Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.
Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.
Amayed
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Dynamique du Cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurale Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France.
Carlier
M F
MF
Pantaloni
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D
D000818:Animals; D002417:Cattle; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D050777:Stathmin; D014364:Tryptophan; D014404:Tubulin / Q000378:metabolism*; D014461:Ultracentrifugation
D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D010750:Phosphoproteins; D050777:Stathmin; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan