Probing the native structure of stathmin and its interaction domains with tubulin. Combined use of limited proteolysis, size exclusion chromatography, and mass spectrometry.
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 time-of-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 alpha-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.
Redeker
V
V
Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, Neurobiologie et Diversité Cellulaire, CNRS UMR 7637, 10 Rue Vauquelin, 75005 Paris, France.
Lachkar
S
S
Siavoshian
S
S
Charbaut
E
E
Rossier
J
J
Sobel
A
A
Curmi
P A
PA
D002850:Chromatography, Gel; D013058:Mass Spectrometry; D008868:Microtubule Proteins; D008870:Microtubules / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D012996:Solutions; D050777:Stathmin; D014404:Tubulin / Q000737:chemistry*
D008868:Microtubule Proteins; D010750:Phosphoproteins; D011108:Polymers; D012996:Solutions; D050777:Stathmin; D014404:Tubulin