Stathmin:  A Tubulin-Sequestering Protein Which Forms a Ternary T₂S Complex with Two Tubulin Molecules

Abstract

Stathmin is an important regulatory protein thought to control the dynamics of microtubules through the cell cycle in a phosphorylation-dependent manner. Here we show that stathmin interacts with two molecules of dimeric alphabeta-tubulin to form a tight ternary T2S complex, sedimenting at 7.7 S. This complex appears in slow association-dissociation equilibrium in the analytical ultracentrifuge. The T2S complex is formed under a variety of ionic conditions, either from GTP- or GDP-tubulin or from the tubulin-colchicine complex. The S16/25/38/63E mutated stathmin in contrast is in rapid equilibrium with tubulin in the T2S complex. The T2S complex cannot polymerize in microtubules nor in ring oligomers. Stathmin acts as a pure tubulin-sequestering protein via formation of the T2S complex. It does not act directly on microtubule ends to promote catastrophe nor enhance microtubule dynamics.

Probing the Native Structure of Stathmin and Its Interaction Domains with Tubulin

“…Previous in vitro studies have shown that depolymerization of microtubules can be explained by stathmin sequestration of free tubulin in a T2S complex (16,26). We also have measured that stathmin alters neither the steady state GTPase activity of microtubules nor the critical concentration, all results consistent with a pure tubulin sequestering activity (26).…”

“…Experiments were carried out in 50 mM MES-KOH, pH 6.8, 30% glycerol, 0.5 mM EGTA, 6 mM MgCl 2 , and 0.5 mM GTP (buffer M). Critical concentration plots representing the amount of polymerized tubulin observed at steady state versus the total concentration of tubulin, measured in the absence or presence of stathmin and stathmin fragments, were constructed as described (26).…”

“…Previous in vitro studies have shown that depolymerization of microtubules can be explained by stathmin sequestration of free tubulin in a T2S complex (16,26). We also have measured that stathmin alters neither the steady state GTPase activity of microtubules nor the critical concentration, all results consistent with a pure tubulin sequestering activity (26). On the other hand, Howell et al (27) have proposed that stathmin may be a dual potential protein as follows: part of the N-terminal stathmin region could promote catastrophes, whereas tubulin sequestration could be supported by the rest of the molecule.…”

SIAH-1 interacts with α-tubulin and degrades the kinesin Kid by the proteasome pathway during mitosis

“…pME-KID was kindly provided by Dr N Tokai (Kyoto-Japan). Puri®ed tubulin was prepared as described (Jourdain et al, 1997) and kindly provided by Dr MF Carlier. Puri®ed SIAH-1 was prepared by thrombin cleavage of the GST fusion protein (1 U/ml Pharmacia Biotech).…”

Identification of novel targets for cancer therapy using expression proteomics

“…42 Op18 has been shown to destabilize microtubules in vitro, at least in part due to tubulin sequestration and microtubule catastrophe-promoting activities. [43][44][45][46] Op18 is expressed in proliferating cells of most cell lineages 47 and its level increases significantly during the S phase of the cell cycle. 48,49 The functionality of Op18 is also tightly controlled by phosphorylation during the cell cycle, 50 with its activity being down-regulated by increased phosphorylation on four serine (S16, S25, S38 and S63) residues.…”