuka A.,
    10. Morii N. and
    11. Narumiya S.
    (1996). The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J 15, 1885–1893
    OpenUrlPubMedWeb of Science
    1. Itoh M.,
    2. Yonemura S.,
    3. Nagafuchi A.,
    4. Tsukita Sa. and
    5. Tsukita Sh
    (1991). A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites. J. Cell Biol 115, 1449–1462
    OpenUrlAbstract/FREE Full Text
    1. Jainchill J. L.,
    2. Aaronson S. A. and
    3. Todaro G. J.
    (1969). Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol 4, 549–553
    OpenUrlAbstract/FREE Full Text
    1. Kimura G.,
    2. Itagaki A. and
    3. Summers J.
    (1975). Rat cell line 3y1 and its virogenic polyoma-and sv40-transformed derivatives. Int. J. Cancer 15, 694–706
    OpenUrlCrossRefPubMedWeb of Science
    1. Kondo T.,
    2. Takeuchi K.,
    3. Doi Y.,
    4. Yonemura S.,
    5. Nagata S.,
    6. Tsukita Sh. and
    7. Tsukita Sa
    (1997). ERM (ezrin/radixin/moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J. Cell Biol 139, 749–758
    OpenUrlAbstract/FREE Full Text
    1. Krieg J. and
    2. Hunter T.
    (1992). Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J. Biol Chem 267, 19258–19265
    OpenUrlAbstract/FREE Full Text
    1. Lankes W.,
    2. Griesmacher A.,
    3. Grunwald J.,
    4. Schwartz-Albiez R. and
    5. Keller R.
    (1988). A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation. Biochem J 251, 831–842
    OpenUrlAbstract/FREE Full Text
    1. Lankes W. T. and
    2. Furthmayr H.
    (1991). Moesin: a member of the protein 4.1-talin-ezrin family of proteins. Proc. Nat. Acad. Sci. USA 88, 8297–8301
    OpenUrlAbstract/FREE Full Text
    1. Leong S. P. L.,
    2. Cooperband S. R.,
    3. Deckers P. J.,
    4. Sutherland C. M.,
    5. Cesare J. F. and
    6. Krementz E. T.
    (1979). Effect of different fixatives on the localization of human melanoma antigens by immunofluorescence. Oncology 36, 202–207
    OpenUrlPubMed
    1. Leung T.,
    2. Manser E.,
    3. Tan L. and
    4. Lim L.
    (1995). A novel serine/threoninekinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol Chem 270, 29051–29054
    OpenUrlAbstract/FREE Full Text
    1. Magendantz M.,
    2. Henry M. D.,
    3. Lander A. and
    4. Solomon F.
    (1995). Interdomain interactions of radixin in vitro. J. Biol Chem 270, 25324–25327
    OpenUrlAbstract/FREE Full Text
    1. Martin M.,
    2. Andreoli C.,
    3. Sahuquet A.,
    4. Montcourrier P.,
    5. Algrain M. and
    6. Mangeat P.
    (1995). Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain. J. Cell Biol 128, 1081–1093
    OpenUrlAbstract/FREE Full Text
    1. Matsui T.,
    2. Amano M.,
    3. Yamamoto T.,
    4. Chihara K.,
    5. Nakafuku M.,
    6. Ito M.,
    7. Nakano T.,
    8. Okawa K.,
    9. Iwamatsu A. and
    10. Kaibuchi K.
    (1996). Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J 15, 2208–2216
    OpenUrlPubMedWeb of Science
    1. Matsui T.,
    2. Maeda M.,
    3. Doi Y.,
    4. Yonemura S.,
    5. Amano M.,
    6. Kaibuchi K.,
    7. Tsukita Sa. and
    8. Tsukita Sh
    (1998). Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol 140, 647–657
    OpenUrlAbstract/FREE Full Text
    1. Nakamura F.,
    2. Amieva M. R. and
    3. Furthmayr H.
    (1995). Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J. BiolChem 270, 31377–31385
    OpenUrlAbstract/FREE Full Text
    1. Nakamura F.,
    2. Amieva M. R.,
    3. Hirota C.,
    4. Mizuno Y. and
    5. Furthmayr H.
    (1996). Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res.Commun 226, 650–656
    OpenUrlCrossRefPubMed
    1. Oshiro N.,
    2. Fukata Y. and
    3. Kaibuchi K.
    (1998). Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol Chem 273, 34663–34666
    OpenUrlAbstract/FREE Full Text
    1. Pakkanen R.,
    2. Hedman K.,
    3. Turunen O.,
    4. Wahlstrom T. and
    5. Vaheri A.
    (1987). Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells. J. Histochem. Cytochem 35, 809–816
    OpenUrlAbstract/FREE Full Text
    1. Pestonjamasp K.,
    2. Amieva M. R.,
    3. Strassel C. P.,
    4. Nauseef W. M.,
    5. Furthmayr H. and
    6. Luna E. J.
    (1995). Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. Mol. Biol Cell 6, 247–259
    OpenUrlAbstract/FREE Full Text
    1. Pietromonaco S. F.,
    2. Simons P. C.,
    3. Altman A. and
    4. Elias L.
    (1998). Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J. Biol Chem 273, 7594–7603
    OpenUrlAbstract/FREE Full Text
    1. Sato N.,
    2. Yonemura S.,
    3. Obinata T.,
    4. Tsukita Sa. and
    5. Tsukita Sh
    (1991). Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis. J. Cell Biol 113, 321–330
    OpenUrlAbstract/FREE Full Text
    1. Sato N.,
    2. Funayama N.,
    3. Nagafuchi A.,
    4. Yonemura S.,
    5. Tsukita Sa. and
    6. Tsukita Sh
    (1992). A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J. Cell Sci 103, 131–143
    OpenUrlAbstract/FREE Full Text
    1. Serrador J. M.,
    2. Alonso-Lebrero J. L.,
    3. del Pozo M. A.,
    4. Furthmayr H.,
    5. Schwartz-Albiez R.,
    6. Calvo J.,
    7. Lozano F. and
    8. Sanchez-Madrid F.
    (1997). Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. J. Cell Biol 138, 1409–1423
    OpenUrlAbstract/FREE Full Text
    1. Schwartz-Albiez R.,
    2. Merling A.,
    3. Spring H.,
    4. Moller P. and
    5. Koretz K.
    (1995). Differential expression of the microspike-associated protein moesin in human tissues. Eur. J. Cell Biol 67, 189–198
    OpenUrlPubMedWeb of Science
    1. Shaw R. J.,
    2. Henry M.,
    3. Solomon F. and
    4. Jacks T.
    (1998). RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol. Biol. Cell 9, 403–419
    OpenUrlAbstract/FREE Full Text
    1. Takeuchi K.,
    2. Sato N.,
    3. Kasahara H.,
    4. Funayama N.,
    5. Nagafuchi A.,
    6. Yonemura S.,
    7. Tsukita Sa. and
    8. Tsukita Sh
    (1994). Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J. Cell Biol 125, 1371–1384
    OpenUrlAbstract/FREE Full Text
    1. Tsukita Sa.,
    2. Hieda Y. and
    3. Tsukita Sh
    (1989). A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization. J. Cell Biol 108, 2369–2382
    OpenUrlAbstract/FREE Full Text
    1. Tsukita Sa.,
    2. Oishi K.,
    3. Sato N.,
    4. Sagara J.,
    5. Kawai A. and
    6. Tsukita Sh
    (1994). ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J. Cell Biol 126, 391–401
    OpenUrlAbstract/FREE Full Text
    1. Tsukita Sa.,
    2. Yonemura S. and
    3. Tsukita Sh
    (1997). ERM proteins: head-to-tail regulation of actin-plasma membrane interaction. Trends Biochem Sci 22, 53–58
    OpenUrlCrossRefPubMedWeb of Science
    1. Tsukita Sa.,
    2. Yonemura S. and
    3. Tsukita Sh
    (1997). ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction. Curr. Opin. Cell Biol 9, 70–75
    OpenUrlCrossRefPubMedWeb of Science
    1. Turunen O.,
    2. Wahlstrom T. and
    3. Vaheri A.
    (1994). Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J.Cell Biol 126, 1445–1453
    OpenUrlAbstract/FREE Full Text
    1. Urushidani T.,
    2. Hanzel D. K. and
    3. Forte J. G.
    (1989). Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation. Am. J.Physiol 256, 1070–.
    OpenUrl
    1. Yonemura S.,
    2. Hirao M.,
    3. Doi Y.,
    4. Takahashi N.,
    5. Kondo T.,
    6. Tsukita Sa. and
    7. Tsukita Sh
    (1998). 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. J. Cell Biol 140, 885–895
    OpenUrlAbstract/FREE Full Text
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Journal Articles
Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues
K. Hayashi, S. Yonemura, T. Matsui, S. Tsukita
Journal of Cell Science 1999 112: 1149-1158;
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