Alkylation of beta-tubulin on Glu 198 by a microtubule disrupter.
We have shown that beta-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 beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60:985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-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 beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of beta-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 beta-tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament.
Bouchon
Bernadette
B
UMR484, INSERM-Université d'Auvergne, BP184, Rue Montalembert, F-63005 Clermont-Fd, France.
Chambon
Christophe
C
Mounetou
Emmanuelle
E
Papon
Janine
J
Miot-Noirault
Elisabeth
E
Gaudreault
René C
RC
Madelmont
Jean-Claude
JC
Degoul
Françoise
F
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D045744:Cell Line, Tumor; D051379:Mice; D008870:Microtubules / Q000187:drug effects*; D008969:Molecular Sequence Data; D010671:Phenylurea Compounds / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D018906:Antineoplastic Agents, Alkylating; D010671:Phenylurea Compounds; D014404:Tubulin; C084239:4-tert-butyl-(3-(2-chloroethyl)ureido)benzene