The stathmin/tubulin interaction in vitro.
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-A 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 microM 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.
Curmi
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PA
INSERM U440, 17 rue du Fer à Moulin, 75005 Paris, France. curmi@infobiogen.fr
Andersen
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SS
Lachkar
S
S
Gavet
O
O
Karsenti
E
E
Knossow
M
M
Sobel
A
A
D000818:Animals; D015374:Biosensing Techniques; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008868:Microtubule Proteins; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D050777:Stathmin; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D008868:Microtubule Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D050777:Stathmin; D014404:Tubulin; D012694:Serine