From the 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
Received for publication, January 24, 2003
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ABSTRACT |
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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 Topoisomerases alter DNA topology for the efficient processing of
genetic material (1-3). These enzymes play a pivotal role in the
maintenance of genomic integrity and are essential for many chromosomal
functions including DNA replication and recombination, transcription,
and chromosome segregation (1-3). Topoisomerases regulate various
cellular processes by relaxing and untangling the intertwined strands
of DNA. Two major categories of topoisomerases, type I and type II,
have been characterized. The type II enzymes, which consist of two
highly homologous isoforms in humans, topoisomerase (topo)1 II The essential nature of topo II Several different mechanisms, including post-translational modification
via phosphorylation, regulate topo II activity. Site-specific phosphorylation of topo II Despite the extensive knowledge of sites in topo II Materials--
The topo II-targeting drugs VP-16 and m-AMSA were
obtained from Sigma and the NCI, National Institutes of Health,
respectively. Stock solutions of these drugs were prepared in dimethyl
sulfoxide (Sigma) and stored frozen at Cell Culture--
Cultures of wild type HL-60 cells (HL-60/S)
were maintained in RPMI 1640 supplemented with 10% fetal bovine serum
and 2 mM L-glutamine (BioWhittaker,
Gaithersburg, MD) at 37 °C in a humidified 5% CO2 plus
95% air atmosphere. The resistant subline of HL-60 developed by
culturing the wild type cells in increasing concentrations of
0.025-0.05 µg/ml DOX has been described previously (10). Following
in vitro selection, the DOX-resistant subline (HL-60/R), which is 40-fold resistant to the cytotoxic effects of VP-16 compared with the HL-60/S cells, was routinely cultured in the absence of DOX.
Under these conditions the drug-resistant phenotype of HL-60/R cells
was not altered following in vitro culture for at least 3 months.
Human Topoisomerase II Expression of Human Topoisomerase II Metabolic Labeling with [32P]Orthophosphoric
Acid--
HL-60 or BJ201 yeast cells were metabolically labeled with
[32P]orthophosphoric acid to obtain in vivo
32P-labeled phosphorylated topo II Purification of Human Topo II
Recombinant topo II Phosphopeptide Mapping of Topo
II N-terminal Edman Sequencing and Mass Spectrometry--
CNBr
phosphopeptides were separated by gel electrophoresis, transferred to a
PVDF membrane, and stained with Coomassie Brilliant Blue R-250. The
stained bands were excised and subjected to N-terminal Edman sequencing
(27, 28). The N-terminal sequencing of the peptide fragments was
carried out on a Procise, model 492, protein sequencer (Applied
Biosystems, Foster City, CA) fitted with a 140c microgradient system,
785A programmable absorbance detector, and a 610A (version 2.1) data
analysis system. The internal peptide sequences were searched and
identified by BLASTP program of the NCBI. To determine whether the
sequenced peptides were phosphorylated, the membrane was
autoradiographed before and after cutting out the stained bands.
LC-tandem MS analysis was carried out following tryptic digestion of
the 170-kDa topo II
The data were analyzed using CID spectra to search NCBI nonredundant
data base with the search program TurboSequest. Spectra from samples
analyzed by MALDI-TOF were internally calibrated using trypsin
autolysis peptides, giving mass accuracy that was generally better than
25 ppm. These spectra were used for data base searches with the program MASCOT.
Decatenation of Kinetoplast DNA (kDNA) by Topo
II Precipitation of Covalent Topo II Drug Sensitivity Test in Yeast JN394t2-4 Strain--
S.
cerevisiae strain JN394 t2-4 transformed with either WT or
S1106A-MT topo II CNBr Peptide 34 Is Hypophosphorylated in HL-60/S Cells
Treated with BAPTA-AM and in HL-60/R Cells--
Previously
we have demonstrated the presence of two hypophosphorylated tryptic
peptides in a derivative of HL-60 cells (HL-60/R) that are resistant to
the topo II-targeting drug, VP-16 (10, 11). However, the location of
the phosphorylation site(s) in topo II Serine 1106 Is the Major Phosphorylation Site Present in CNBr
Peptide 34--
To determine the specific hypophosphorylation site in
peptide 34, we performed LC-tandem mass spectrometry. This analysis led
to the identification of a phosphorylated serine, Ser-1106, located in
the catalytic domain of topo II Hypophosphorylated Site in CNBr Peptide 34 and in Tryptic Peptides
2 and 3 Corresponds to Ser-1106--
Because Ser-1106 was identified
as the major phosphorylation site in peptide 34, we determined the
effect of mutation of this site to alanine on the phosphorylation of
CNBr peptide 34 and tryptic peptides 2 and 3. Although the mammalian
system was the basis of our findings, the facile expression of human
topo II S1106A-MT Topo II
Because the S1106A-MT protein was enzymatically less active than the WT
protein, we next determined whether these proteins also differed in
their ability to form DNA-cleavable complex in the absence or presence
of drugs. In the presence of 1 mM ATP, the level of topo II
DNA-cleavable complex formation with the S1106A-MT topo II S1106A-MT Topo II Post-translational modification of topo II Our previous observation demonstrating that specific sites in topo
II Previous studies have identified several phosphorylation sites in topo
II Resistance of tumor cells to topo II-targeting drugs have primarily
focused on (a) overexpression of MDR1, which encodes
P-glycoprotein resulting in enhanced drug efflux, and (b)
point mutations or truncation in the topo II Ser-1106 is flanked by acidic amino acids, thereby fulfilling the
consensus sequence requirement for both casein kinase I and casein
kinase II (37). Thus these enzymes could serve as potential physiologic
kinases regulating phosphorylation of Ser-1106. Although casein kinase
II has been shown to phosphorylate several sites in the C-terminal
region of topo II In summary, results from the present study demonstrate the significance
of Ser-1106 phosphorylation in regulating topo II. 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and topo II
with molecular masses of 170 and 180 kDa, respectively, catalyze
the ATP-dependent transport of one intact DNA double helix
through another by creating a transient double-stranded break (1).
during cell proliferation and its
ability to cleave DNA in a reversible manner makes topo II
an ideal
target for agents that poison the enzyme (4, 5). In the presence of
DNA-damaging topo II-targeting drugs, topo II is converted to a
nuclease that irreversibly cleaves duplex DNA. The "poisoning" of
the enzyme is via the trapping of the transient reaction intermediate,
termed a cleavable complex, which is composed of topo II bound
covalently to the 5' end of the cleaved DNA strands. This leads to DNA
damage, apoptosis, genomic instability, and cell death. Several
clinically effective cancer chemotherapeutic agents, daunorubicin,
doxorubicin (DOX), amsacrine (m-AMSA), and etoposide (VP-16), stabilize
the topo II-DNA cleavable complex and prevent religation of DNA (5).
However, a major factor limiting success of topo-directed
chemotherapeutic regimens is the development of resistance to topo
II-targeting drugs, which is frequently observed clinically and in
tumor model systems. Thus, an understanding of the mechanism(s) that
lead to development of resistance to drugs that poison topo II is
essential for improving the therapeutic potential of these agents.
, which occurs in a cell cycle
phase-dependent manner (6-9), also modulates drug
sensitivity to topo II-targeting drugs (10, 11). Whereas Takano
et al. (12) reported hyperphosphorylation of topo II
in
etoposide-resistant cells, most other studies (9, 10, 13) have
demonstrated a correlation between hypophosphorylation of this enzyme
and resistance to the topo II-targeting drugs, DOX, m-AMSA, or VP-16.
Hypophosphorylation of topo II
is observed in resistant cells in the
absence or presence of multidrug resistance gene (MDR1) overexpression
and in cells expressing decreased levels of protein kinase C (9, 10,
13). Decreasing intracellular calcium transients, which mimics the
resistant phenotype, also results in site-specific hypophosphorylation
of topo II
(9, 10). A majority of the phosphorylation sites in topo
II
protein is located within the C-terminal region (3). Casein
kinase II has been recognized as the major kinase interacting with and phosphorylating several sites in topo II
, including Ser-1342, Ser-1376, Ser-1469, and Ser-1524 in human topo II
(14-21). In addition to casein kinase II, protein kinase C has been shown to
phosphorylate Ser-29 (8) and a proline-directed kinase phosphorylates Ser-1212, Ser-1246, Ser-1353, Ser-1360, and Ser-1392 (9).
that are
phosphorylated, very little is known about the significance of these
phosphorylation sites in regulating topo II
function. In this study,
we employed a combination of in vivo phosphorylation and
proteomic approaches to identify the site-specific phosphorylation of
topo II
, which affects enzymatic activity and confers resistance to
topo II-targeting drugs. Our results demonstrate that Ser-1106 is a
major phosphorylation site in the catalytic domain of topo II
,
hypophosphorylation of which correlates with drug resistance in the
human leukemia cell line, HL-60. Mutation of serine 1106 to alanine
leads to a decrease in the enzymatic activity and sensitivity to topo
II-targeting drugs, thereby establishing the functional significance of
the Ser-1106 phosphorylation site in topo II
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 or 4 °C.
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) was obtained from Calbiochem. 5'-Fluoroorotic acid was
obtained from Sigma.
Plasmids--
Two plasmids, pHT212
(22) and pYEpWob6 (23), containing wild type human topo II
sequences
were employed. The plasmid pHT212 also contains a c-myc and
hexahistidine tag in the C terminus (22), and the plasmid pYEpWob6 has
a URA3 selection marker (23). For mutating Ser-1106 to
alanine (S1106A) in topo II
in these plasmids, site-directed
mutagenesis was carried out using the Quikchange Site-directed
mutagenesis kit (Stratagene Inc., La Jolla, CA). The primers used for
mutation are as follows: 5'-CA GAT GAA GAA GAA AAT GAA GAG GCT GAC AAC
GAA AAG GAA ACT G-3' and 5'-C AGT TTC CTT TTC GTT GTC AGC CTC TTC ATT
TTC TTC TTC ATC TG-3'. The site-directed mutagenesis of S1106A was
confirmed by PCR and DNA sequence analysis.
in Yeast Cells--
Wild
type (WT) or S1106A mutant (S1106A-MT) human topo II
was expressed
in two Saccharomyces cerevisiae yeast strains, BJ201 (22)
and JN394t2-4 (24). The BJ201 strain (Mat
ura3 leu2 pep4::HIS3 prb1 top2::TRP1
can1) carries a disrupted endogenous TOP2 gene but is
maintained by plasmid pSp100 (URA3) carrying the
Schizosaccharomyces pombe TOP2 gene. BJ201 yeast
cells were transformed with the human topo II
(WT or S1106A mutant)
pHT212 plasmids using the Yeastmaker, lithium acetate yeast
transformation system (Clontech, Palo Alto, CA).
Following transformation, cells carrying Leu selection
marker and human topo II sequence were selected on leucine-lacking
plates. The pSp100 plasmid encoding TOP2 S. pombe gene was
then "chased out" of the yeast by counter-selection for URA3
growth on 5'-fluoroorotic acid-containing plates. Transformed BJ201
cells were cultured overnight at 30 °C in leucine-deficient medium
with shaking (250 rpm), centrifuged, and incubated with shaking at
30 °C in YPDA (yeast extract, peptone, dextrose, and adenine) medium
supplemented with 8% glucose to a cell density of 2 × 107 cells/ml (A600 = 0.6).
S. cerevisiae strain JN394 t2-4 (Mat
ade1 ura3-52
trp1 tyr1 his7 top2-4 rad52::LEU2 ISE2),
kindly provided by Dr. J. L. Nitiss (St. Jude Children's Research
Hospital, Memphis, TN), expresses a temperature-sensitive mutant topo
II protein and is capable of growth only at 25 °C (24). This yeast
strain when transformed with either wild type pYEpWob6 (23) or S1106A pYEpWob6 mutant plasmid expresses human topo II
and grows at 35 °C in synthetic dropout medium without uracil
(URA).
protein. Log phase
cultures of HL-60/S or HL-60/R cells were first incubated in
phosphate-free RPMI supplemented with 10% dialyzed fetal bovine serum
and 2 mM L-glutamine for 1 h at 37 °C.
Cells were then labeled with 100 µCi/ml of carrier-free
[32P]orthophosphoric acid (PerkinElmer Life Sciences) for
an additional 2 h. In experiments involving treatment with
BAPTA-AM, HL-60/S cells were incubated with 20 µM
BAPTA-AM during the 2-h labeling period. The BJ201 yeast cells,
expressing WT or S1106A-MT human topo II
protein, were incubated
overnight at 30 °C with shaking (250 rpm) in synthetic dropout
liquid medium lacking leucine. The overnight cultures were transferred
to YPDA without phosphate medium and incubated at 30 °C with shaking
to a cell density corresponding to 0.6 units
(A600). Following centrifugation, cells were
resuspended into 20 ml of YPDA medium without phosphate containing 5 mCi of [32P]orthophosphoric acid and incubated at
30 °C for 1 h with shaking.
--
HL-60 cells were lysed at
4 °C for 30 min in RIPA buffer (50 mM Tris-HCl, pH 8.0, 425 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%
SDS, and 10 mM 2-mercaptoethanol) supplemented with a
mixture of proteases and phosphatase inhibitors (10, 11). The lysate was centrifuged at 100,000 × g for 30 min, and topo
II
present in the supernatant was incubated overnight at 4 °C
with a topo II
-specific polyclonal antibody (25) and protein
A-agarose. The antigen-antibody complex was dissociated in
lithium dodecyl sulfate sample buffer (Invitrogen), and topo
II
was purified by gel electrophoresis in Tris acetate gels.
Following transfer to nitrocellulose membrane (0.45 µM),
the topo II
protein band, visualized by staining with 0.025%
Coomassie Brilliant Blue R-250, was excised and used for phosphopeptide mapping.
from yeast cells was isolated after freezing the
cells in liquid nitrogen. The frozen cell pellet stored at
80 °C
was lysed by gentle rotation at 4 °C for 40 min in 2-3 volumes of
Y-PER lysis buffer (Pierce) supplemented with 40 mM imidazole, 20 mM
-mercaptoethanol, and a mixture of
protease and phosphatase inhibitors. The cell lysate was centrifuged at 13,000 × g for 10 min at 4 °C, and topo II
protein in the supernatant was purified by
Ni2+-nitrilotriacetic acid (Ni-NTA)-agarose column (Qiagen,
Valencia, CA) chromatography. After the column was washed twice with
wash buffer (40 mM imidazole, 20 mM sodium
phosphate, 0.5 M NaCl, pH 7.4), the protein was eluted with
six successive 1-ml portions of the elution buffer (200 mM
imidazole, 20 mM sodium phosphate, 0.5 M NaCl,
pH 7.4). The fractions from eluates 2-4 were pooled and concentrated
at 3000 × g for 15 min using an Amicon Ultra-4, 30K
NMWL concentrator (Millipore Inc., Milford, MA). The retentate was
recovered and stored at
20 °C in 40% glycerol. Total protein content was determined using the Bio-Rad protein assay reagent (Bio-Rad), and purity was assessed by SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250. To determine the relative amount
of WT and S1106A-MT protein present in purified preparations, Western
blot analysis was also carried out using polyclonal topo II
-specific
antibody and horseradish peroxidase-labeled secondary antibody.
--
32P-Labeled topo II
from HL-60 or BJ201
cells was purified by SDS-PAGE and transferred to nitrocellulose
membrane as described above. The 170-kDa band of topo II
on the
nitrocellulose membrane, identified by staining with Coomassie
Brilliant Blue R-250, was excised and used for proteolysis by CNBr or
trypsin. CNBr digestion was carried out at 47 °C for 90 min in the
presence of 250-400 µl of a solution containing 160 µg/ml CNBr in
70% formic acid. Following the incubation, the peptides released into
the supernatant were concentrated by evaporation in a Savant SpeedVac
and separated by one-dimensional gel electrophoresis on Tris-Tricine
peptide gels. The separated peptides were transferred to a PVDF
membrane and subjected to autoradiography or Cyclone image analysis
(PerkinElmer Life Sciences) to compare the phosphopeptide profiles of
topo II
obtained from HL-60 cells treated under different conditions or yeast cells transformed with WT or S1106A-MT topo II
. For tryptic
digestion, the 170-kDa topo II protein bound to the nitrocellulose membrane was incubated overnight with
L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin
(0.5 µg) in 50 µl of 1% ammonium bicarbonate. Two-dimensional
phosphopeptide mapping (11, 18, 26) was carried out by electrophoresis
with pH 1.9 buffer in the horizontal dimension and chromatography in
the vertical dimension using phosphochromatography buffer
(n-butyl alcohol/pyridine/acetic acid/deionized
water, 6.4:5:1:4, v/v)). Image density of the phosphopeptides was
quantified with a Cyclone imager.
protein or CNBr peptides of interest, which were
excised from SDS-polyacrylamide gels (29, 30). This procedure was
accomplished using a Finnigan LCQ-Deca ion trap mass spectrometry
system equipped with an electrospray ion source interfaced to a
10-cm × 50-µm inner diameter C18 capillary high pressure liquid
chromatography column. The digests were analyzed by LC-tandem MS with
data-dependent acquisition methods to map as many peptides
as possible from the protein. In these analyses, the LC-tandem MS data
were searched versus the topoisomerase sequence using the
program Sequest. Phosphopeptides were recognized by combinations of
characteristic additions of 80-Da multiples (depending on the degree of
phosphorylation) to peptide molecular weights and/or characteristic
neutral losses of 98 Da for H3PO4 from
phosphoserine and phosphothreonine in the collisionally induced
dissociation (CID) spectra. The MALDI-TOF analyses were carried out
using a Micromass TofSpec 2E matrix-assisted laser desorption
ionization-time-of-flight (MALDI-TOF) mass spectrometry system. Digests
desalted using ZipTips were eluted with a solution of
-cyano-4-hydroxycinnamic acid as the matrix before spotting on the
target plate.
--
Topo II
enzymatic activity was assayed by measuring the
decatenation (31) of kDNA (Topogen, Columbus, OH). A standard assay carried out in a total volume of 20 µl included 50 mM
Tris-HCl, pH 7.9, 88 mM KCl, 10 mM
MgCl2, 0.5 mM EDTA, 10 mM ATP, 10 mM dithiothreitol, 100 µg/ml bovine serum albumin, and
300 ng of kDNA. The reaction mixture containing varying
amounts of WT or S1106A-MT topo II
was incubated at 37 °C, and at
timed intervals the reaction was stopped by the addition of 5 µl of
stop solution (5% SDS, 25% Ficoll, and 0.05% bromphenol blue). The
samples were resolved by electrophoresis at 115 V using a 1% agarose
gel in Tris acetate EDTA buffer. Following electrophoresis, the gel was stained with ethidium bromide and photographed under UV illumination, and the amount of decatenated minicircles of kDNA was
quantified using an AlphaInnotech Image analyzer (AlphaInnotech Corp.,
San Leandro, CA).
-DNA Complex--
Formation
of covalent topo II-DNA complex by WT or S1106A-MT topo II
was
determined by the precipitation of 3'-end-labeled 32P-pcDNA3 in a cell-free system as described by
Zwelling et al. (32). Briefly, pcDNA3 was linearized
with EcoRI and 3'-end-labeled with [32P]dATP.
The 3'-end-labeled pcDNA3 was incubated with 10-200 ng of WT or
S1106A-MT topo II
in the absence or presence of 1 mM ATP. To determine drug-stabilized DNA-cleavable complex formation in
the presence of 1 mM ATP, 25 or 100 ng of WT or S1106A-MT
topo II
was incubated with 1-100 µM VP-16 for 30 min
at 37 °C (10, 11, 32). The reaction was stopped by the addition of
SDS, and the protein-DNA complex was precipitated by the addition of KCl (10, 11, 32). The precipitate was washed twice with 100 mM KCl and dissolved in water at 65 °C, and the solution
was added to Ecolume (ICN Pharmaceutical, Costa Mesa, CA). The level of
radiolabel was then determined using a liquid scintillation counter.
pYEpWob6 (23) plasmid was cultured at 30 °C in
synthetic dropout liquid medium without uracil. The cells were
collected by centrifugation and resuspended in YPDA medium. After
adjusting the cell number to 2 × 106 cells/ml, cells
were treated for 24 h at 35 °C with VP-16 (0-200 µM) or m-AMSA (0.5-25 µM) at a final
concentration of 2% Me2SO. Following treatment, the
control or treated cultures were diluted 1000-fold with sterile water
and plated in triplicate using YPD agar in 100 × 15-mm Petri
dishes. After incubation for 3-4 days at 35 °C, the number of
colonies on the control and treated drug plates were counted in an
AlphaInnotech image analyzer (AlphaImagerTM, Alpha Innotech
Corp., San Leandro, CA). We have confirmed that the JN394t2-4 yeast
cells are not viable at 35 °C and growth at 35 °C occurs only
following transformation with WT pYEpWob6 or S1106A-MT pYEpWob6
plasmid. Colony growth of the JN394t2-4 on YPDA is also similar
following transformation of the WT pYEpWob6 or S1106A-MT pYEpWob6
plasmid. Topo II
protein levels in the JN394t2-4 cells transformed
with WT pYEpWob6 or S1106A-MT pYEpWob6 plasmid were determined in cell
lysates following SDS-PAGE and staining with Coomassie Brilliant Blue
R-250 and Western blotting with a topo II
-specific antibody (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was not defined. To identify
the region in topo II
that contained these sites, we compared the
one-dimensional maps of CNBr phosphopeptides generated from
32P-labeled topo II
immunoprecipitated from HL-60/S
cells, HL-60/S cells treated with BAPTA-AM, and HL-60/R cells. Our
results demonstrated that a CNBr phosphopeptide of an approximate
molecular mass of 12 kDa was hypophosphorylated in HL-60/S cells
treated with BAPTA-AM and HL-60/R cells as compared with HL-60/S cells
(Fig. 1). Although the decrease in
phosphorylation of this peptide was modest, it was consistently lower
(30% when normalized to the phosphorylated peptide migrating at 25 kDa) than that observed for other peptides. The 12-kDa
hypophosphorylated peptide was identified by N-terminal Edman
sequencing to correspond to CNBr peptide 34 (amino acids 1041-1131) of
a calculated molecular mass of 10.4 kDa. This peptide is located in the
catalytic domain of topo II
. Two-dimensional phosphopeptide maps of
complete tryptic digests of topo II
protein from HL-60/S cells or
HL-60/S cells treated with BAPTA-AM revealed hypophosphorylation of two
peptides (peptide 2 and 3, Fig. 1B) in
HL-60/S cells treated with BAPTA-AM compared with HL-60/S cells. These
two hypophosphorylated peptides are identical to those observed in
HL-60/R cells and are also present in tryptic digests of peptide 34 (data not shown).
View larger version (53K):
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Fig. 1.
Hypophosphorylation of CNBr peptide 34 and
tryptic peptides 2 and 3 in HL-60/S cells treated with BAPTA-AM and
HL-60/R cells. Topo II present in extracts of HL-60 cells,
metabolically labeled with [32P]orthophosphoric acid, was
immunoprecipitated and subjected to SDS-PAGE. Following transfer of the
SDS-polyacrylamide gel to a nitrocellulose membrane, the stained topo
II
band was excised and digested with either CNBr or trypsin.
A, samples of CNBr digests of topo II
purified from
HL-60/S cells (lane 1), HL-60/S cells treated with 20 µM BAPTA-AM (lane 2), or HL-60/R cells
(lane 3) were electrophoresed on SDS-polyacrylamide gels
(10-20% Tris-Tricine gels), transferred to a PVDF membrane, and
autoradiographed. B, samples of tryptic digests of topo
II
purified from HL-60/S cells or HL-60/S cells treated with 20 µM BAPTA-AM were analyzed by two-dimensional
phosphopeptide mapping and autoradiography.
. The Ser-1106 phosphorylation site
was detected initially in the 12-kDa CNBr fragment of topo II
by
data-dependent analysis. The CID spectrum of the peptide, VPDEEENEEpSDNEK is shown in Fig. 2. In addition, another
peptide, VPDEEENEEpSDNEKETEK, was
detected. The detection of two phosphorylated peptides containing
Ser-1106 (generated because of partial proteolysis) provides an
explanation for the presence of two hypophosphorylated peptides in the
two-dimensional tryptic phosphopeptide maps (Fig. 1B). The
CID spectrum of these peptides is typical for a serine or threonine
phosphopeptide, with a high abundance ion due to the loss of
H3PO4 and low abundance (but significant)
sequence ions. Based on this spectrum, a highly specific, selected,
reaction-monitoring scheme was designed and used to detect this
phosphopeptide in the more complex digest of intact topo II
.
View larger version (12K):
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Fig. 2.
Identification of Ser-1106 phosphorylation
site by mass spectrometry. An in-gel tryptic digest of CNBr
peptide 34 was analyzed using capillary column LC-tandem mass
spectrometry. The data were analyzed using CID spectra to search NCBI
nonredundant data base. The CID spectrum of the phosphopeptide
containing Ser-1106 shows the facile loss of
H3PO4 to produce the doubly charged base peak
in the spectrum (m/z 823.4). The peptide sequence
and the site of phosphorylation (VPDEEENEEpSDNEK) were
subsequently established by interpretation of the low abundance
fragment ions.
and the absence of topo II
in yeast cells made this
system an attractive model to carry out comparative studies of WT and
S1106A-MT topo II
protein. BJ201 yeast cells expressing WT or
S1106A-MT topo II
protein were metabolically labeled with
[32P]orthophosphoric acid, and recombinant topo II
protein purified from these cells was digested with CNBr or trypsin.
The maps of the CNBr digests characterized by one-dimensional SDS-PAGE
(Fig. 3A) showed that peptide
34 was phosphorylated only in digests of WT topo II
protein but not
of S1106A-MT protein. Comparison of the tryptic digests (Fig.
3B) by two-dimensional phosphopeptide mapping revealed the
presence of the phosphorylated peptides 2 and 3 (inset) in
the WT topo II
protein sample but not in the S1106A-MT protein
sample. This finding indicates that hypophosphorylation of CNBr peptide
34 and of tryptic peptides 2 and 3, observed in HL-60 cells resistant
to topo II-targeting drugs, occurs at Ser-1106. It is interesting to
note that the CNBr and tryptic phosphopeptide maps of human topo II
from HL-60 and yeast cells (Figs. 1 and 3) are similar, although the
relative intensities of individual phosphopeptides varies depending on
the source of the topo II
protein. The only major difference is the
absence of a 4-kDa phosphopeptide in the CNBr maps and of
phosphopeptide 1 in the tryptic maps of the recombinant protein (WT or
S1106A-MT) expressed in yeast, as compared with native topo II
expressed in HL-60 cells. This discrepancy is due to differential
migration of the C-terminal CNBr or tryptic peptide, which is larger in
the recombinant protein (due to the presence of additional amino acids
transcribed from linker sequences) compared with the native protein.
Indeed the C-terminal CNBr peptide has a molecular mass of 4 kDa and
the C-terminal tryptic peptide phosphorylated at Ser-1524 corresponds to peptide 1 (18).
View larger version (51K):
[in a new window]
Fig. 3.
Differential phosphorylation of CNBr peptide
34 and tryptic peptides 2 and 3 generated from WT and S1106A-MT topo
II protein expressed in BJ201 yeast
cells. BJ201 yeast cells expressing WT or S1106A-MT protein were
metabolically labeled with [32P]orthophosphoric acid. The
recombinant protein from these cells was purified by
Ni2+-NTA column chromatography and subjected to SDS-PAGE.
Following transfer to a nitrocellulose membrane, the stained topo II
band was excised and digested with CNBr or trypsin. A,
samples of CNBr digests of WT (lane 1) or S1106A-MT
(lane 2) topo II
were electrophoresed on
SDS-polyacrylamide gels (10-20% Tris-Tricine gels), transferred to
PVDF membrane, and autoradiographed. B, samples of
tryptic digests of WT or S1106A-MT topo II
protein were analyzed by
two-dimensional phosphopeptide mapping and autoradiography.
Exhibits Reduced Enzymatic Activity and
Formation of Protein-DNA Cleavable Complex--
We next examined the
functional role of Ser-1106 phosphorylation by comparing the catalytic
activity and level of drug-stabilized, DNA-cleavable complex formation
of recombinant WT or S1106A-MT topo II
. For these studies the WT and
S1106A-MT topo II
proteins were expressed in the BJ201 yeast strain
and purified by metal ion affinity chromatography using
Ni2+-NTA columns. This procedure led to significant
purification of topo II
protein, which was present in equivalent
amounts in the WT and S1106-MT protein preparation, as judged by
Coomassie Brilliant Blue R-250 staining and Western blot analysis (Fig.
4). The catalytic activity of equivalent
amounts of purified WT or S1106A-MT topo II
protein was determined
using 300 ng of kDNA as the substrate (31). As seen in Fig.
5, 4-fold more S1106A-MT topo II
protein, as compared with WT topo II
protein, was required to
produce equivalent levels of decatenated minicircles from
kDNA (Fig. 5, A and B). Comparison of
the time course of relative decatenation activity by WT and S1106A-MT
topo II
revealed a 4-fold higher rate of reaction for WT, compared
with S1106A-MT, topo II
protein, based on the initial slope of the
reaction curves (Fig. 5C). In these experiments (Fig.
5C), to observe measurable decatenation in the linear range
of the slope, 50 ng of the mutant protein and 12.5 ng of the WT protein
were required.
View larger version (21K):
[in a new window]
Fig. 4.
Purification of WT and S1106A-MT topo
II from BJ201 yeast cells. Extracts of
yeast cells expressing WT or S1106A-MT topo II
were purified by
Ni2+-NTA column chromatography. An aliquot (1.2 µg) of
the purified preparation of WT (lanes 1 and 3) or
S1106A-MT (lanes 2 and 4) topo II
was
subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane
was stained with Coomassie Brilliant Blue R-250 (lanes 1 and
2) or immunoblotted with topo II
-specific antibody
(lanes 3 and 4).
View larger version (27K):
[in a new window]
Fig. 5.
Comparison of the enzymatic activity of WT
and S1106A-MT topo II protein. Varying
concentrations (200 ng, lane 1; 100 ng, lane 2;
50 ng, lane 3; 25 ng, lane 4; 12.5 ng, lane
5; 6.25 ng, lane 6; 3.125 ng, lane 7) of WT
or S1106A-MT topo II
protein were incubated with kinetoplast DNA
(kDNA). A, an aliquot of the reaction
mixture was electrophoresed on a 1% agarose gel to separate the
kDNA substrate from decatenated (minicircles) DNA, and the
DNA bands were visualized by UV illumination of ethidium
bromide-stained gels. The relative intensity of the bands was
determined using an AlphaInnotech image analyzer. B,
the percent decatenation, calculated as the ratio of the intensity of
the decatenated band to the total intensity of the substrate band plus
the decatenated band, was plotted versus concentration of WT
(
) or S1106A-MT (
) topo II
. C, time course of
percent relative decatenation/ng of purified WT (
) or S1106A-MT
(
) topo II
.
was
2-fold reduced compared with that of the WT protein (Fig.
6A). No difference was
observed in the absence of ATP (Fig. 6A). In the presence of
ATP and the topo II-targeting drug, VP-16, a significant
(p = 0.01) 2-4-fold decrease in drug-stabilized, DNA-cleavable complex formation was observed with the S1106A-MT compared with WT topo II
protein (Fig. 6B). This
difference was seen using two different concentrations of the topoII
protein (25 and 100 ng as indicated above the panels in Fig.
6B).
View larger version (25K):
[in a new window]
Fig. 6.
Comparison of the level of formation of
DNA-cleavable complex by WT and S1106-MT topo II.
A, varying concentrations of purified WT (closed
symbol) or S1106A-MT (open symbol) topo II were
incubated with 32P-labeled pcDNA3 in the absence (
,
) or presence of 1 mM ATP (
,
). B,
WT (
) or S1106A-MT (
) topo II
protein (25 or 100 ng) was
incubated with 32P-labeled pcDNA3, 1 mM
ATP, and varying concentrations of VP-16. The protein-DNA complex was
precipitated by the addition of KCl, and the counts/min present in the
precipitate were determined by liquid scintillation counting.
Transformed in the JN394t2-4 Yeast Strain Is
Resistant to the Cytotoxic Effects of the Topo II-targeting Drugs,
VP-16 and m-AMSA--
To confirm that hypophosphorylation of topoII
at Ser-1106 confers a resistant phenotype in vivo, we tested
drug sensitivity of yeast cells expressing WT or S1106A-MT topo II
protein. Sensitivity to two topo II-targeting drugs, VP-16 and m-AMSA,
was determined in the yeast strain JN394t2-4 transformed with the
pYepWob6 construct of WT or S1106A-MT human topo II
protein. At
35 °C JN394t2-4 yeast cells do not grow unless the human recombinant
topo II
is expressed. The survival data in Table
I demonstrate that JN394t2-4 cells
transformed with S1106A-MT topo II
are more resistant to the
cytotoxic effects of different concentrations of VP-16 (up to 12-fold
higher survival) and m-AMSA (up to 20-fold higher survival) than
JN394t2-4 cells transformed with WT topo II
. This survival difference is not due to differential cellular expression of the WT and
S1106A-MT protein, because extracts made from identical numbers of
yeast cells transformed with the pYEpWob6 construct of human WT or
S1106A-MT topo II
express equivalent amounts of topo II
protein
(data not shown).
Survival of JN394t2-4 yeast cells expressing WT or S1106A-MT topo
II following treatment with VP-16 or m-AMSA for 24 h
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by reversible
phosphorylation is a key mechanism regulating its function. In this study, we identified a novel phosphorylation site, Ser-1106, in the
catalytic domain of topo II
. Mutation of this site to alanine leads
to a reduction in the catalytic activity of topo II
and level of
formation of enzyme-DNA-cleavable complex. This results in decreased
sensitivity to topo II-targeting drugs in vivo. To our
knowledge this is the first report identifying a phosphorylation site
in the catalytic domain of topo II
that is capable of modulating its function.
protein were hypophosphorylated in cells resistant to topo
II-targeting drugs (10, 11) provided the impetus for defining
phosphorylation site(s) that were functionally relevant for topo II
function, in particular regulation of sensitivity to topo II-targeting
drugs. By using an integrated approach involving CNBr and tryptic
phosphopeptide mapping of in vivo 32P-labeled
topo II
, N-terminal Edman sequencing of CNBr peptides, and mass
spectrometry, we were initially able to locate a region in the
catalytic domain of the enzyme (amino acids 1041-1131) that harbored
the hypophosphorylated site. Phosphorylation of this region could also
be manipulated by altering intracellular Ca2+ transients
using BAPTA-AM, which mimicked the resistant phenotype (10, 11). Mass
spectrometric analysis of this region led to the identification of
Ser-1106 as a major phosphorylation site. The functional significance
of this site was established by comparing the activity of WT and
Ser-1106-MT topo II
protein in vitro and drug sensitivity
of yeast cells transformed with these proteins in vivo.
, primarily in the C-terminal region. Consistent with the
physiologic function of topo II
, phosphorylation of several sites is
cell cycle phase-regulated (6-9). Although several kinases have been
shown to phosphorylate topo II
, casein kinase II has emerged as the
major kinase interacting with and phosphorylating several sites,
including Ser-1342, Ser-1367, Ser-1469, and Ser-1524 in human topo
II
(14-21). In addition to casein kinase II, protein kinase C has
been shown to phosphorylate Ser-29 (8), and a proline-directed kinase
phosphorylates Ser-1212, Ser-1246, Ser-1353, Ser-1360, and Ser-1392
(9). Identification of most of these sites was based on in
vitro studies employing purified protein kinases. However,
in vivo phosphorylation of these sites was confirmed by
matching tryptic phosphopeptides generated following phosphorylation in vitro and in vivo (8, 9). Despite these
extensive studies, the functional significance of phosphorylation at
these sites in vivo remains unclear. Although casein kinase
II is capable of modulating the activity of mammalian topo II
, the
mechanism by which this occurs is thought to involve stabilization of
topo II
but not phosphorylation per se (33, 34). Indeed,
it has been shown that the C-terminal domain is not important for
enzymatic activity, because deletion of this region or mutation of
Ser-1376 and/or -1524 does not render the enzyme inactive (34). Rather, it has been proposed that phosphorylation at these sites may be important for subcellular localization of topo II
(34). Furthermore, phosphorylation of Ser-29 does not affect the ATPase activity of topo
II
, which is localized to the N-terminal region and required for the
final religation step in the enzymatic reaction (35).
gene (5). Indeed, point
mutations identified in model systems resistant to topo II-targeting
drugs have been shown to confer drug resistance when tested in the
JN394t2-4 yeast system (24). The functional role of site-specific
phosphorylation on sensitivity of topo II
to DNA-cleavable complex
formation by topo II-targeting drugs in vitro or in
vivo has not been addressed. In general, hypophosphorylated (10,
13) or hyperphosphorylated (12, 36) topo II
has been correlated with
drug insensitivity. Our results on resistance to topo II-targeting
drugs in vitro and in vivo with S1106A-MT topo
II
provide evidence for a regulatory role of site-specific
phosphorylation in sensitivity to topo II-targeting drugs. Thus,
hypophosphorylation of topo II
may be responsible for the lack of
response of cancer patients to treatment with topo II-targeting drugs.
Unlike S1106A-MT, the double mutant S1376A and S1524A topo II
enzyme
that is enzymatically active (34) is not resistant to the topo
II-targeting drugs VP-16 or m-AMSA when tested in the JN394t2-4 yeast
system (data not shown).
, the role of casein kinase I has not been
evaluated, despite the presence of several casein kinase I consensus
sites. Both casein kinase I and casein kinase II play an important role
in regulating numerous cellular events. However, the presence of
different isoforms of casein kinase I allows for a more diverse
mechanism by which this enzyme can mediate signaling events. Of
particular relevance to this study is the ability of two isoforms of
casein kinase I, casein kinase I
and casein kinase I
(but not
casein kinase II), to be activated by
Ca2+-dependent dephosphorylation or proteolysis
(38, 39). Indeed, it has been reported that
Ca2+-dependent dephosphorylation of casein
kinase I
by calcineurin regulates phosphorylation and activation of
DARP32 by metabotropic glutamate receptors in neostriatal neurons (40,
41). A mechanism similar to this could provide for an explanation for
Ca2+-dependent phosphorylation of Ser-1106 by
casein kinase I
or casein kinase I
, which is supported by our
preliminary data demonstrating decreased phosphorylation of CNBr and
tryptic peptides containing Ser-1106 by two inhibitors of casein kinase
I, CKI-7 and IC-261.2 However
more detailed studies are required for identifying the physiologic
kinases(s) required for phosphorylation of Ser-1106.
function,
viz. decatenation of DNA and formation of drug-stabilized DNA cleavable complex. Although the S1106A mutant topo II
protein exhibits decreased enzymatic activity, it is able to complement growth
of yeast cells. This observation suggests that the presence of adequate
cellular levels of S1106A-MT topo II
protein can compensate for
attenuation in enzymatic activity. Alternatively it is possible that
other mechanisms in conjunction with Ser-1106 phosphorylation may be
involved in regulating topo II
function. Our results implicating the
importance of Ser-1106 phosphorylation in modulating drug sensitivity
suggest that mechanisms upstream of Ser-1106 phosphorylation may be
altered in clinical resistance to topo II-targeting drugs. Thus a
future challenge will be the identification of the relevant kinase(s)
and/or phosphatase(s) involved in regulating phosphorylation of this
site, which could aid in the design of novel treatment strategies for
tumors resistant to topo II-targeting drugs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. L. Nitiss (St. Jude Children's Research Hospital, Memphis, TN) for kindly providing the S. cerevisiae strain JN394 t2-4 and Dr. David B. Wilson (Institute for Comparative and Environmental Toxicology and the Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY) and Dr. Kunio Misono (Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH) for helpful suggestions during the developmental stages of this work. We also thank Jim Reed (Art Medical Illustrations and Photography Department) for skillful preparation of the artwork.
![]() |
FOOTNOTES |
---|
* 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.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M300837200
2 K. Chikamori, D. R. Grabowski, M. Kinter, B. B. Willard, S. Yadav, R. H. Aebersold, R. M. Bukowski, I. D. Hickson, A. H. Andersen, R. Ganapathi, and M. K. Ganapathi, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: topo, topoisomerase; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; DOX, doxorubicin; m-AMSA, amsacrine; WT, wild type; VP-16, etoposide; PVDF, polyvinylidene difluoride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Ni-NTA, Ni2+-nitrilotriacetic acid; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption-ionization-time-of-flight; CID, collisionally induced dissociation; LC-tandem MS, liquid chromatography-tandem/mass spectrometry; kDNA, kinetoplast DNA.
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