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. 2004 Mar 15;378(Pt 3):877–888. doi: 10.1042/BJ20031413

A synergistic relationship between three regions of stathmin family proteins is required for the formation of a stable complex with tubulin.

Isabelle Jourdain 1, Sylvie Lachkar 1, Elodie Charbaut 1, Benoit Gigant 1, Marcel Knossow 1, André Sobel 1, Patrick A Curmi 1
PMCID: PMC1224029  PMID: 14670078

Abstract

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3(SLD) were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.

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Selected References

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  1. Amayed Phedra, Pantaloni Dominique, Carlier Marie-France. The effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration. J Biol Chem. 2002 Apr 15;277(25):22718–22724. doi: 10.1074/jbc.M111605200. [DOI] [PubMed] [Google Scholar]
  2. Belmont L. D., Mitchison T. J. Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell. 1996 Feb 23;84(4):623–631. doi: 10.1016/s0092-8674(00)81037-5. [DOI] [PubMed] [Google Scholar]
  3. Beretta L., Boutterin M. C., Sobel A. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines. Endocrinology. 1988 Jan;122(1):40–51. doi: 10.1210/endo-122-1-40. [DOI] [PubMed] [Google Scholar]
  4. Brattsand G. Correlation of oncoprotein 18/stathmin expression in human breast cancer with established prognostic factors. Br J Cancer. 2000 Aug;83(3):311–318. doi: 10.1054/bjoc.2000.1264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlier M. F., Pantaloni D. Kinetic analysis of cooperativity in tubulin polymerization in the presence of guanosine di- or triphosphate nucleotides. Biochemistry. 1978 May 16;17(10):1908–1915. doi: 10.1021/bi00603a017. [DOI] [PubMed] [Google Scholar]
  6. Charbaut E., Curmi P. A., Ozon S., Lachkar S., Redeker V., Sobel A. Stathmin family proteins display specific molecular and tubulin binding properties. J Biol Chem. 2001 Feb 15;276(19):16146–16154. doi: 10.1074/jbc.M010637200. [DOI] [PubMed] [Google Scholar]
  7. Chneiweiss H., Cordier J., Sobel A. Stathmin phosphorylation is regulated in striatal neurons by vasoactive intestinal peptide and monoamines via multiple intracellular pathways. J Neurochem. 1992 Jan;58(1):282–289. doi: 10.1111/j.1471-4159.1992.tb09308.x. [DOI] [PubMed] [Google Scholar]
  8. Curmi P. A., Andersen S. S., Lachkar S., Gavet O., Karsenti E., Knossow M., Sobel A. The stathmin/tubulin interaction in vitro. J Biol Chem. 1997 Oct 3;272(40):25029–25036. doi: 10.1074/jbc.272.40.25029. [DOI] [PubMed] [Google Scholar]
  9. Curmi P. A., Maucuer A., Asselin S., Lecourtois M., Chaffotte A., Schmitter J. M., Sobel A. Molecular characterization of human stathmin expressed in Escherichia coli: site-directed mutagenesis of two phosphorylatable serines (Ser-25 and Ser-63). Biochem J. 1994 Jun 1;300(Pt 2):331–338. doi: 10.1042/bj3000331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Curmi P. A., Noguès C., Lachkar S., Carelle N., Gonthier M. P., Sobel A., Lidereau R., Bièche I. Overexpression of stathmin in breast carcinomas points out to highly proliferative tumours. Br J Cancer. 2000 Jan;82(1):142–150. doi: 10.1054/bjoc.1999.0891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deléage G., Roux B. An algorithm for protein secondary structure prediction based on class prediction. Protein Eng. 1987 Aug-Sep;1(4):289–294. doi: 10.1093/protein/1.4.289. [DOI] [PubMed] [Google Scholar]
  12. Detrich H. W., 3rd, Williams R. C. Reversible dissociation of the alpha beta dimer of tubulin from bovine brain. Biochemistry. 1978 Sep 19;17(19):3900–3907. doi: 10.1021/bi00612a002. [DOI] [PubMed] [Google Scholar]
  13. Di Paolo G., Lutjens R., Pellier V., Stimpson S. A., Beuchat M. H., Catsicas S., Grenningloh G. Targeting of SCG10 to the area of the Golgi complex is mediated by its NH2-terminal region. J Biol Chem. 1997 Feb 21;272(8):5175–5182. doi: 10.1074/jbc.272.8.5175. [DOI] [PubMed] [Google Scholar]
  14. Doye V., Boutterin M. C., Sobel A. Phosphorylation of stathmin and other proteins related to nerve growth factor-induced regulation of PC12 cells. J Biol Chem. 1990 Jul 15;265(20):11650–11655. [PubMed] [Google Scholar]
  15. Gavet O., Ozon S., Manceau V., Lawler S., Curmi P., Sobel A. The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network. J Cell Sci. 1998 Nov;111(Pt 22):3333–3346. doi: 10.1242/jcs.111.22.3333. [DOI] [PubMed] [Google Scholar]
  16. Ghosh P. K., Anderson J., Cohen N., Takeshita K., Atweh G. F., Lebowitz P. Expression of the leukemia-associated gene, p18, in normal and malignant tissues; inactivation of expression in a patient with cleaved B cell lymphoma/leukemia. Oncogene. 1993 Oct;8(10):2869–2872. [PubMed] [Google Scholar]
  17. Gigant B., Curmi P. A., Martin-Barbey C., Charbaut E., Lachkar S., Lebeau L., Siavoshian S., Sobel A., Knossow M. The 4 A X-ray structure of a tubulin:stathmin-like domain complex. Cell. 2000 Sep 15;102(6):809–816. doi: 10.1016/s0092-8674(00)00069-6. [DOI] [PubMed] [Google Scholar]
  18. Hailat N., Strahler J., Melhem R., Zhu X. X., Brodeur G., Seeger R. C., Reynolds C. P., Hanash S. N-myc gene amplification in neuroblastoma is associated with altered phosphorylation of a proliferation related polypeptide (Op18). Oncogene. 1990 Nov;5(11):1615–1618. [PubMed] [Google Scholar]
  19. Holmfeldt P., Larsson N., Segerman B., Howell B., Morabito J., Cassimeris L., Gullberg M. The catastrophe-promoting activity of ectopic Op18/stathmin is required for disruption of mitotic spindles but not interphase microtubules. Mol Biol Cell. 2001 Jan;12(1):73–83. doi: 10.1091/mbc.12.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horwitz S. B., Shen H. J., He L., Dittmar P., Neef R., Chen J., Schubart U. K. The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation. J Biol Chem. 1997 Mar 28;272(13):8129–8132. doi: 10.1074/jbc.272.13.8129. [DOI] [PubMed] [Google Scholar]
  21. Howell B., Larsson N., Gullberg M., Cassimeris L. Dissociation of the tubulin-sequestering and microtubule catastrophe-promoting activities of oncoprotein 18/stathmin. Mol Biol Cell. 1999 Jan;10(1):105–118. doi: 10.1091/mbc.10.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jourdain L., Curmi P., Sobel A., Pantaloni D., Carlier M. F. Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules. Biochemistry. 1997 Sep 9;36(36):10817–10821. doi: 10.1021/bi971491b. [DOI] [PubMed] [Google Scholar]
  23. Larsson N., Marklund U., Gradin H. M., Brattsand G., Gullberg M. Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis. Mol Cell Biol. 1997 Sep;17(9):5530–5539. doi: 10.1128/mcb.17.9.5530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Larsson N., Segerman B., Howell B., Fridell K., Cassimeris L., Gullberg M. Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities. J Cell Biol. 1999 Sep 20;146(6):1289–1302. doi: 10.1083/jcb.146.6.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marklund U., Larsson N., Gradin H. M., Brattsand G., Gullberg M. Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics. EMBO J. 1996 Oct 1;15(19):5290–5298. [PMC free article] [PubMed] [Google Scholar]
  26. Maucuer A., Doye V., Sobel A. A single amino acid difference distinguishes the human and the rat sequences of stathmin, a ubiquitous intracellular phosphoprotein associated with cell regulations. FEBS Lett. 1990 May 21;264(2):275–278. doi: 10.1016/0014-5793(90)80266-l. [DOI] [PubMed] [Google Scholar]
  27. Melhem R. F., Zhu X. X., Hailat N., Strahler J. R., Hanash S. M. Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia. J Biol Chem. 1991 Sep 25;266(27):17747–17753. [PubMed] [Google Scholar]
  28. Müller D. R., Schindler P., Towbin H., Wirth U., Voshol H., Hoving S., Steinmetz M. O. Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal Chem. 2001 May 1;73(9):1927–1934. doi: 10.1021/ac001379a. [DOI] [PubMed] [Google Scholar]
  29. Ozon S., Byk T., Sobel A. SCLIP: a novel SCG10-like protein of the stathmin family expressed in the nervous system. J Neurochem. 1998 Jun;70(6):2386–2396. doi: 10.1046/j.1471-4159.1998.70062386.x. [DOI] [PubMed] [Google Scholar]
  30. Ozon S., Maucuer A., Sobel A. The stathmin family -- molecular and biological characterization of novel mammalian proteins expressed in the nervous system. Eur J Biochem. 1997 Sep 15;248(3):794–806. doi: 10.1111/j.1432-1033.1997.t01-2-00794.x. [DOI] [PubMed] [Google Scholar]
  31. Redeker V., Lachkar S., Siavoshian S., Charbaut E., Rossier J., Sobel A., Curmi P. A. Probing the native structure of stathmin and its interaction domains with tubulin. Combined use of limited proteolysis, size exclusion chromatography, and mass spectrometry. J Biol Chem. 2000 Mar 10;275(10):6841–6849. doi: 10.1074/jbc.275.10.6841. [DOI] [PubMed] [Google Scholar]
  32. Sackett D. L. Natural osmolyte trimethylamine N-oxide stimulates tubulin polymerization and reverses urea inhibition. Am J Physiol. 1997 Aug;273(2 Pt 2):R669–R676. doi: 10.1152/ajpregu.1997.273.2.R669. [DOI] [PubMed] [Google Scholar]
  33. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  35. Sobel A., Boutterin M. C., Beretta L., Chneiweiss H., Doye V., Peyro-Saint-Paul H. Intracellular substrates for extracellular signaling. Characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin). J Biol Chem. 1989 Mar 5;264(7):3765–3772. [PubMed] [Google Scholar]
  36. Sobel A. Stathmin: a relay phosphoprotein for multiple signal transduction? Trends Biochem Sci. 1991 Aug;16(8):301–305. doi: 10.1016/0968-0004(91)90123-d. [DOI] [PubMed] [Google Scholar]
  37. Stein R., Mori N., Matthews K., Lo L. C., Anderson D. J. The NGF-inducible SCG10 mRNA encodes a novel membrane-bound protein present in growth cones and abundant in developing neurons. Neuron. 1988 Aug;1(6):463–476. doi: 10.1016/0896-6273(88)90177-8. [DOI] [PubMed] [Google Scholar]
  38. Steinmetz M. O., Jahnke W., Towbin H., García-Echeverría C., Voshol H., Müller D., van Oostrum J. Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin. EMBO Rep. 2001 Jun;2(6):505–510. doi: 10.1093/embo-reports/kve105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steinmetz M. O., Kammerer R. A., Jahnke W., Goldie K. N., Lustig A., van Oostrum J. Op18/stathmin caps a kinked protofilament-like tubulin tetramer. EMBO J. 2000 Feb 15;19(4):572–580. doi: 10.1093/emboj/19.4.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsao K. L., DeBarbieri B., Michel H., Waugh D. S. A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli. Gene. 1996 Feb 22;169(1):59–64. doi: 10.1016/0378-1119(95)00762-8. [DOI] [PubMed] [Google Scholar]
  41. Wallon G., Rappsilber J., Mann M., Serrano L. Model for stathmin/OP18 binding to tubulin. EMBO J. 2000 Jan 17;19(2):213–222. doi: 10.1093/emboj/19.2.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]

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