THE STATHMIN TUBULIN INTERACTION IN-VITRO

Stathmin is a highly conserved ubiquitous cytoplasmic protein, phospho rylated in response to extracellular signals and during the cell cycle , Stathmin has recently been shown to destabilize microtubules, but th e molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin, We assessed the condit ions of this interaction and determined some its quantitative paramete rs using plasmon resonance, gel filtration chromatography, and analyti cal ultracentrifugation. The stathmin/tubulin interaction leads to the formation of a 7.7 S complex with a 60-Angstrom Stokes radius, associ ating one stathmin with two tubulin heterodimer molecules as determine d by direct quantification by Western blotting, This interaction is se nsitive to pH and ionic environment. Its equilibrium dissociation cons tant, determined by plasmon resonance measurement of kinetic constants , has an optimum value of 0.5 mu M 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. F inally, analysis of microtubule dynamics by video microscopy shows tha t, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results sug gest that the stathmin destabilizing activity on microtubules is relat ed to tubulin sequestration by stathmin.