Abstract 2526: Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity

Topoisomerase (topo) II catalyzes topological changes in DNA. Although, both human isozymes, topo IIα and β, are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin we detected four +80-Da modified sites, tyrosine 656, serine 1395, threonine 1426 and serine 1545. Phosphorylation at serine 1395, threonine 1426 and serine 1545 was established based on neutral loss of H 3 PO 4 (−98 Da) in the collision-induced dissociation (CID) spectra and on differences in 2D-phosphopeptide maps of 32 P-labeled wild type (WT) and serine1395alanine or threonine1426alanine/serine1545alanine mutant topo IIβ. However, phosphorylation at tyrosine 656 could not be verified by 2D-phosphopeptide mapping of 32 P-labeled WT and tyrosine656phenylalanine mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyrosine 656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions (characteristic of bromination) in CID spectra of two peptides containing tyrosine 656 and tyrosine 711, confirming bromination. Interestingly, mutation of only tyrosine 656, but not serine 1395, threonine 1426 or serine 1545, decreased topo IIβ activity suggesting a functional role for tyrosine 656. Homology modeling of the region of topo IIβ (amino acids 449-1108) containing these two reactive tyrosine residues revealed that tyrosine 711 is buried, whereas tyrosine 656 is highly exposed on the surface. This region is within, or very close to the dimerization interface, which suggests that these amino acids may be critical for topo IIβ dimer formation. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2526. doi:10.1158/1538-7445.AM2011-2526