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Alkylation of -Tubulin on Glu 198 by a Microtubule Disrupter

Unité Mixte de Recherche 484, Institut National de la Santé et de la Recherche Médicale-Université d'Auvergne, Clermont-Ferrand, France (B.B., E.M., J.P., E.M.-N., J.-C.M., F.D.); Plate-forme Protéomique, Institut National de la Recherche Agronomique de Theix, St-Genès-Champanelle, France (C.C.); and Centre de Recherche, Centre Hospitalier Universitaire de Québec, Hôpital St-François d'Assise, Québec City, Québec, Canada (R.C.G.)

We have shown that -tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl-N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tert-butyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of -tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of -tubulin, but this binding site was not clearly confirmed (Cancer Res 60:985-992, 2000). We have isolated and analyzed -tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated -tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[¹²⁵I]CEU was effectively bound to the modified -tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of -tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of -tubulin confirmed binding of iodophenylethylureido moiety to peptides [175-213] or [197-208] respectively. Fragmentation analyses by electrospray mass spectrometry using triply charged ions of peptide [175-213] identified a glutamic acid at position 198 as target for alkylation via an ester bond with ICEU. This amino acid located in the intermediate domain of the -tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament.

Address correspondence to: Françoise Degoul, UMR484, INSERM-Université d'Auvergne, BP184, Rue Montalembert, F-63005 Clermont-Fd, France. E-mail: degoul{at}inserm484.u-clermont1.fr

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