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Amino Acids

, Volume 41, Issue 4, pp 781–787 | Cite as

S100A6 mediates nuclear translocation of Sgt1: a heat shock-regulated protein

  • Wiktor Prus
  • Anna FilipekEmail author
Original Article

Abstract

Sgt1 was originally identified in yeast as a suppressor of the Skp1 protein. Later, it was found that Sgt1 is present in plant and mammalian organisms and that it binds other ligands such as S100A6, a calcium-binding protein. In this work we show that in HEp-2 cells Sgt1 translocates to the nucleus due to heat shock. We also found that in HEp-2 cells with diminished level of S100A6, due to stable transfection with siRNA against S100A6, such translocation occurred at a much smaller scale in comparison with cells expressing a normal level of S100A6. Moreover, translocation of Sgt1 was observed in HEp-2 cells treated with thapsigargin instead of heat shock. In contrast thapsigargin was ineffective in cells with diminished level of S100A6. Thus, our results suggest that increase in intracellular concentration of Ca2+, transduced by S100A6, is necessary for nuclear translocation of the Sgt1 protein.

Keywords

Sgt1 S100A6 (calcyclin) Heat shock proteins Nuclear translocation 

Abbreviations

AM

Acetoxymethyl ester

BAPTA

Bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid

BSA

Bovine serum albumin

[Ca2+]i

Intracellular calcium concentration

DMEM

Dulbecco’s Modified Eagle’s Medium

DTT

Dithiothreitol

EDTA

Ethylene diamine tetraacetic acid

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

HDAC1

Histone deacetylase 1

PBS

Phosphate buffered saline

PAGE

Polyacrylamide gel electrophoresis

SDS

Sodium dodecyl sulfate

TBS

Tris buffered saline

Notes

Acknowledgments

We thank Drs W. Lesniak and G. Schneider for helpful discussion and critical reading of the manuscript. This work was supported by a grant to A. Filipek (No. N301 293637) from the Ministry of Science and Higher Education of Poland, by Polish Network for Visualization of Biomedical events and by statutory funds from the Nencki Institute of Experimental Biology.

References

  1. Ali MM, Roe SM, Vaughan CK, Meyer P, Panaretou B, Piper PW, Prodromou C, Pearl LH (2006) Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440:1013–1017PubMedCrossRefGoogle Scholar
  2. Arya R, Mallik M, Lakhotia SC (2007) Heat shock genes—integrating cell survival and death. J Biosci 32:595–610PubMedCrossRefGoogle Scholar
  3. Catelli MG, Binart N, Jung-Testas I, Renoir JM, Baulieu EE, Feramisco JR, Welch WJ (1985) The common 90-kDa protein component of non-transformed ‘8S’ steroid receptors is a heat-shock protein. EMBO J 4:3131–3135PubMedGoogle Scholar
  4. da Silva Correia J, Miranda Y, Leonard N, Ulevitch R (2007) SGT1 is essential for Nod1 activation. Proc Natl Acad Sci USA 104:6764–6769PubMedCrossRefGoogle Scholar
  5. Daniel S, Bradley G, Longshaw VM, Söti C, Csermely P, Blatch GL (2008) Nuclear translocation of the phosphoprotein Hop (Hsp70/Hsp90 organizing protein) occurs under heat shock, and its proposed nuclear localization signal is involved in Hsp90 binding. Biochim Biophys Acta 1783:1003–1014PubMedCrossRefGoogle Scholar
  6. Galigniana MD, Harrell JM, Housley PR, Patterson C, Fisher SK, Pratt WB (2004) Retrograde transport of the glucocorticoid receptor in neurites requires dynamic assembly of complexes with the protein chaperone hsp90 and is linked to the CHIP component of the machinery for proteasomal degradation. Brain Res Mol Brain Res 123:27–36PubMedCrossRefGoogle Scholar
  7. Hubert DA, He Y, McNulty BC, Tornero P, Dangl JL (2009) Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation. Proc Natl Acad Sci USA 106:9556–9563PubMedCrossRefGoogle Scholar
  8. Kadota Y, Amigues B, Ducassou L, Madaoui H, Ochsenbein F, Guerois R, Shirasu K (2008) Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants. EMBO Rep 9:1209–1215PubMedCrossRefGoogle Scholar
  9. Khandjian EW, Rose TM (2000) Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40. Biochem Cell Biol 78:437–445PubMedCrossRefGoogle Scholar
  10. Kitagawa K, Skowyra D, Elledge SJ, Harper JW, Hieter P (1999) SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol Cell 4:21–33PubMedCrossRefGoogle Scholar
  11. Knudsen NØ, Andersen SD, Lützen A, Nielsen FC, Rasmussen LJ (2009) Nuclear translocation contributes to regulation of DNA excision repair activities. DNA Repair 8:682–689PubMedCrossRefGoogle Scholar
  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  13. Lee YT, Jacob J, Michowski W, Nowotny M, Kuznicki J, Chazin WJ (2004) Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain. J Biol Chem 279:16511–16517PubMedCrossRefGoogle Scholar
  14. Leśniak W, Słomnicki ŁP, Filipek A (2009) S100A6 – New facts and features. Biochem Biophys Res Commun 390:1087–1092Google Scholar
  15. Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55:1151–1191PubMedCrossRefGoogle Scholar
  16. Martins T, Maia AF, Steffensen S, Sunkel CE (2009) Sgt1, a co-chaperone of Hsp90 stabilizes Polo and is required for centrosome organization. EMBO J 28:234–247PubMedCrossRefGoogle Scholar
  17. Mayor A, Martinon F, De Smedt T, Pétrilli V, Tschopp J (2007) A crucial function of SGT1 and HSP90 in inflammasome activity links mammalian and plant innate immune responses. Nat Immunol 8:497–503PubMedCrossRefGoogle Scholar
  18. Meyer P, Prodromou C, Liao C, Hu B, Roe SM, Vaughan CK, Vlasic I, Panaretou B, Piper PW, Pearl LH (2004) Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. EMBO J 23:1402–1410PubMedCrossRefGoogle Scholar
  19. Nadeau K, Sullivan MA, Bradley M, Engman DM, Walsh CT (1992) 83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases. Protein Sci 1:970–979PubMedCrossRefGoogle Scholar
  20. Nadeau K, Das A, Walsh CT (1993) Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. J Biol Chem 268:1479–1487PubMedGoogle Scholar
  21. Nowotny M, Spiechowicz M, Jastrzebska B, Filipek A, Kitagawa K, Kuznicki J (2003) Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins. J Biol Chem 278:26923–26928PubMedCrossRefGoogle Scholar
  22. Passinen S, Valkila J, Manninen T, Syvälä H, Ylikomi T (2001) The C-terminal half of Hsp90 is responsible for its cytoplasmic localization. Eur J Biochem 268:5337–5342PubMedCrossRefGoogle Scholar
  23. Perdew GH, Hord N, Hollenback CE, Welsh MJ (1993) Localization and characterization of the 86- and 84-kDa heat shock proteins in Hepa 1c1c7 cells. Exp Cell Res 209:350–356PubMedCrossRefGoogle Scholar
  24. Picard D (2008) A stress protein interface of innate immunity. EMBO Rep 9:1193–1195PubMedCrossRefGoogle Scholar
  25. Pratt WB, Galigniana MD, Morishima Y, Murphy PJ (2004) Role of molecular chaperones in steroid receptor action. Essays Biochem 40:41–58PubMedGoogle Scholar
  26. Sharma D, Masison DC (2009) Hsp70 structure, function, regulation and influence on yeast prions. Protein Pept Lett 16:571–581PubMedCrossRefGoogle Scholar
  27. Shirasu K (2009) The HSP90-SGT1 chaperone complex for NLR immune sensors. Annu Rev Plant Biol 60:139–164PubMedCrossRefGoogle Scholar
  28. Silver PA (1991) How proteins enter the nucleus. Cell 64:489–497PubMedCrossRefGoogle Scholar
  29. Słomnicki ŁP, Nawrot B, Leśniak W (2009) S100A6 binds p53 and affects its activity. Int J Biochem Cell Biol 41:784–790PubMedCrossRefGoogle Scholar
  30. Spiechowicz M, Zylicz A, Bieganowski P, Kuznicki J, Filipek A (2007) Hsp70 is a new target of Sgt1—an interaction modulated by S100A6. Biochem Biophys Res Commun 357:1148–1153PubMedCrossRefGoogle Scholar
  31. Steensgaard P, Garrè M, Muradore I, Transidico P, Nigg EA, Kitagawa K, Earnshaw WC, Faretta M, Musacchio A (2004) Sgt1 is required for human kinetochore assembly. EMBO Rep 5:626–631PubMedCrossRefGoogle Scholar
  32. Stevenson MA, Calderwood SK, Hahn GM (1986) Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock. Biochem Biophys Res Commun 137:826–833PubMedCrossRefGoogle Scholar
  33. van Bergen en Henegouwen PM, Berbers G, Linnemans WA, van Wijk R (1987) Subcellular localization of the 84,000 Dalton heat-shock protein in mouse neuroblastoma cells: evidence for a cytoplasmic and nuclear location. Eur J Cell Biol 43:469–478PubMedGoogle Scholar
  34. Zabka M, Leśniak W, Prus W, Kuźnicki J, Filipek A (2008) Sgt1 has co-chaperone properties and is up-regulated by heat shock. Biochem Biophys Res Commun 370:179–183PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Nencki Institute of Experimental BiologyWarsawPoland

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