Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site

Proteomics. 2011 Mar;11(5):829-42. doi: 10.1002/pmic.201000194. Epub 2011 Jan 31.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Phospho-Specific / metabolism
  • Artifacts*
  • Biocatalysis
  • Blotting, Western
  • Circular Dichroism
  • Cyanogen Bromide / chemistry
  • DNA / metabolism
  • DNA Topoisomerases, Type II / genetics
  • DNA Topoisomerases, Type II / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • HL-60 Cells
  • Halogenation
  • Humans
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Models, Molecular
  • Mutation
  • Phosphorylation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae
  • Serine / genetics
  • Serine / metabolism
  • Threonine / genetics
  • Threonine / metabolism
  • Trypsin / metabolism
  • Tyrosine / genetics
  • Tyrosine / metabolism*


  • Antibodies, Phospho-Specific
  • DNA-Binding Proteins
  • Isoenzymes
  • Recombinant Proteins
  • Threonine
  • Tyrosine
  • Serine
  • DNA
  • Trypsin
  • DNA Topoisomerases, Type II
  • Cyanogen Bromide