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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.

Received for publication June 6, 2005.

Accepted for publication August 12, 2005.

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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