RT Journal Article
SR Electronic
T1 Characterization of the Covalent Binding of <em>N</em>-Phenyl-<em>N'</em>-(2-chloroethyl)ureas to β-tubulin: Importance of Glutamic Acid 198 in Microtubule Stability
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP jpet.110.171082
DO 10.1124/jpet.110.171082
A1 Sebastien Fortin
A1 Bernadette Bouchon
A1 Christophe Chambon
A1 Jacques Lacroix
A1 Emmanuel Moreau
A1 Jean-Michel Chezal
A1 Francoise Degoul
A1 Rene C.-Gaudreault
YR 2010
UL http://jpet.aspetjournals.org/content/early/2010/10/26/jpet.110.171082.abstract
AB N-Phenyl-N'-(2-chloroethyl)ureas (CEU) are antimicrotubule agents interacting covalently with β-tubulin near the colchicine-binding site (C-BS). Glutamyl-198 residue in β-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic Cys239 and Cys354 residues, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). Using mass spectrometry, we have now identified residues in β-tubulin that become irreversibly modified by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU) and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to β-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs were then used as molecular probes to assess in mouse B16F0 and human MDA-MB-231 cells the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation; as observed also with other microtubule depolymerizing agents. Therefore, CEU affect the stability and the dynamics of microtubule likewise a Glu198Gly mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular crosslink between Cys239 and Cys354 of β-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.