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J. Biol. Chem., Vol. 278, Issue 15, 12696-12702, April 11, 2003
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From the 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
Phosphorylation of Serine 1106 in the Catalytic Domain of
Topoisomerase II
Regulates Enzymatic Activity and Drug
Sensitivity*
,,,
,, and
Experimental Therapeutics Program, Taussig
Cancer Center, § Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, Ohio 44195, ¶ Institute for Systems
Biology, Seattle, Washington 98103, Cancer Research UK
Oxford Cancer Centre, Oxford OX3 9DS, United Kingdom, and the
** Department of Molecular Biology, Aarhus University,
DK-8000 Aarhus C, Denmark
. This effect
can be mimicked in sensitive cells treated with the intracellular Ca2+ 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. 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 containing a S1106A substitution is
4-fold less active than wild type topo II 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 protein are more resistant to the cytotoxic
effects of etoposide or amsacrine. These results demonstrate that
Ca2+-regulated phosphorylation of Ser-1106 in the catalytic
domain of topo II modulates the enzymatic activity of this protein
and sensitivity to topo II-targeting drugs.
*
This work was supported by United States Public Health
Service Grants RO1 CA74939 and RO1 DK56917.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Taussig Cancer
Center, R40, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8416; Fax: 216-444-7115; E-mail:
ganapam@ccf.org.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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