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LIM domain protein TES changes its conformational states in different cellular compartments

Molecular and Cellular Biochemistry volume 320, Article number: 85 (2009) Cite this article

Abstract

The human TESTIN (TES) is a putative tumor suppressor and localizes to the cytoplasm as a component of focal adhesions and cell contacts. TES contains a PET domain in the NH2-terminus and three tandem LIM domains in the COOH-terminus. It has been hypothesized that interactions between two termini of TES might lead to a “closed” conformational state of the protein. Here, we provide evidence for different conformational states of TES. We confirmed that the NH2-terminus of TES can interact with its third LIM domain in the COOH-terminus by GST pull-down assays. In addition, antisera against the full-length or two truncations of TES were prepared to examine the relationship between the conformation and cellular distribution of the protein. We found that these antisera recognize different regions of TES and showed that TES is co-localised with the marker protein B23 in nucleolus, in addition to its localization in endoplasmic reticulum (ER). Furthermore, our co-immunoprecipitation (co-IP) analysis of TES and B23 demonstrated their co-existence in the same complex. Taken together, our results suggest that TES has different conformational states in different cellular compartments, and a “closed” conformational state of TES may be involved in nucleolar localization.

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References

  1. Tobias ES, Hurlstone AF, MacKenzie E, McFarlane R, Black DM (2001) The TES gene at 7q31.1 is methylated in tumours and encodes a novel growth-suppressing LIM domain protein. Oncogene 20:2844–2853. doi:10.1038/sj.onc.1204433

    Article  CAS  PubMed  Google Scholar 

  2. Tatarelli C, Linnenbach A, Mimori K, Croce CM (2000) Characterization of the human TESTIN gene localized in the FRA7G region at 7q31.2. Genomics 68:1–12. doi:10.1006/geno.2000.6272

    Article  CAS  PubMed  Google Scholar 

  3. Freyd G, Kim SK, Horvitz HR (1990) Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11. Nature 344:876–879. doi:10.1038/344876a0

    Article  CAS  PubMed  Google Scholar 

  4. Karlsson O, Thor S, Norberg T, Ohlsson H, Edlund T (1990) Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature 344:879–882. doi:10.1038/344879a0

    Article  CAS  PubMed  Google Scholar 

  5. Michelsen JW, Sewell AK, Louis HA, Olsen JI, Davis DR, Winge DR et al (1994) Mutational analysis of the metal sites in an LIM domain. J Biol Chem 269:11108–11113

    CAS  PubMed  Google Scholar 

  6. Dawid IB, Toyama R, Taira M (1995) LIM domain proteins. C R Acad Sci III 318:295–306

    CAS  PubMed  Google Scholar 

  7. Li B, Zhuang L, Trueb B (2004) Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1. J Biol Chem 279:20401–20410. doi:10.1074/jbc.M310304200

    Article  CAS  PubMed  Google Scholar 

  8. Sadler I, Crawford AW, Michelsen JW, Beckerle MC (1992) Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol 119:1573–1587. doi:10.1083/jcb.119.6.1573

    Article  CAS  PubMed  Google Scholar 

  9. Salgia R, Li JL, Lo SH, Brunkhorst B, Kansas GS, Sobhany ES et al (1995) Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J Biol Chem 270:5039–5047. doi:10.1074/jbc.270.10.5039

    Article  CAS  PubMed  Google Scholar 

  10. Shen YL, Xia XX, Zhang Y, Liu JW, Wei DZ, Yang SL (2003) Refolding and purification of Apo2l/TRAIL produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. Biotechnol Lett 25:2097–2101. doi:10.1023/B:BILE.0000007072.56577.0c

    Article  CAS  PubMed  Google Scholar 

  11. Vallenius T, Luukko K, Makela TP (2000) CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4. J Biol Chem 275:11100–11105. doi:10.1074/jbc.275.15.11100

    Article  CAS  PubMed  Google Scholar 

  12. Chew CS, Chen X, Parente JA Jr, Tarrer S, Okamoto C, Qin HY (2002) Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo. J Cell Sci 115:4787–4799. doi:10.1242/jcs.00174

    Article  CAS  PubMed  Google Scholar 

  13. Coutts AS, MacKenzie E, Griffith E, Black DM (2003) TES is a novel focal adhesion protein with a role in cell spreading. J Cell Sci 116:897–906. doi:10.1242/jcs.00278

    Article  CAS  PubMed  Google Scholar 

  14. Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Kocher T et al (2003) The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions. J Cell Biol 161:33–39. doi:10.1083/jcb.200211015

    Article  CAS  PubMed  Google Scholar 

  15. Griffith E, Coutts AS, Black DM (2004) Characterisation of chicken TES and its role in cell spreading and motility. Cell Motil Cytoskeleton 57:133–142. doi:10.1002/cm.10162

    Article  CAS  PubMed  Google Scholar 

  16. Schwartz LB, Bradford TR, Lee DC, Chlebowski JF (1990) Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase. J Immunol 144:2304–2311

    CAS  PubMed  Google Scholar 

  17. Skirgello OE, Balyasnikova IV, Binevski PV, Sun ZL, Baskin II, Palyulin VA et al (2006) Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action. Biochemistry 45:4831–4847. doi:10.1021/bi052591h

    Article  CAS  PubMed  Google Scholar 

  18. Yakimenko EF, Yazova AK, Goussev AI, Abelev GI (2001) Epitope mapping of human alpha-fetoprotein. Biochemistry (Mosc) 66:524–530. doi:10.1023/A:1010258902421

    Article  CAS  Google Scholar 

  19. Boulter JM, Glick M, Todorov PT, Baston E, Sami M, Rizkallah P et al (2003) Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng 16:707–711. doi:10.1093/protein/gzg087

    Article  CAS  PubMed  Google Scholar 

  20. Hu YX, Guo JY, Shen L, Chen Y, Zhang ZC, Zhang YL (2002) Get effective polyclonal antisera in one month. Cell Res 12:157–160. doi:10.1038/sj.cr.7290122

    Article  PubMed  Google Scholar 

  21. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379. doi:10.1016/0003-2697(87)90587-2

    Article  CAS  PubMed  Google Scholar 

  22. Asai K, Hirabayashi T, Houjou T, Uozumi N, Taguchi R, Shimizu T (2003) Human group IVC phospholipase A2 (cPLA2gamma). Roles in the membrane remodeling and activation induced by oxidative stress. J Biol Chem 278:8809–8814. doi:10.1074/jbc.M212117200

    Article  CAS  PubMed  Google Scholar 

  23. Deng Y, Bennink JR, Kang HC, Haugland RP, Yewdell JW (1995) Fluorescent conjugates of Brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells. J Histochem Cytochem 43:907–915

    CAS  PubMed  Google Scholar 

  24. Sarti M, Sevignani C, Calin GA, Aqeilan R, Shimizu M, Pentimalli F et al (2005) Adenoviral transduction of TESTIN gene into breast and uterine cancer cell lines promotes apoptosis and tumor reduction in vivo. Clin Cancer Res 11:806–813

    CAS  PubMed  Google Scholar 

  25. Imai H, Ochs RL, Kiyosawa K, Furuta S, Nakamura RM, Tan EM (1992) Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies. Am J Pathol 140:859–870

    CAS  PubMed  Google Scholar 

  26. Yung BY, Chan PK (1987) Identification and characterization of a hexameric form of nucleolar phosphoprotein B23. Biochim Biophys Acta 925:74–82

    CAS  PubMed  Google Scholar 

  27. Yung BY, Busch H, Chan PK (1985) Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis. Biochim Biophys Acta 826:167–173

    CAS  PubMed  Google Scholar 

  28. Boeda B, Briggs DC, Higgins T, Garvalov BK, Fadden AJ, McDonald NQ et al (2007) Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding. Mol Cell 28:1071–1082

    Article  CAS  PubMed  Google Scholar 

  29. Griffith E, Coutts AS, Black DM (2005) RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation. Cell Motil Cytoskeleton 60:140–152. doi:10.1002/cm.20052

    Article  CAS  PubMed  Google Scholar 

  30. Zheng Q, Zhao Y (2007) The diverse biofunctions of LIM domain proteins: determined by subcellular localization and protein–protein interaction. Biol Cell 99:489–502. doi:10.1042/BC20060126

    Article  CAS  PubMed  Google Scholar 

  31. Kadrmas JL, Beckerle MC (2004) The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol 5:920–931. doi:10.1038/nrm1499

    Article  CAS  PubMed  Google Scholar 

  32. Cattaruzza M, Lattrich C, Hecker M (2004) Focal adhesion protein zyxin is a mechanosensitive modulator of gene expression in vascular smooth muscle cells. Hypertension 43:726–730. doi:10.1161/01.HYP.0000119189.82659.52

    Article  CAS  PubMed  Google Scholar 

  33. Song Y, Maul RS, Gerbin CS, Chang DD (2002) Inhibition of anchorage-independent growth of transformed NIH3T3 cells by epithelial protein lost in neoplasm (EPLIN) requires localization of EPLIN to actin cytoskeleton. Mol Biol Cell 13:1408–1416. doi:10.1091/mbc.01-08-0414

    Article  CAS  PubMed  Google Scholar 

  34. Crawford AW, Beckerle MC (1991) Purification and characterization of zyxin, an 82,000-dalton component of adherens junctions. J Biol Chem 266:5847–5853

    CAS  PubMed  Google Scholar 

  35. Crawford AW, Michelsen JW, Beckerle MC (1992) An interaction between zyxin and alpha-actinin. J Cell Biol 116:1381–1393. doi:10.1083/jcb.116.6.1381

    Article  CAS  PubMed  Google Scholar 

  36. Degenhardt YY, Silverstein S (2001) Interaction of zyxin, a focal adhesion protein, with the e6 protein from human papillomavirus type 6 results in its nuclear translocation. J Virol 75:11791–11802. doi:10.1128/JVI.75.23.11791-11802.2001

    Article  CAS  PubMed  Google Scholar 

  37. Louis HA, Pino JD, Schmeichel KL, Pomies P, Beckerle MC (1997) Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression. J Biol Chem 272:27484–27491. doi:10.1074/jbc.272.43.27484

    Article  CAS  PubMed  Google Scholar 

  38. Arber S, Caroni P (1996) Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Dev 10:289–300. doi:10.1101/gad.10.3.289

    Article  CAS  PubMed  Google Scholar 

  39. Cyert MS (2001) Regulation of nuclear localization during signaling. J Biol Chem 276:20805–20808. doi:10.1074/jbc.R100012200

    Article  CAS  PubMed  Google Scholar 

  40. Szebeni A, Olson MO (1999) Nucleolar protein B23 has molecular chaperone activities. Protein Sci 8:905–912

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (Nos. 20335020, 30470945, 30571005), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0445), the 973 project of Ministry of Science and Technology of China (No. 2005CB522505 and 2006CB0F0306), and the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (No. 705041).

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  1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Science, Hunan Normal University, Changsha, Hunan, 410081, People’s Republic of China

    Yingli Zhong, Jiaolian Zhu, Yan Wang, Jianlin Zhou, Kaiqun Ren, Xiaofeng Ding & Jian Zhang

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  1. Yingli Zhong
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  2. Jiaolian Zhu
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Correspondence to Jian Zhang.

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Yingli Zhong, Jiaolian Zhu, and Yan Wang contributed equally to the work.

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Zhong, Y., Zhu, J., Wang, Y. et al. LIM domain protein TES changes its conformational states in different cellular compartments. Mol Cell Biochem 320, 85 (2009). https://doi.org/10.1007/s11010-008-9901-7

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  • DOI: https://doi.org/10.1007/s11010-008-9901-7

Keywords

  • TES
  • Conformational state
  • Nucleolus
  • Cellular localization