Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity.
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.
Chikamori
Kenichi
K
Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Grabowski
Dale R
DR
Kinter
Michael
M
Willard
Belinda B
BB
Yadav
Satya
S
Aebersold
Ruedi H
RH
Bukowski
Ronald M
RM
Hickson
Ian D
ID
Andersen
Anni H
AH
Ganapathi
Ram
R
Ganapathi
Mahrukh K
MK
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
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