Molecular and Cellular Biochemistry

, Volume 262, Issue 1–2, pp 187–193

Ribosomal protein S18 identified as a cofilin-binding protein by using phage display library

  • Kaoru Kusui
  • Haruyo Sasaki
  • Reiko Adachi
  • Sachiko Matsui
  • Kazuo Yamamoto
  • Teruhide Yamaguchi
  • Tadashi Kasahara
  • Kazuhiro Suzuki


We previously reported that an actin-binding protein, cofilin, is involved in superoxide production, phagocytosis, and chemotaxis in activated phagocytes through cytoskeletal reorganization. To elucidate the functions of cofilin in greater detail we tried to identify cofilin-binding proteins by using a phage-displayed cDNA library constructed from human brain mRNAs. Several phage clones capable of binding to cofilin were obtained, and the phage with the strongest binding affinity contained the C-terminal half of ribosomal protein S18. To confirm the interaction between the S18 protein and cofilin, we investigated whether cofilin would bind to His-tagged S18 protein immobilized in Ni-NTA-agarose gel. Cofilin and the S18 protein co-eluted with a low pH (4.5) buffer, suggesting that the proteins interact with each other. Preincubation of cofilin with actin abrogated the binding to protein S18, indicating that cofilin interacts with S18 protein at the actin-binding site, and cofilin co-immunoprecipitated with FLAG-tagged S18 protein expressed in COS-7 cells. These results suggest that some cofilin molecules bind the ribosomal S18 protein under physiological conditions. (Mol Cell Biochem 262: 187–193, 2004)

actin cofilin phage display ribosomal protein S18 


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  1. 1.
    Edwards SW: Biochemistry and physiology of the neutrophil.Cambridge University Press, Cambridge, UK, 1994Google Scholar
  2. 2.
    Suzuki K, Yamaguchi T, Oshizawa T, Yamamoto Y, Nishimaki-Mogami T, Hayakawa T, Takahashi A: Okadaic acid induces both augmentation and inhibition of opsonized zymosan-stimulated superoxide production by differentiated HL-60 cells. Possible involvement of dephosphorylation of a cytosolic 21K protein in respiratory burst. Biochim Biophys Acta 1266: 261–267, 1995CrossRefPubMedGoogle Scholar
  3. 3.
    Suzuki K, Yamaguchi T, Tanaka T, Kawanishi T, Nishimaki-Mogami T, Yamamoto K, Tsuji T, Irimura T, Hayakawa T, Takahashi A: Activation induces dephosphorylation of cofilin and its translocation to plasma membranes in neutrophil-like differentiated HL-60 cells. J Biol Chem 270: 19551–19556, 1995CrossRefPubMedGoogle Scholar
  4. 4.
    Nishida E, Maekawa S, Sakai H: Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin. Biochemistry 23: 5307–5313, 1984PubMedGoogle Scholar
  5. 5.
    Bamburg JR: Proteins of the ADF/cofilin family: Essential regulators of actin dynamics. Annu Rev Cell Dev Biol 15: 185–230, 1999CrossRefPubMedGoogle Scholar
  6. 6.
    Okada K, Takano-Ohmuro H, Obinata T, Abe H: Dephosphorylation of cofilin in polymorphonuclear leukocytes derived from peripheral blood.Exp Cell Res 227: 116–122, 1996CrossRefPubMedGoogle Scholar
  7. 7.
    Djafarzadeh S, Niggli V: Signaling pathways involved in dephosphorylation and localization of the actin-binding protein cofilin in stimulated human neutrophils. Exp Cell Res 236: 427–435, 1997CrossRefPubMedGoogle Scholar
  8. 8.
    Heyworth PG, Robinson JM, Ding J, Ellis BA, Badwey JA: Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation.Histochem Cell Biol 108: 221–233, 1997PubMedGoogle Scholar
  9. 9.
    Nagaishi K, Adachi R, Matsui S, Yamaguchi T, Kasahara T, Suzuki K: Herbimycin A inhibits both dephosphorylation and translocation of cofilin induced by opsonized zymosan in macrophagelike U937 cells. J Cell Physiol 180: 345–354, 1999PubMedGoogle Scholar
  10. 10.
    Matsui S, Adachi R, Kusui K, Yamaguchi T, Kasahara T, Hayakawa T, Suzuki K: U73122 inhibits the dephosphorylation and translocation of cofilin in activated macrophage-like U937 cells. Cell Signal 13: 17–22, 2001CrossRefPubMedGoogle Scholar
  11. 11.
    Nagaishi K, Adachi R, Kawanishi T, Yamaguchi T, Kasahara T, Hayakawa T, Suzuki K: Participation of cofilin in opsonized 193 zymosan-triggered activation of neutrophil-like HL-60 cells through rapid dephosphorylation and translocation to plasma membranes. J Biochem 125: 891–898, 1999PubMedGoogle Scholar
  12. 12.
    Matsui S, Matsumoto S, Adachi R, Kusui K, Hirayama A, Watanabe H, Ohashi K, Mizuno K, Yamaguchi T, Kasahara T, Suzuki K: LIM kinase 1 modulates opsonized zymosan-triggered activation of macrophagelike U937 cells. Possible involvement of phosphorylation of cofilin and reorganization of actin cytoskeleton. J Biol Chem 277: 544–549, 2002CrossRefPubMedGoogle Scholar
  13. 13.
    Adachi R, Takeuchi K, Suzuki K: Antisense oligonucleotide to cofilin enhances respiratory burst and phagocytosis in opsonized zymosanstimulated mouse macrophage J774.1 cells. J Biol Chem 277: 45566–45571, 2002CrossRefPubMedGoogle Scholar
  14. 14.
    Adachi R, Matsui S, Kinoshita M, Nagaishi K, Sasaki H, Kasahara T, Suzuki K: Nitric oxide induces chemotaxis of neutrophil-like HL-60 cells and translocation of cofilin to plasma membranes. Int J Immunopharmacol 22: 855–864, 2000CrossRefPubMedGoogle Scholar
  15. 15.
    Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, Caroni P: Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393: 805–809, 1998CrossRefPubMedGoogle Scholar
  16. 16.
    Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, Nishida E, Mizuno K: Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393: 809–812, 1998CrossRefPubMedGoogle Scholar
  17. 17.
    Niwa R, Nagata-Ohashi K, Takeichi M, Mizuno K, Uemura T: Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108: 233–246, 2002CrossRefPubMedGoogle Scholar
  18. 18.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970PubMedGoogle Scholar
  19. 19.
    Smith GP, Scott JK: Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol 217: 228–257, 1993CrossRefPubMedGoogle Scholar
  20. 20.
    Hesketh J: Translation and the cytoskeleton: A mechanism for targeted protein synthesis. Mol Biol Rep 19: 233–243, 1994PubMedGoogle Scholar
  21. 21.
    Bassell G, Singer RH: mRNA and cytoskeletal filaments. Curr Opin Cell Biol 9: 109–115, 1997CrossRefPubMedGoogle Scholar
  22. 22.
    Shestakova EA, Motuz LP, Minin AA, Gelfand VI, Gavrilova LP: Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts. Cell Biol Int Rep 15: 75–84, 1991PubMedGoogle Scholar
  23. 23.
    Gavrilova LP, Rutkevitch NM, Gelfand VI, Motuz LP, Stahl J, Bommer UA, Bielka H: Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts. Cell Biol Int Rep 11: 745–753, 1987PubMedGoogle Scholar
  24. 24.
    Yang F, Demma M, Warren V, Dharmawardhane S, Condeelis J: Identification of an actin-binding protein from Dictyostelium as elongation factor 1a. Nature 347: 494–496, 1990CrossRefPubMedGoogle Scholar
  25. 25.
    Liu G, Tang J, Edmonds BT, Murray J, Levin S, Condeelis J: F-actin sequesters elongation factor 1 alpha from interaction with aminoacyltRNA in a pH-dependent reaction. J Cell Biol 135: 953–963, 1996CrossRefPubMedGoogle Scholar
  26. 26.
    Edmonds BT, Wyckoff J, Yeung YG, Wang Y, Stanley ER, Jones J, Segall J, Condeelis J: Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma. J Cell Sci 109: 2705–2714, 1996PubMedGoogle Scholar
  27. 27.
    Hovland R, Campbell G, Pryme I, Hesketh J: The mRNAs for cyclin A: c-myc and ribosomal proteins L4 and S6 are associated with cytoskeletal-bound polysomes in HepG2 cells. Biochem J 310: 193–196, 1995PubMedGoogle Scholar
  28. 28.
    Izawa T, Fukata Y, Kimura T, Iwamatsu A, Dohi K, Kaibuchi K: Elongation factor-1 alpha is a novel substrate of rho-associated kinase. Biochem Biophys Res Commun 278: 72–78, 2000PubMedGoogle Scholar
  29. 29.
    Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumiya S: Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.Science 285: 895–898, 1999CrossRefPubMedGoogle Scholar
  30. 30.
    Pfannstiel J, Cyrklaff M, Habermann A, Stoeva S, Griffiths G, Shoeman R, Faulstich H: Human cofilin forms oligomers exhibiting actin bundling activity. J Biol Chem 276: 49476–49484, 2001CrossRefPubMedGoogle Scholar
  31. 31.
    Lee K, Jung J, Kim M, Guidotti G: Interaction of the alpha subunit of Na,K-ATPase with cofilin. Biochem J 353: 377–385, 2001CrossRefPubMedGoogle Scholar
  32. 32.
    Birkenfeld J, Betz H, Roth D: Identification of cofilin and LIM-domaincontaining protein kinase 1 as novel interaction partners of 14-3-3 zeta.Biochem J 369: 45–54, 2003PubMedGoogle Scholar
  33. 33.
    Jung J, Yoon T, Choi EC, Lee K: Interaction of cofilin with triosephosphate isomerase contributes glycolytic fuel for Na,K-ATPase via Rho-mediated signaling pathway. J Biol Chem 277: 48931–48937, 2002CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Kaoru Kusui
    • 1
  • Haruyo Sasaki
    • 1
  • Reiko Adachi
    • 1
  • Sachiko Matsui
    • 2
    • 3
  • Kazuo Yamamoto
    • 4
  • Teruhide Yamaguchi
    • 1
  • Tadashi Kasahara
    • 3
  • Kazuhiro Suzuki
    • 2
  1. 1.National Institute of Health SciencesKamiyoga, Setagaya-ku, TokyoJapan
  2. 2.National Institute of Health SciencesKamiyoga, Setagaya-ku, TokyoJapan
  3. 3.Kyoritsu College of PharmacyShibakoen, Minato-ku, TokyoJapan
  4. 4.Graduate School of Frontier SciencesUniversity of TokyoChibaJapan

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