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Abstract
Stathmin is a phosphorylation-regulated tubulin-binding protein. In vitro and in vivo studies using nonphosphorylatable and pseudophosphorylated mutants of stathmin have questioned the view that stathmin might act only as a tubulin-sequestering factor. Stathmin was proposed to effectively regulate microtubule dynamic instability by increasing the frequency of catastrophe (the transition from steady growth to rapid depolymerization), without interacting with tubulin. We have used a noninvasive method to measure the equilibrium dissociation constants of the T(2)S complexes of tubulin with stathmin, pseudophosphorylated (4E)-stathmin, and diphosphostathmin. At both pH 6.8 and pH 7.4, the relative sequestering efficiency of the different stathmin variants depends on the concentration of free tubulin, i.e. on the dynamic state of microtubules. This control is exerted in a narrow range of tubulin concentration due to the highly cooperative binding of tubulin to stathmin. Changes in pH affect the stability of tubulin-stathmin complexes but do not change stathmin function. The 4E-stathmin mutant mimics inactive phosphorylated stathmin at low tubulin concentration and sequesters tubulin almost as efficiently as stathmin at higher tubulin concentration. We propose that stathmin acts solely by sequestering tubulin, without affecting microtubule dynamics, and that the effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration.
Highlights
Microtubules are dynamic polymers that play a role in cell morphology and cell division
We propose that stathmin acts solely by sequestering tubulin, without affecting microtubule dynamics, and that the effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration
We tentatively propose that the changes in microtubule dynamics during the cell cycle are associated with variations in the concentration of free tubulin that coexists with microtubules
Summary
Microtubules are dynamic polymers that play a role in cell morphology and cell division. The steady-state concentration of dimeric GTP-tubulin allows equal net rates of assembly at the plus end and disassembly from the minus end. When the fraction of free minus ends increases, e.g. by detachment of microtubules from centrosomes, the concentration of free GTPtubulin is expected to increase from a value close to the critical concentration of the plus end to a value closer to the critical concentration of the minus end This shift has been observed [18], supporting the view that the concentration of GTP-tubulin may vary in vivo. In contrast with regulatory factors that control microtubule assembly dynamics, tubulin-sequestering factors bind tubulin in a nonpolymerizable complex These proteins establish a pool of unassembled tubulin, built at the expense of the microtubule pool and in equilibrium with free tubulin at its steady-state concentration. It is phosphorylated to a low basal level in interphase and becomes hyperphosphorylated by cyclin-
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