Abstract 4496: Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources.

Abstract

Abstract Distinct α- and β-tubulin isotypes are present in different quantities in diverse eukaryotic cells. The tritium-labeled photoaffinity Taxol analog, 2-(m-azidobenzoyl)Taxol, was used to photolabel tubulins prepared from HeLa cells, bovine brain and chicken erythrocytes. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of tubulin by >90%, demonstrating the specificity of this photolabeling. Tubulin isotype content varies from one source to another, particularly for chicken erythrocytes that contain only one α-tubulin and one β-tubulin (α1 and βVI). 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulin was carried out in the presence of six different microtubule stabilizing agents, including Taxol, epothilone B, discodermolide, ixabepilone, laulimalide and peloruside A. The inhibitory effects elicited by Taxol, epothilone B, discodermolide and ixabepilone, at a 5-fold molar excess, on photolabeling are different for tubulins prepared from different sources. Taxol had a minimal inhibitory effect on HeLa and bovine brain tubulin, but a strong inhibitory effect on chicken erythrocyte tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. This is most likely due to the presence of distinct tubulin isotypes in different sources. However, laulimalide and peloruside A that are known to bind to a different site than Taxol in β-tubulin, stimulated photolabeling of tubulins from all three sources. Stathmin (S) plays an important role in the regulation of microtubule dynamics through sequestering tubulin (T) into a T2S complex. When stathmin was incubated, in the presence of 1 mM CaCl2, with chicken erythrocyte tubulin at a tubulin:stathmin ratio of 2:1, followed by incubation with tritium-labeled 2-(m-azidobenzoyl)Taxol in a buffer containing EGTA, photolabeling of β-tubulin was markedly reduced, suggesting that stathmin inhibited binding of Taxol to tubulin. Radiolabeled photoaffinity drug analogs are useful tools for determining drug binding affinities for the different tubulin isotypes. We are currently analyzing binding affinities of 2-(m-azidobenzoyl)Taxol to different bovine brain tubulin isotypes resolved by isoelectrofocusing. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2013-4496