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 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. 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.Stathmin (1, 2), also designated Op18, p18, p19, prosolin, and metablastin (3-6), is a ubiquitous cytosolic phosphoprotein highly conserved in vertebrates (7,8) and specifically abundant in neurons (9 -11). Expression and phosphorylation of stathmin are modulated in various situations related to the control of cellular activities, and it has been proposed that it may act as a relay integrating various intracellular signaling pathways (1). Expression of stathmin was shown to be regulated in vivo during development (7,(12)(13)(14), during tissue regeneration (15, 16), and in cell culture by cell/cell interactions (17). Stathmin is also up-regulated in many malignant cell types and tumors (5,18,19). Phosphorylation of stathmin is observed in response to hormones (20), cytokines (21), neurotransmitters (22), and growth and differentiation factors (23). Moreover, progression through the cell cycle appears to require multisite phosphorylation of stathmin (24). Actually, overexpression of a nonphosphorylatable mutant of stathmin resulted in a large population of cells blocked in G 2 /M with a high DNA content (24,25). Finally, stathmin is the generic element of a protein family whose other members most probably play distinct roles related to the control of neuronal differentiation or to the expression of neuron-specific traits (8, 26).The molecular mechanism(s) by which stathmin acts in these processes remain largely unknown. Two domains can be distinguished in the primary structure of stathmin, an N-terminal "regulatory" domain that contains the four phosphorylation sites that account for all of the electrophoretic f...

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Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics which is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. Here we have investigated the role of serine-threonine phosphatases in the control of microtubule dynamics using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles.

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