title: Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.
abstract: 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.
authors.affiliation: Clinical Pharmacology Program, Taussig Cancer Institute, Cleveland, OH, USA.
authors.forename: Adrian G
authors.initials: AG
authors.lastname: Grozav
authors.forename: Belinda B
authors.initials: BB
authors.lastname: Willard
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kozuki
authors.forename: Kenichi
authors.initials: K
authors.lastname: Chikamori
authors.forename: Marius A
authors.initials: MA
authors.lastname: Micluta
authors.forename: Andrei-Jose
authors.initials: AJ
authors.lastname: Petrescu
authors.forename: Michael
authors.initials: M
authors.lastname: Kinter
authors.forename: Ram
authors.initials: R
authors.lastname: Ganapathi
authors.forename: Mahrukh K
authors.initials: MK
authors.lastname: Ganapathi
mesh_terms: D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D016477:Artifacts; D055162:Biocatalysis; D015153:Blotting, Western; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018922:HL-60 Cells; D054879:Halogenation; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
chemical_list: D045163:Antibodies, Phospho-Specific; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D004247:DNA; D014357:Trypsin; D004250:DNA Topoisomerases, Type II; D003488:Cyanogen Bromide