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Journal of Muscle Research & Cell Motility

, Volume 26, Issue 4–5, pp 175–181 | Cite as

Small heat shock protein with apparent molecular mass 20 kDa (Hsp20, HspB6) is not a genuine actin-binding protein

  • Olesya V. Bukach
  • Steven B. Marston
  • Nikolai B. Gusev
Article

Abstract

The interaction of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20, HspB6) with actin was investigated. Wild type Hsp20 and its S16D mutant mimicking phosphorylation of Hsp20 by cyclic nucleotide-dependent protein kinases do not affect the rate and extent of actin polymerization. Ultracentrifugation of the mixture of Hsp20 (or its S16D mutant) with isolated F-actin or F-actin containing tropomyosin, calponin or α-actinin resulted in co-sedimentation of less than 0.04 mol of Hsp20 monomer per mol of actin. Myofibrils of skeletal, cardiac or smooth muscle bound less than 0.04 mol of Hsp20 monomer per mol of actin and this stoichiometry was independent of phosphorylation or mutation of Ser16 of Hsp20. Since Hsp20 is not a genuine actin-binding protein, the earlier described correlation between Hsp20 phosphorylation and smooth muscle relaxation cannot be explained by direct interaction of Hsp20 with actin.

Keywords

Actin Polymerization Apparent Molecular Mass Small Heat Shock Protein Hsp20 Phosphorylation S16D Mutant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

Hsp20

recombinant human heat shock protein with apparent molecular mass 20 kDa

S16D mutant of Hsp20

mutant with replacing S16 with aspartic acid

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Notes

Acknowledgements

The authors are grateful to M.V. Kim (Department of Biochemistry, School of Biology, Moscow State University) for her help in performing experiments on actin polymerization. This investigation was supported by the grants from the Russian Foundation for Basic Research (04-04-48404) and by The Wellcome Trust (CRIG 064581).

References

  1. Beall AC, Kato K, Goldenring JR, Rasmussen H, Brophy CM, (1997) Cyclic nucleotide-dependent vasorelaxation is associated with the phosphorylation of a small heat shock-related protein J Biol Chem 272: 11283–11287PubMedGoogle Scholar
  2. Beall A, Bagwell D, Woodrum D, Stoming TA, Kato K, Suzuki A, Rasmussen H, Brophy CM, (1999) The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation J Biol Chem 274: 11344–11351CrossRefPubMedGoogle Scholar
  3. Benndorf R, Sun X, Gilmont RR, Biderman KJ, Molloy MP, Goodmurphy CW, Cheng H, Andrews PC, Welsh MJ, (2001) HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 (3DHSP27) J Biol Chem 276: 26753–26761CrossRefPubMedGoogle Scholar
  4. Brophy CM, Lamb S, Graham A, (1999) The small heat shock-related protein-20 is an actin-associated protein J Vasc Surg 29: 326–333PubMedCrossRefGoogle Scholar
  5. Bukach OV, Seit-Nebi AS, Marston SB, Gusev NB, (2004) Some properties of human small heat shock protein Hsp20 (HspB6) Eur J Biochem 271: 291–302CrossRefPubMedGoogle Scholar
  6. Chu G, Egnaczyk GF, Zhao W, Jo S-H, Fan G-C, Maggio JE, Xiao R-P, Kranias EG, (2004) Phosphoproteome analysis of cardiomyocytes subjected to β-adrenergic stimulation. Identification and characterization of cardiac heat shock protein p20 Circ Res 94: 184–193CrossRefPubMedGoogle Scholar
  7. Dreiza CM, Brophy CM, Komalavilas P, Furnish EJ, Joshi L, Pallero MA, Murphy-Ullrich JE, von Rechenberg M, Ho YS, Richardson B, Xu N, Zhen Y, Peltier JM, Panitch A, (2005) Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics FASEB J 19: 261–263PubMedGoogle Scholar
  8. Ehrnsperger M, Lilie H, Gaestel M, Buchner J, (1999) The dynamics of Hsp25 quaternary structure. Structure and function of different oligomeric species J Biol Chem 274: 14867–14874CrossRefPubMedGoogle Scholar
  9. Fan G-C, Chu G, Mitton B, Song Q, Yuan Q, Kranias EG, (2004) Small heat-shock protein Hsp20 phosphorylation inhibits β-agonist-induced cardiac apoptosis Circ Res 94: 1474–1482CrossRefPubMedGoogle Scholar
  10. Flynn CR, Komalavilas P, Tessier D, Thresher J, Niederkofler EE, Dreiza CM, Nelson RW, Panitch A, Joshi L, Brophy CM, (2003) Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle FASEB J 17: 1358–1360PubMedGoogle Scholar
  11. Franck E, Madsen O, van Rheede T, Ricard G, Huynen MA, de Jong WW, (2004) Evolutionary diversity of vertebrate small heat shock proteins J Mol Evol 59: 792–805CrossRefPubMedGoogle Scholar
  12. Kato K, Goto S, Inaguma Y, Hasegawa K, Morishita R, Asano T, (1994) Purification and characterization of a 20-kDa protein that is highly homologous to αB crystallin J Biol Chem 269: 15302–15309PubMedGoogle Scholar
  13. Kouyama T, Mihashi K, (1981) Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structure change of actin protomer both on polymerization and on binding of heavy meromyosin Eur J Biochem 114: 33–38PubMedCrossRefGoogle Scholar
  14. Laemmli UK, (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227: 680–685CrossRefPubMedGoogle Scholar
  15. Lin P, Luby-Phels K, Stull JT, (1997) Binding of myosin light chain kinase to cellular actin-myosin filaments J Biol Chem 272: 7412–7420CrossRefPubMedGoogle Scholar
  16. Lynch W, Bretscher A, (1986) Purification of caldesmon Methods Enzymol 124: 37–42CrossRefGoogle Scholar
  17. Matsuno H, Ishisaki A, Nakajima K, Kato K, Kozawa O, (2003) A peptide isolated from αB-crystallin is a novel and potent inhibitor of platelet aggregation via dual prevention of PAR-1 and GPIb/V/IX J Thromb Haemost 1: 2636–2642CrossRefPubMedGoogle Scholar
  18. Panasenko OO, Gusev NB, (2001) Mutual effects of α-actinin, calponin and filamin on actin binding Biochim Biophys Acta 1544: 393–405PubMedGoogle Scholar
  19. Panasenko OO, Kim MV, Marston SB, Gusev NB, (2003) Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin Eur J Biochem 270: 892–901CrossRefPubMedGoogle Scholar
  20. Pardee JD, Spudich JA, (1982) Purification of muscle actin Methods Enzymol 85: 164–181PubMedGoogle Scholar
  21. Pipkin W, Johnson JA, Creazzo TL, Burch J, Komalavilas P, Brophy C, (2003) Localization, macromolecular associations, and function of the small heat shock-related protein HSP20 in rat heart Circulation 107: 469–476CrossRefPubMedGoogle Scholar
  22. Rembold CM, Foster DB, Strauss JD, Wingard CJ, van Eyk JE, (2000) cGMP-mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation in swine carotid artery J Physiol 524: 865–878CrossRefPubMedGoogle Scholar
  23. Rembold CM, O’Connor M, Clarkson M, Wardle RL, Murphy RA, (2001) Selected contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone J Appl Physiol 91: 1460–1466PubMedGoogle Scholar
  24. Rembold CM, Zhang E, (2001) Localization of heat shock protein 20 in swine carotid artery BMC Physiol 1: 10CrossRefPubMedGoogle Scholar
  25. Taussky HH, Schorr E, (1953) A microscopic method for the determination of inorganic phosphorus J Biol Chem 202: 675–685PubMedGoogle Scholar
  26. Tessier DJ, Komalavilas P, Panitch A, Joshi L, Brophy CM, (2003) The small heat shock protein (HSP) 20 is dynamically associated with the actin cross-linking protein actinin J Surg Res 111: 152–157CrossRefPubMedGoogle Scholar
  27. Tessier DJ, Komalavilas P, Liu B, Kent CK, Thresher JS, Dreiza CM, Panitch A, Joshi L, Furnish E, Stone W, Fowl R., Brophy CM, (2004) Transduction of peptide analogs of the small heat shock-related protein HSP20 inhibits intimal hyperplasia J Vasc Surg 40: 106–114CrossRefPubMedGoogle Scholar
  28. van de Klundert FAJM, Smulders RHPH, Gijsen MLJ, Lindner RA, Jaenicke R, Carver JA, de Jong WW, (1998) The mammalian small heat shock protein Hsp20 forms dimers and is a poor chaperone Eur J Biochem 258: 1014–1021CrossRefPubMedGoogle Scholar
  29. Wang Y, Xu A, Ye J, Kraegen EW, Tse CA, Cooper GJS, (2001) Alteration in phosphorylation of P20 is associated with insulin resistance Diabetes 50: 1821–1827PubMedCrossRefGoogle Scholar
  30. Woodrum D, Pipkin W, Tessier D, Komalavilas P, Brophy CM, (2003) Phosphorylation of the heat shock-related protein, HSP20, mediates cyclic nucleotide-dependent relaxation J Vasc Surg 37: 874–881PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Olesya V. Bukach
    • 1
  • Steven B. Marston
    • 2
  • Nikolai B. Gusev
    • 1
  1. 1.Department of Biochemistry, School of BiologyMoscow State UniversityMoscowRussia
  2. 2.Imperial College LondonNational Heart and Lung InstituteLondonUK

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