title: Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity.
abstract: Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.
authors.affiliation: Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Chikamori
authors.forename: Dale R
authors.initials: DR
authors.lastname: Grabowski
authors.forename: Michael
authors.initials: M
authors.lastname: Kinter
authors.forename: Belinda B
authors.initials: BB
authors.lastname: Willard
authors.forename: Satya
authors.initials: S
authors.lastname: Yadav
authors.forename: Ruedi H
authors.initials: RH
authors.lastname: Aebersold
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Bukowski
authors.forename: Ian D
authors.initials: ID
authors.lastname: Hickson
authors.forename: Anni H
authors.initials: AH
authors.lastname: Andersen
authors.forename: Ram
authors.initials: R
authors.lastname: Ganapathi
authors.forename: Mahrukh K
authors.initials: MK
authors.lastname: Ganapathi
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm; D047390:Casein Kinase II; D047388:Casein Kinases; D020134:Catalytic Domain; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004317:Doxorubicin / Q000633:toxicity; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000951:Antigens, Neoplasm; D002614:Chelating Agents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D010768:Phosphoserine; D012694:Serine; D004533:Egtazic Acid; D004317:Doxorubicin; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II; D000409:Alanine