Download Creatine Kinase B Is a Target Molecule of Reactive Oxygen Species

Mol. Cells, Vol. 12, No. 3, pp. 412-417
Communication
Molecules
and
Cells
KSMCB 2001
Creatine Kinase B Is a Target Molecule of Reactive Oxygen
Species in Cervical Cancer
Hyun Choi1, Chang Soo Park, Byoung Gie Kim, Jae Won Cho2, Jong-Bae Park3, Yun Soo Bae1, and
Duk Soo Bae*
Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul 135-710,
Korea;
1
Division of Molecular Life Sciences, Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea;
2
Department of General Surgery, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul 135-710, Korea;
3
Department of Molecular Life Sciences, Pohang University of Science and Technolgy, Pohang 790-784, Korea.
(Received November 1, 2001; Accepted November 12, 2001)
Recently, a procedure for detecting ROS-sensitive proteins that contain active cysteine residues was developed. The method is based on the fact that biotinconjugated iodoacetamide (BIAM) and ROS competitively and selectively react with the active cysteine
residues in ROS-sensitive proteins. To investigate the
role of ROS in cervical cancer, BIAM labeling on cytosolic proteins in normal and cancer tissues was performed, respectively. The BIAM labeling proteins are
separated by 2-dimensional electrophoresis, and then
identified by MALDI-TOF mass analysis. ROSsensitive protein is identified as creatine kinase B containing cysteine residue in active center. Activity of
creatine kinase B in normal tissue is higher than that
of oxidized form in cervical cancer tissues. The result
suggests that ROS play an important role in metabolic
regulation in cervical cancer cells. However, molecular
mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular transformation remain to be elucidated.
Keywords: 2-Dimensional Gel Electrophoresis (2DE);
Biotinylation; Cervical Cancer; Creatine Kinase (CK);
Hydrogen Peroxide; MALDI; Reactive Oxygen Species
(ROS).
Introduction
Most reactive oxygen species (ROS) in cell are generated
* To whom correspondence should be addressed.
Tel: 82-2-3410-3511; Fax: 82-2-3410-0044
E-mail: [email protected]
from results of aerobic respiration and various oxidase
actions such as lipoxygenase and xanthin oxidase. Furthermore, many cell types transiently produce ROS in
response to a variety of extracellular stimuli including
growth factors and hormones (Finkel et al., 1998; Rhee et
al., 2000). The term ROS encompasses many species including singlet oxygen, the superoxide anion radical (O2•), H2O2, lipid peroxides, nitric oxide (NO), peroxynitrite
(ONOO-), the thiyl peroxyl radical (RSOO• ), the ferryl
radical (FeO2+) and the hydroxyl radical (OH• ) (Yim et al.,
1994). Although the chemical nature of ROS generated in
response to the activation of various receptors has not
been well characterized, H2O2 was shown to be a major
component of ROS in cells (Bae et al., 1997; Sundaresan
et al., 1995). Hydrogen peroxide is thought to contribute
to various cellular functions, including the activation of
transcription factors, phospholipases, and protein tyrosine
kinases as well as the inactivation of protein tyrosine
phosphatases and serine-threonine phosphatases (Bae et
al., 1997; Kamata et al., 1999; Schreck et al., 1991;
Sundaresan et al., 1995).
A number of methods have been developed for the detection of such irreversibly oxidized products, including
lipid-derived malondialdehyde (Luo et al., 1995), carbonyl group-containing proteins (Levine et al., 1995), 4hydroxynonenal protein (Yoritaka et al., 1996), and 8hydroxy-2′-deoxyguanosine derived from DNA (Teixeira
et al., 1995). Recently, a procedure for identifying proteins
Abbreviations: 2DE, 2-dimensional gel electrophoresis; BIAM,
biotin-conjugated iodoacetamide; CK, creatine kinase; Cr,
creatine; DTT, dithiothreitol; MALDI-TOF, Matrix-assisted
laser desorption/ionization-time of flight; ROS, reactive oxygen
species.
Hyun Choi et al.
teins that contain H2O2-sensitive Cys residues has been
developed (Kim et al., 2000). This procedure is based on
the facts that H2 O2 selectively oxidized Cys residues with
a low acid constant (pKa), that these Cys residues are also
selectively labeled at pH 6.5 with biotin-conjugated iodoacetamide (BIAM), that oxidized cysteines are not susceptible to labeling with BIAM, and then the decrease in
BIAM labeling of proteins that results from oxidation can
be monitored by streptavidin blot analysis. The method
provides systematic attempts to identify such target proteins of H2O2 in cells.
Creatine kinase (CK) catalyzes the reversible transfer of
γ-phosphate group of ATP to the guanidine group of
creatine (Cr) to produce ADP and phosphorylcreatine
(PCr). To date, four different isozymes of CK are identified in mammalian tissues (Wyss et al., 2000). Muscle CK
(M-CK) and brain CK (B-CK) are located in cytosol and
two mitochondrial CK isoforms (ubiquitous Mi-CK and
sarcomeric Mi-CK) are located in mitochondrial intermembrane space. CK isozymes were known to contain a
single active sulfhydryl group (Cys-278 in Mi-CK and
Cys-283 in cytosolic CK) (Furter et al., 1993). Modification of sulfhydryl residue by ROS and NO resulted in a
decrease of CK activity (Kaneko et al., 1993; Mekhfi et
al., 1996; Suzuki et al., 1992). The result indicates that
the active sulfhydryl group in CK plays an important role
for catalytic activity.
In this paper, it is demonstrated that the ROS target
molecules in normal and cervical cancer tissue are identified using BIAM labeling method and MALDI-TOF mass
analysis. Moreover, the modification of sulfhydryl group
in CK by ROS resulted in a decrease of their activity. The
result suggests that the inactivation of CK by ROS might
be involved in energy metabolic regulation in cancer.
Materials and Methods
Materials and reagents Fresh cancer tissue and adjacent normal tissue from surgical specimens were divided and immediately frozen in liquid nitrogen and stored at −70°C until analyzed. N-(biotinoyl)-N′-(iodoacetyl)ethylenediamine (BIAM) was
obtained from Molecular Probes. Horseradish peroxidase (HRP)conjugated antibodies to mouse and rabbit immunoglobulin G
were from Upstate biotechnology. Enhanced chemiluminescence
(ECL) reagents were from NEN. Horseradish peroxidase
(HRP)-conjugated streptavidin was from Amersham. Rabbit anti
creatine kinase-BB isoenzyme was from Fitzgerald. Aprotinin,
leupeptin, 4-(2-aminoethyl)-benzene-sulfonyl fluoride hydrochloride were from ICN.
Labeling with BIAM Human endometrial cervical cancer tissues were homogenated with lysis buffer [0.2 M Mes-NaOH
(pH 6.5), 1 mM EDTA, aprotinin (1 µg/ml), leupeptin (1 µg/ml),
413
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (1
µg/ml) and 50 µM BIAM] (Kim et al., 2000). Bubbling with
nitrogen gas at low flow rate for 15 min eliminated the oxygen
from buffer. The cell lysates were incubated for 1 h at room
temperature with nitrogen gas flush. The labeling reaction was
terminated by the addition of β-mercaptoethanol to a final concentration of 50 mM. The reaction mixture was centrifuged
40,000 rpm for 1 h at 4°C. The supernatant was subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), and the separated proteins were transferred to a nitrocellulose membrane.
Proteins labeled with BIAM were detected with HRPconjugated streptavidin and ECL.
2-Dimensional gel electrophoresis The supernatant was mixed
for 2 h at room temperature with sample buffer (9.5 M urea, 2%
Triton X-100, 5% β-mercaptoethanol) and electrofocused in 7
cm ImmobilineR DryStrips (pH 4−7) with the Amersham
IPGphor. The following focusing protocol was used: 50 µA per
strip at 20°C; (1) rehydration for 13 h; (2) 500 V for 1 h (step
and hold); (3) 1000 V for 1 h (step and hold); (4) 8000 V for 3 h
(step and hold). After electrofocusing, the strips were either
stored at −70°C or shaken for 15 min with equilibration buffer
(1.5 M Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 10
mg/ml DTT) and subjected to 10% SDS-PAGE.
Protein identification by peptide mass fingerprinting analysis Protein peptide fingerprinting analysis was performed as
described (Jensen et al., 1996). In brief, the candidate band was
excised from the gel and digested with trypsin. One µl aliquot of
the total digest (total volume 30 µl) was used for peptide mass
fingerprinting. The masses of the tryptic peptides were measured with a Bruker Reflex III mass spectrometer. Matrixassisted laser desorption/ionization (MALDI) was performed
with α-cyano-4-hydroxycinnamic acid as the matrix. Trypsin
autolysis products were used for internal calibration. Accuracy
in peptide masses was achieved with better than 50 ppm on average. Comparison of the mass values against the SWISS-PROT
database was performed by Profound.
Creatine kinase activity of tissues and cells Tissues were
chopped and homogenized with PBS (pH 7.4) containing
aprotinin (1 µg/ml), leupeptin (1 µg/ml), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (1 µg/ml), and were
centrifuged at 40,000 rpm for 1 h at 4°C. The creatine kinase
activities of supernatants were measured using CK kit according
to the manufacturer’s instructions (Diagnostica Merck) (Alterio
et al., 1990).
Results and Discussion
Identification of ROS-sensitive molecules in cervical
cancer tissues A few proteins are identified as a ROS
target protein. Those proteins contain cystein thiolate anion (Cys-S-) form in active center. The cystein thiolate
414
ROS-Target Molecules
A
A
B
B
Fig. 1. Detection of ROS target molecules. A. Human cervical
cancer and normal tissues were homogenated with lysis buffer
containing BIAM. The labeling reaction was terminated by the
addition of β-mercaptoethanol to a final concentration of 50 mM.
The reaction mixture was centrifuged 40,000 rpm for 1 h at 4°C.
The supernatant was subjected to 2-dimensional gel electrophoresis, and the separated proteins were transferred to a nitrocellulose membrane. Proteins labeled with BIAM were detected with
HRP-conjugated streptavidin and ECL. Detection of ROS target
molecules in normal tissue (left, inset) and cervical cancer tissue
(right, inset). B. Coomasie staining of normal (left) and cervical
cancer tissue (right) extracts of 2-D electrophoresis.
anion is more readily oxidized by ROS than is the cysteine sulfhydryl group (Cys-SH). Moreover, sulfhydryl
modifying agent, biotin-conjugated iodoacetamide
(BIAM) is also more reactive with cystein thiolate ion
than sulfhydryl cystein residues (Kim et al., 2000). However, the oxidized cystein residues (sulfenic or sulfinic
form) by ROS do not react with BIAM. Therefore, BIAM
labeling amount of cysteine residues might be decreased
in cells after exposure to ROS. The extent of BIAM labeling on proteins can be readily measured by SDS-PAGE
and blot analysis of the biotinylated proteins with HRPconjugated streptavidin and ECL. We investigated
whether differential BIAM labeling of protein in between
normal and cervical cancer tissues.
Figure 1A showed that BIAM labeling of one spot (approximately 42 kDa, and PI point is 5.3) is markedly decreased. The protein spot from 2-D gel electrophoresis are
subjected into trypsin digestion and tryptic peptides are
analyzed by MALDI-TOF mass spectroscopy (Jensen et
al., 1996). Mass spectrum is analyzed by using of database of peptide map from University of Rockefeller. Nine
peptides are matched with creatine kinase B (Fig. 2 and
Fig. 2. MALDI-TOF mass analysis of ROS target molecule. A.
The mass spectrum was produced using the peptide supernatant
obtained after ‘in -gel’ digestion of the excised band with trypsin
as described under “Experimental Procedure”. Database search
for the measured tryptic peptide masses uniquely identified as
creatine kinase. The peaks labelled with an arrow matched the
calculated tryptic peptide masses from creatine kinase within 50
ppm. B. The sequences of peptides derived from MALDI-TOF
mass analysis are contained within a creatine kinase B sequence.
Table 1). Result from 2-D electrophoresis is also similar
to physicochemical parameter of creatine kinase B. Recently, Kim et al. (2000) demonstrated that creatine
kinase B identified as a ROS-sensitive molecule and
Cys283 of the enzyme is the major site of modification by
BIAM at pH 6.5 as well as the site of oxidation by H2O2
in PC12 cells (Kim et al., 2000).
Effect of oxidation on creatine kinase B activity Several lines of evidence indicate that ROS can oxidize active
cysteine residue in proteins to regulate their activity (Kim
et al., 2000; Rhee et al., 2000). To investigate the effect
of oxidation on creatine kinase B activity, the activity
assay of total cytosolic fraction from cancer and normal
tissues was performed, respectively. Although normal and
cancer tissues contain similar amount of creatine kinase B,
the enzyme activity from cytosolic fraction of normal
tissue is higher than that of cancer tissues (Fig. 3). HeLa
cells contain high creatine kinase B activity and expression level. Creatine kinase B isozymes were known to
contain a single active sulfhydryl group (Cys-283 in cytosolic CK) (Furter et al., 1993). The cysteine residue is
essential for catalytic activity. The result suggests that the
regulation of creatine kinase B by ROS might be involved
in energy metabolism in cancer cells.
Next we explored whether the oxidatively inactivated
creatine kinase B by ROS is restored by the addition of
reducing agent, DTT. Reduction of crude extracts from
Hyun Choi et al.
415
Table 1. MALDI-TOF mass spectrometry identification of the peptide.
Mass (m/z)
(theoretical)
Mass (m/z)
(observed)
Difference
(theoretical-observed)
Amino acid residues
1030.547
1231.608
1302.718
1585.830
1656.825
1863.964
1963.923
2120.024
1030.547
1231.604
1302.722
1585.873
1656.904
1864.024
1963.949
2120.050
0.000
0.004
-0.005
-0.043
-0.079
-0.059
-0.027
-0.026
LLIEMEQR366
DLFDPIIEDR96
33
VLTPELYAELR43
157
LAVEALSSLDGDLAGR172
224
TFLVWVNEEDHLR236
342
LGFSEVELVQMVVDGVK358
321
GTGGVDTAAVGGVFDVSNADR341
320
RGTGGVDTAAVGGVGGVFDVSNADR341
359
87
A
B
Fig. 4. Effect of DTT on creatine kinase B activity in cervical
tissues. Tissue extracts were preincubated with 500 µM DTT for
10 min on ice, and measured kinase activity according to the
manufacturer’s instructions (Diagnostica Merck).
Fig. 3. Creatine kinase activity in extract of normal and tumor
tissues, respectively. A. Tissues were homogenized with phosphate-buffered saline (pH 7.4) containing aprotinin (1 µg/ml),
leupeptin (1 µg/ml), 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride (1 µg/ml) and then were centrifuged at 40,000
rpm for 1 h at 4°C. The creatine kinase activity of supernatants
was measured using CK kit according to the manufacturer’s
instructions (Diagnostica Merck). B. Immunoblot analysis of
cell extracts with antibody against creatine kinase B or actin.
Tissue extracts were subjected to SDS-PAGE on 10% gel, transferred to a nitrocellulose membrane, and incubated with antibody to creatine kinase B or actin.
cervical cancer tissues with DTT did not affect the
creatine kinase B activity (Fig. 4). It is likely that the failure of activation by DTT suggests the irreversible oxidation of creatine kinase B by ROS in tumor cells. Cys sulfhydryl groups (-SH) oxidized to sulfenic (-SOH) or disulfide (-S-S-) can be reduced back to sulfhydryl groups by
the reduction of DTT. Further oxidation of sulfenic acid to
sulfinic (-SOOH) and sulfonic acid (-SOOOH) by ROS
such as hydrogen peroxide has been well characterized
(Davis et al., 1981). Due to the irreversible nature of such
oxidation, proteins underwent that kind of severe oxidation become inactivated and their activity cannot be recovered even by the treatment of strong reductant such as
DTT.
Creatine kinase B is expressed in a wide range of tissues, such as brain, cervix, kidney, and prostate. Several
lines of evidence indicate that the enzyme might be involved in the metabolic events that take place in oncogenic activation (Lillie et al., 1993). The highest CK levels were found in mesotheliomas, brain tumors, and small
cell lung cancer cells, whereas the lowest levels are observed in kidney cancer, lymphomas, and non-small cell
lung cancer cells. The tumors that show low CK activity
were not able to form colonies in soft agar. This result
suggested that the enzyme might be required for tumor
establishment.
It is known that the infection of papilloma virus to normal cervical cells is a major risk factor for cellular trans-
416
ROS-Target Molecules
formation. It has been reported that E6 protein from papilloma virus promotes the degradation of p53 tumor suppressor protein resulting in increased creatine kinase B
expression (Tamir et al, 1998). The pathway suggested
that the enzyme is an important factor for the metabolic
process after oncogenic process. In our results, creatine
kinase B expression in tumor tissues comparing to normal
tissues is very similar or lower rather than overexpression.
It is well known that growth of cancer cells is very rapid
compared to that of normal cells, because cancerous cells
produce various growth factors and angiogenesis-related
factors. These factors stimulated the generation of ROS in
cells, resulting in the production of large amounts of ROS
in several human cancer cell lines (Shackelford et al.,
2000). Furthermore, activity of antioxidants such as catalase and superoxide dismutase in cancer cells is very low
compared to normal cells (Mates et al., 1999). Although
many efforts for the understanding of ROS function have
been done, precise role of ROS in cancer cells is still unclear. Our results suggest that intracellular generated ROS
in tumor cells inhibit creatine kinase activity resulting in
the regulation of energy metabolism. However, molecular
mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular
transformation remain to be elucidated.
Acknowledgments We thank Drs. Chae HZ and Ryu SH for
valuable discussions. This work is supported by Center for Excellence Grant 2001G0202 (to Y.S.B) and the Basic Research
Program (1999-1-207-005-3 to Y.S.B) of Korea Science and
Engineering Foundation. C H. is a scholorship recipient of
BK21 program.
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