Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway

The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S- transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5- PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism.

[1]  K. Fujisawa,et al.  Protein Kinase N (PKN) and PKN-Related Protein Rhophilin as Targets of Small GTPase Rho , 1996, Science.

[2]  K. Kaibuchi,et al.  Identification of a Putative Target for Rho as the Serine-Threonine Kinase Protein Kinase N , 1996, Science.

[3]  S. Zigmond,et al.  Signal transduction and actin filament organization. , 1996, Current opinion in cell biology.

[4]  M. Amieva,et al.  Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets (*) , 1995, The Journal of Biological Chemistry.

[5]  C. Andreoli,et al.  Identification of a phosphatidylinositol‐4,5‐bisphosphate‐binding domain in the N‐terminal region of ezrin , 1995, FEBS letters.

[6]  A. Bretscher,et al.  Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis , 1995, The Journal of cell biology.

[7]  P. Janmey,et al.  Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets , 1995, Cell.

[8]  J. Cohn,et al.  Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Bretscher,et al.  Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. , 1995, Molecular biology of the cell.

[10]  T. Sasaki,et al.  Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. , 1995, Oncogene.

[11]  T. Sasaki,et al.  Rho as a regulator of the cytoskeleton. , 1995, Trends in biochemical sciences.

[12]  F. Solomon,et al.  Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains , 1995, The Journal of cell biology.

[13]  M. Zerial,et al.  A GDP/GTP exchange-stimulatory activity for the Rab5-RabGDI complex on clathrin-coated vesicles from bovine brain , 1995, The Journal of Biological Chemistry.

[14]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[15]  L. Lim,et al.  The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts , 1995, Molecular and cellular biology.

[16]  P. Mangeat,et al.  Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain , 1995, The Journal of cell biology.

[17]  K. Pestonjamasp,et al.  Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. , 1995, Molecular biology of the cell.

[18]  L. Chong,et al.  The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells , 1994, Cell.

[19]  M. Symons,et al.  Activation of rat liver phospholipase D by the small GTP-binding protein RhoA. , 1994, The Journal of biological chemistry.

[20]  A. Vaheri,et al.  Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family , 1994, The Journal of cell biology.

[21]  P. Mangeat,et al.  Ezrin has properties to self-associate at the plasma membrane. , 1994, Journal of cell science.

[22]  N. Sato,et al.  ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons , 1994, The Journal of cell biology.

[23]  A. Kawashima,et al.  Structural diversity of band 4.1 superfamily members. , 1994, Journal of cell science.

[24]  N. Sato,et al.  Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members , 1994, The Journal of cell biology.

[25]  T. Soldati,et al.  Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange , 1994, Nature.

[26]  P. Novick,et al.  No exchange without receipt , 1994, Nature.

[27]  T. Sasaki,et al.  rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells , 1994, Molecular and cellular biology.

[28]  M. Zerial,et al.  Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange , 1994, Nature.

[29]  M. Arpin,et al.  Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1. , 1994, Current opinion in cell biology.

[30]  S. Narumiya,et al.  A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs , 1993, Zygote.

[31]  J. Zhang,et al.  Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. , 1993, The Journal of biological chemistry.

[32]  J. Lambeth,et al.  Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein. , 1993, The Journal of biological chemistry.

[33]  A. Bretscher,et al.  Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. , 1993, Journal of cell science.

[34]  S. Pulst,et al.  Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2 , 1993, Nature.

[35]  A. Bretscher,et al.  Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable. , 1993, Journal of cell science.

[36]  J. Haines,et al.  A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor , 1993, Cell.

[37]  T. Sasaki,et al.  Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) , 1993, The Journal of cell biology.

[38]  N. Sato,et al.  Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons , 1993, The Journal of cell biology.

[39]  M. Arpin,et al.  Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker , 1993, The Journal of cell biology.

[40]  S. Tsukita,et al.  Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions. , 1992, Current opinion in cell biology.

[41]  T. Takenawa,et al.  Requirement of phosphatidylinositol 4,5-bisphosphate for α-actinin function , 1992, Nature.

[42]  N. Sato,et al.  A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. , 1992, Journal of cell science.

[43]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992, Cell.

[44]  Anne J. Ridley,et al.  The small GTP-binding protein rac regulates growth factor-induced membrane ruffling , 1992, Cell.

[45]  N. Sato,et al.  Radixin is a novel member of the band 4.1 family , 1991, The Journal of cell biology.

[46]  H. Furthmayr,et al.  Moesin: a member of the protein 4.1-talin-ezrin family of proteins. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[47]  E. Nishida,et al.  A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin. , 1991, The Journal of biological chemistry.

[48]  H. Liao,et al.  The transmembrane hyaluronate receptor (CD44): multiple functions, multiple forms. , 1991, Cancer cells.

[49]  A. Bretscher,et al.  The secretion‐stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli. , 1991, The EMBO journal.

[50]  N. Tonks,et al.  Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4.1, ezrin, and talin. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[51]  J. York,et al.  Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[52]  N. Sato,et al.  Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis [published erratum appears in J Cell Biol 1991 Sep;114(5):1101-3] , 1991, The Journal of cell biology.

[53]  K. Kaibuchi,et al.  Tissue and subcellular distributions of an inhibitory GDP/GTP exchange protein (GDI) for the rho proteins by use of its specific antibody. , 1991, Biochemical and biophysical research communications.

[54]  T. Südhof,et al.  gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. , 1990, Science.

[55]  Sarah E. Ades,et al.  Sequence and domain structure of talin , 1990, Nature.

[56]  K. Kaibuchi,et al.  Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. , 1990, Oncogene.

[57]  A. Hall,et al.  The cellular functions of small GTP-binding proteins. , 1990, Science.

[58]  Y. Hata,et al.  Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. , 1990, The Journal of biological chemistry.

[59]  J. Vandekerckhove.  Actin-binding proteins. , 1990, Current opinion in cell biology.

[60]  M. Telen,et al.  CD44--a molecule involved in leukocyte adherence and T-cell activation. , 1989, Immunology today.

[61]  M. Itoh,et al.  A new 400-kD protein from isolated adherens junctions: its localization at the undercoat of adherens junctions and at microfilament bundles such as stress fibers and circumferential bundles , 1989, The Journal of cell biology.

[62]  A. Bretscher,et al.  cDNA cloning and sequencing of the protein‐tyrosine kinase substrate, ezrin, reveals homology to band 4.1. , 1989, The EMBO journal.

[63]  D. Goeddel,et al.  Molecular cloning and expression of Pgp-1. The mouse homolog of the human H-CAM (Hermes) lymphocyte homing receptor. , 1989, Journal of immunology.

[64]  K. Grzeschik,et al.  Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization. , 1989, The Journal of biological chemistry.

[65]  S. Tsukita,et al.  A new 82-kD barbed end-capping protein (radixin) localized in the cell- to-cell adherens junction: purification and characterization , 1989, The Journal of cell biology.

[66]  L. Picker,et al.  A human lymphocyte homing receptor, the Hermes antigen, is related to cartilage proteoglycan core and link proteins , 1989, Cell.

[67]  V. Bennett,et al.  The spectrin-actin junction of erythrocyte membrane skeletons. , 1989, Biochimica et biophysica acta.

[68]  J. Vandekerckhove,et al.  Purification of the 22 kDa protein substrate of botulinum ADP‐ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP‐binding protein highly homologous to the rho gene product , 1989, FEBS letters.

[69]  S. Tsukita,et al.  Isolation of cell-to-cell adherens junctions from rat liver , 1989, The Journal of cell biology.

[70]  S. Narumiya,et al.  Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product. , 1988, The Journal of biological chemistry.

[71]  Y. Takai,et al.  ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1. , 1988, The Journal of biological chemistry.

[72]  W. Carter,et al.  The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa , 1988, The Journal of cell biology.

[73]  A. Griesmacher,et al.  A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation. , 1988, The Biochemical journal.

[74]  K. Aktories,et al.  Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. , 1988, European journal of biochemistry.

[75]  A. Vaheri,et al.  Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells. , 1987, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[76]  J. Conboy,et al.  Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[77]  Jonathan A. Cooper,et al.  The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein , 1986, The Journal of cell biology.

[78]  U. Lindberg,et al.  Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin , 1985, Nature.

[79]  A. Bretscher.  Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells , 1983, The Journal of cell biology.

[80]  M. Kirschner,et al.  Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. , 1977, The Journal of biological chemistry.

[81]  H. Pelham,et al.  An efficient mRNA-dependent translation system from reticulocyte lysates. , 1976, European journal of biochemistry.

[82]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[83]  N. Divecha,et al.  Phospholipid signaling , 1995, Cell.

[84]  M. Amieva,et al.  Radixin is a component of hepatocyte microvilli in situ. , 1994, Experimental cell research.

[85]  P. Kincade,et al.  CD44 and its interaction with extracellular matrix. , 1993, Advances in immunology.

[86]  P. Janmey,et al.  Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate , 1987, Nature.

[87]  S. Jalkanen,et al.  A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man , 1986, European journal of immunology.

[88]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.