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Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80‐Da modification at this site
Author(s) -
Grozav Adrian G.,
Willard Belinda B.,
Kozuki Toshiyuki,
Chikamori Kenichi,
Micluta Marius A.,
Petrescu AndreiJose,
Kinter Michael,
Ganapathi Ram,
Ganapathi Mahrukh K.
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000194
Subject(s) - phosphopeptide , phosphorylation , chemistry , cyanogen bromide , trypsin , mutant , enzyme , biochemistry , tyrosine , tyrosine phosphorylation , microbiology and biotechnology , blot , peptide , peptide sequence , biology , gene
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: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H 3 PO 4 (−98 Da) in the CID spectra and on differences in 2‐D‐phosphopeptide maps of 32 P‐labeled wild‐type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2‐D‐phosphopeptide mapping of 32 P‐labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine‐specific antibodies. Since the +80‐Da modification on tyr656 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 in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.