The Stathmin/Tubulin Interaction in Vitro*
- Patrick A. Curmi‡§,
- Søren S. L. Andersen¶,
- Sylvie Lachkar‡,
- Olivier Gavet‡,
- Eric Karsenti¶,
- Marcel Knossow‖ and
- André Sobel‡
- From ‡INSERM U440, 17 rue du Fer à Moulin, 75005 Paris, France, ¶EMBL, Cell Biology Program, D-69012 Heidelberg, Germany, and ‖Laboratoire d’Enzymologie et Biologie Structurale UPR 9063 CNRS, 91198 Gif sur Yvette, France
Abstract
Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/tubulin interaction leads to the formation of a 7.7 S complex with a 60-Å Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic constants, has an optimum value of 0.5 μm at pH 6.5. The affinity was lowered with a fully “pseudophosphorylated” 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin.
Footnotes
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↵* This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, l’Association Française contre les Myopathies, l’Association pour la Recherche contre le Cancer, and the Ligue Nationale Française Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: INSERM U440, 17 rue du Fer à Moulin, 75005 Paris, France. Tel.: 33 1 45 87 61 30; Fax: 33 1 45 87 61 32; E-mail: curmi{at}infobiogen.fr.
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↵1 The abbreviations used are: MT, microtubule; BSA, bovine serum albumin; Fc, flow cell; f cat, catastrophe frequency; R S, Stokes radius; RU, resonance unit; Vg, growth rate; Pipes, 1,4-piperazinediethanesulfonic acid; WT, recombinant wild type; bWT, boiled recombinant wild type; FPLC, fast protein liquid chromatography.
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↵2 P. Curmi and V. Manceau, unpublished data.
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- Received June 26, 1997.
- Revision received July 29, 1997.
