ΒI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug–tubulin interactions and microtubule stability. Issue 2 (1st September 2015)

• Record Type: Journal Article
• Authors: Kanakkanthara, Arun; Rowe, Matthew R.; Field, Jessica J.; Northcote, Peter T.; Teesdale-Spittle, Paul H.; Miller, John H.
• Is Part Of: Cancer letters. Volume 365:Issue 2(2015)
• Page Start: 251
• Page End: 260
• Publication Date: 2015-09-01
• Subjects: Microtubule targeting agents -- Peloruside -- Laulimalide -- Paclitaxel -- Tubulin binding sites -- Cancer cell resistance; Cancer -- Periodicals; Neoplasms -- Periodicals; Cancer -- Périodiques; Electronic journals; 616.994
• DOI: 10.1016/j.canlet.2015.06.001
• Languages: English
• ISSNs: 0304-3835

Abstract

Highlights: Roles of specific amino acids in binding of peloruside and laulimalide were determined by site-directed mutagenesis. βI-tubulin residues interact with peloruside and laulimalide at a non-taxoid binding site on the exterior of the microtubule. The first evidence is presented of a βI-tubulin mutation that increases the sensitivity of mammalian cells to peloruside. Abstract: Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to β-tubulin at a site that is different from the classical taxoid site. Multiple βI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M βI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian βI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug–tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on β-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.