The Stathmin/Tubulin Interaction in Vitro

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 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...

Overexpression of the stathmin gene in a subset of human breast cancer

“…More obviously, it has recently been shown that stathmin interferes with the dynamic instability of microtubules by destabilizing them in vitro (Belmont and Mitchison, 1996) and in vivo (Marklund et al, 1996). We demonstrated that this phenomenon is related to a direct interaction of stathmin with tubulin dimers, leading to the sequestration of tubulin in a twotubulin heterodimer-one-stathmin complex (T2S) (Curmi et al, 1997;Jourdain et al, 1997). Furthermore, it has been shown that phosphorylation of stathmin dramatically reduces its affinity for tubulin and its microtubule-destabilizing activity (Marklund et al, 1996;Curmi et al, 1997;Di Paolo et al, 1997;Horwitz et al, 1997;Larsson et al, 1997), giving an additional clue to the mechanisms of the in vivo control of microtubule reorganization during mitosis.…”

“…It was also noticed that the overexpression of a nonphosphorylatable mutant of stathmin resulted in a large population of cells blocked at G2/M with a high DNA content (Marklund et al, 1994b;Larsson et al, 1995;Lawler et al, 1997). To account for these effects, we demonstrated that stathmin directly interacts with, and sequesters, tubulin (Curmi et al, 1997) in a T2S complex (Jourdain et al, 1997), and that this sequestration leads to the displacement of the microtubule/tubulin equilibrium towards depolymerization of microtubules (Jourdain et al, 1997). Importantly, phosphorylation of stathmin altered the affinity of stathmin for tubulin (Marklund et al, 1996;Curmi et al, 1997;Di Paolo et al, 1997;Horwitz et al, 1997;Larsson et al, 1997).…”

“…To account for these effects, we demonstrated that stathmin directly interacts with, and sequesters, tubulin (Curmi et al, 1997) in a T2S complex (Jourdain et al, 1997), and that this sequestration leads to the displacement of the microtubule/tubulin equilibrium towards depolymerization of microtubules (Jourdain et al, 1997). Importantly, phosphorylation of stathmin altered the affinity of stathmin for tubulin (Marklund et al, 1996;Curmi et al, 1997;Di Paolo et al, 1997;Horwitz et al, 1997;Larsson et al, 1997). Together, these results give insight into the observed physiological variations of stathmin phosphorylation during the mitotic cycle (Strahler et al, 1992;Brattsand et al, 1994) and argue for the search for stathmin dysregulations in tumours as well as an understanding of its mechanisms.…”

“…We demonstrated that this phenomenon is related to a direct interaction of stathmin with tubulin dimers, leading to the sequestration of tubulin in a twotubulin heterodimer-one-stathmin complex (T2S) (Curmi et al, 1997;Jourdain et al, 1997). Furthermore, it has been shown that phosphorylation of stathmin dramatically reduces its affinity for tubulin and its microtubule-destabilizing activity (Marklund et al, 1996;Curmi et al, 1997;Di Paolo et al, 1997;Horwitz et al, 1997;Larsson et al, 1997), giving an additional clue to the mechanisms of the in vivo control of microtubule reorganization during mitosis. Finally, the stathmin gene maps to lp35-36.1 (Ferrari et al, 1990), in a region (lp32-lpter) thought to harbour at least one tumoursuppressor gene (Bieche et al, 1994).…”

SCLIP Is Crucial for the Formation and Development of the Purkinje Cell Dendritic Arbor

“…They have been originally identified for their capacity to integrate and relay intracellular signaling pathways (Sobel, 1991), at least in part through tubulin binding and the control of microtubule dynamics (Belmont and Mitchison, 1996;Curmi et al, 1997;Steinmetz et al, 2000;Charbaut et al, 2001;Ravelli et al, 2004). Whereas stathmin is ubiquitous and cytosolic, the other proteins are essentially expressed in the nervous system and localized at the Golgi complex and vesicular structures along neurite shafts and in growth cones (Di Paolo et al, 1997; Gavet et al, 2002;Charbaut et al, 2005).…”