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

Abstract

Stathmin is a cytosoluble phosphoprotein proposed to be a regulatory relay integrating diverse intracellular signaling pathway. Its interaction with tubulin modulates microtubule dynamics by destabilization of assembled microtubules or inhibition of their polymerization from free tubulin. The aim of this study was to probe the native structure of stathmin and to delineate its minimal region able to interact with tubulin. Limited proteolysis of stathmin revealed four structured domains within the native protein, corresponding to amino acid sequences 22-81 (I), 95-113 (II), 113-128 (III), and 128 -149 (IV), which allows us to propose stathmin folding hypotheses. Furthermore, stathmin proteolytic fragments were mixed to interact with tubulin, and those that retained affinity for tubulin were isolated by size exclusion chromatography and identified by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. The results indicate that, to interact with tubulin, a stathmin fragment must span a minimal core region from residues 42 to 126, which interestingly corresponds to the predicted ␣-helical "interaction region" of stathmin. In addition, an interacting stathmin fragment must include a short N-or C-terminal extension. The functional significance of these interaction constrains is further validated by tubulin polymerization inhibition assays with fragments designed on the basis of the tubulin binding results. The present results will help to optimize further stathmin structural studies and to develop molecular tools to target its interaction with tubulin.Stathmin (␣, relay) (1), also referred to as Op 18 (2), is a small ubiquitous cytosolic phosphoprotein that has been proposed to be a relay integrating diverse intracellular signaling pathways (3). It is phosphorylated in parallel with the action of hormones (4 -6), growth factors (7, 8), and neurotransmitters (9), and it interacts with putative downstream protein partners including tubulin (10 -16). Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3, and its two splice variants RB3Ј and RB3Љ (17). A great deal of progress has recently been made in understanding the function of stathmin. It now appears that it may be one of the key regulators of microtubule dynamics, likely implicated in various microtubule-dependent cellular functions in interphase or mitosis (18 -21). Actually, it has been shown that stathmin destabilizes microtubules (15). Semi-quantitative in vitro studies (20, 21) have revealed that stathmin prevents assembly and promotes disassembly of microtubules in a concentration-dependent manner and that inhibition of microtubule assembly is abolished by stathmin phosphorylation in vitro (21) or in vivo (20,22,23). It has been also shown that some of these properties are shared by the other stathmin family proteins (18,24,25).However, although the destabilizing potential of stathmin toward microtubules is firmly established, the mechanism by which it fulfills this function is still ...

Mutational Analysis of Op18/Stathmin-Tubulin-interacting Surfaces

“…18). Moreover, our result is in line with a recent report demonstrating that truncation of both the N-and C-terminal flanks of Op18 abolishes tubulin binding as determined by size exclusion chromatography (23).…”

Thermodynamics of the Op18/Stathmin-Tubulin Interaction

“…1) together with deletion mapping (13,18,19,26,29,41) and mutational studies (40,42) indicate that the helical domain of stathmin accounts predominantly for the binding free energy. Interestingly, the interaction sites of the two ␣/␤-tubulin heterodimers with the RB3-SLD helix (T ␣1␤1 -S h1 and T ␣2␤2 -S h2 ; Fig.…”

“…All these studies indicated that stathmin interacts with two ␣/␤-tubulin heterodimers in the low micromolar range. In analytical ultracentrifugation (21,30) and gel filtration (18,22,32) studies only ternary (denoted T 2 S) but not binary (denoted TS) complexes were isolated. Consistent with this observation, pulldown assays using agarose bead-coupled antibodies against glutathione S-transferase-and FLAG-tagged stathmin variants suggested that the binding of tubulin to stathmin is highly cooperative and that binary TS complexes are labile intermediates (26,28,41).…”

“…Human Op18/stathmin (referred to as stathmin (3)) is an evolutionary well conserved 17-kDa cytoplasmic phosphoprotein. The monomeric protein consists of a N-terminal capping domain (13,14) and a C-terminal helical interaction domain (13,(15)(16)(17)(18)(19). Originally, stathmin was described as a protein that binds to tubulin dimers and increases the catastrophe frequency of MT in vitro (20).…”

Stathmin Family Proteins Display Specific Molecular and Tubulin Binding Properties

“…Stathmin indeed interacts with two ␣ /␤ tubulin heterodimers in vitro to form a T 2 S complex (22,23). The two tubulin heterodimers bind mostly the predicted ␣-helical "interaction" domain of stathmin (24), likely forming a curved complex whose three-dimensional structure has been recently revealed with the stathmin-like domain of RB3 (25). Stathmin displays a microtubule destabilizing activity both in vitro and in vivo (21, 26 -29), which is stoichiometrically accounted for in vitro by a free tubulin sequestration mechanism (22,27), although it has been proposed to be also due in part to a direct catastrophe-promoting activity (21,30,31).…”