From the Department of Pharmacology, Fox Chase Cancer
Center, Philadelphia, Pennsylvania 19111 and the
§ Ruttenberg Cancer Center, Mount Sinai School of Medicine,
New York, New York 10029
Received for publication, February 13, 2001, and in revised form, March 8, 2001
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ABSTRACT |
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c-Jun N-terminal kinase (JNK)-mediated cell
signaling pathways are regulated endogenously in part by
protein-protein interactions with glutathione S-transferase
P1-1 (GSTP1-1) (1). Using purified recombinant proteins, combined
with fluorescence resonance energy transfer technology, we have
found that the C terminus of JNK is critical to the interaction with
GSTP1-1. The apparent Kd for full-length JNK was
188 nM and for a C-terminal fragment (residues 200-424)
217 nM. An N-terminal fragment (residues 1-206) did not bind to GSTP1-1. Increased expression of the C-terminal JNK fragment in a tetracycline-inducible transfected NIH3T3 cell line produced a
concentration-dependent increase in the kinase activity of
JNK under normal, unstressed growth conditions indicating a
dominant-negative effect. This suggests that the fragment can compete
with endogenous full-length functional JNK resulting in dissociation of
the GSTP1-1-JNK interaction and concomitant JNK enzyme
activation. By using an antibody to hemagglutinin-tagged C-JNK, a
concentration-dependent co-immunoprecipitation of GSTP1-1 was
achieved. These data provide evidence for direct interactions between
the C-terminal of JNK and GSTP1-1 and a rationale for considering
GSTP1-1 as a critical ligand-binding protein with a role in regulating
kinase pathways.
c-Jun N-Terminal kinase
(JNK),1 or stress activated
kinase, is a member of the mitogen activated stress kinase family
(MAPK), which also includes extracellular signal regulated kinase and p38-MAPK. JNK activation was initially identified as a cellular response to environmental stresses, proinflammatory cytokines, and
interleukins (2). In addition, the JNK pathway participates in
activating transcription factor 2 (3), ELK-1 (4), and the Sap-1a
transcription factor (5), and JNK may influence p53 (6, 7) and NF- The glutathione S-transferases (GST) are a multigene family
of isozymes that catalyze the nucleophilic attack of the sulfur atom of
glutathione on electrophilic groups of substrate molecules. Although
catalytic efficiencies for many of the substrates are low, their
biological relevance as enzymes has frequently been linked to
pleiotropic substrate specificity. Among this family of isozymes,
GSTP1-1 is generally the most prevalent in mammalian cells. The link
between GST overexpression, especially with respect to GSTP1-1, and
anticancer drug resistance has been extensively studied (11). Because
of the defined role of GST in drug metabolism, elevated expression of
GSTP1-1 in solid tumors or in drug-resistant cells has been associated
frequently with detoxification reactions even in instances where there
is no evidence that the selecting drug is a substrate for GSTP1-1.
More recently, however, the link between the redox active components of
GSTP1-1 and stress-activated kinases, such as JNK, has been redefined
as a non-catalytic, ligand binding activity that mediates both stress
and apoptotic responses (1, 10). In a parallel series of studies,
either GSTµ or thioredoxin has also been identified as a
ligand-binding partner for apoptosis-signaling kinase (ASK1) (12, 13),
extending the role that "redox proteins" may have in kinase
regulation. To elucidate further the mechanism by which GSTP1-1
mediates JNK signal transduction, we have used biophysical methods in
reconstituted protein systems to provide direct evidence for
protein-protein interactions and to measure binding affinities. Our
results demonstrate that GSTP1-1 has significant affinity for the C
terminus of JNK and confirm the ligand-binding regulatory role for this
ubiquitously expressed protein.
Protein Purification--
Recombinant JNK1 protein was expressed
in Escherichia coli. The polymerase chain reaction product
was constructed with a leader His-tag, digested with NdeI
and BamHI, purified, and ligated into NdeI-digested, BamHI-digested, and
phosphatase-treated pET-15b to create JNK.pET-15b. The JNK1 protein was
expressed in E. coli and purified using a standard
Ni2+ column for His-tagged proteins. The purified protein
was homogeneous as judged by the single polypeptide band of the
predicted Mr on silver-stained
SDS-polyacrylamide gels (data not shown).
Both the C-terminal and N-terminal truncated proteins expressed in
E. coli were found mainly in the insoluble fraction of the
bacterial extracts. Thus, inclusion bodies were solubilized in 50 mM CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), pH 11.0, with 0.3% N-lauroylsarcosine supplemented with 1 mM dithiothreitol and slowly diluted to 20 mM
Tris-HCl, pH 8.5, before subjection to Ni2+ column
chromatography. Renaturation was accomplished by dialyzing for 12 h at 4 °C with a change of buffer with dithiothreitol and continued
dialysis for an additional 6 h. To promote refolding, a further
period of dialysis in the above buffer (20 mM Tris, pH 8.5)
without dithiothreitol for the same length of time was enacted.
The GSTP1-1 construct was made by direct digestion of GSTP1-1 from
PCR2.1(+) with HindIII and XhoI followed by
cloning into pET21 vector previously treated with HindIII
and XhoI. This was expressed in E. coli, and
protein was purified using GSH affinity chromatography.
Circular dichroic spectra of the C-terminal and N-terminal JNK1 in 20 mM PO43+ were obtained using an
Aviv 62A DS spectrometer at the Fox Chase Cancer Center core facility.
The corresponding structural elements were calculated using an
algorithm for the estimation of the percentages of protein secondary
structure from UV circular dichroism spectra using a Kohonen neural
network with a two-dimensional output layer using K2D software (14,
15). The program uses a self-organizing neural network to extract the
secondary structure features present in the data from a set of circular
dichroism spectra ranging from 200-241 nm. The result provides an
estimate of percentage helix, Protein Labeling and Reconstitution--
GSTP1-1 was labeled at
a 1:1 molar ratio (probe:protein) with the fluorescence energy transfer
donor 7-methoxycoumarin succinyl ester using previously described
procedures (16). The JNK1 partner proteins (full-length and partial)
were similarly labeled with the non-fluorescence energy transfer
acceptor, 4(dimethylamino) phenylazophenyl-4-sulfonyl chloride succinyl
ester (Molecular Probes Inc., Eugene, OR). The protein:probe labeling
ratios were determined by absorption spectroscopy (17) using the
calculated extinction coefficients for the protein and the coefficients
provided by the probe manufacturer.
Cells and Protein Preparation--
NIH3T3 mouse fibroblast cells
that stably express the pSV40-Hyg plasmid
(CLONTECH, Palo Alto, CA) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and antibiotics (Life Technologies Inc.). Cells were grown
at 37 °C with 5% CO2. The pTet-C-JNK was assembled by
subcloning the cDNA of the C-terminal 224 amino acids of wild type
JNK1 into the tet-regulated promoter of the
pUHD-10 Antibodies, Immunoprecipitation, and
Immunoblotting--
Antibodies to C-JNK and phospho-c-Jun were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and New
England BioLabs (Beverly, MA). Immunoblot analysis was performed using
200 µg of whole cell extract (WCE) separated on SDS-polyacrylamide
gel electrophoresis followed by electrotransfer to a polyvinylidene difluoride membrane in 20 mM CHAPS (3-(3-cholamidopropyl)
dimethylammonio-1-propane-sulfonate)) buffer, pH 11, for 30 min
membrane blocking (5% non-fat milk) and incubating with the respective
antibodies. Reactions were visualized using chemiluminescence (ECL)
reagents. Immunoprecipitations were carried out following the protocol
from the manual provided by Roche Diagnostics Corporation using 1 mg of
WCE and 2 µg of the respective antibodies for 16 h at 4 °C.
Protein G beads (Roche Molecular Biochemicals) were added (15 µl) for 4 h at 4 °C. Immunoprecipitated material was
subjected to immunoblot analysis.
Protein Kinase Assays--
Protein kinase assays were carried
out using JNK kinase assay kits from New England BioLabs according the
to manufacturer's instructions. The kinase reaction was carried out in
the presence of excess unlabeled ATP. c-Jun phosphorylation was
selectively measured using a phospho-c-Jun antibody. This antibody
specifically measures JNK-induced phosphorylation of c-Jun at Ser-63,
an important site for c-Jun-dependent transcriptional activity.
Fluorescence Resonance Energy Transfer
Experiments--
Associations between GSTP1-1 and JNK were measured
by fluorescence resonance energy transfer (FRET) as described
previously (16). Protein concentrations were measured using UV
absorption (A280) and colorimetric assays.
Extinction coefficients for each protein were assessed as described
previously (18). FRET was performed on a PC1 (photon counting)
spectrofluorimeter (ISS, Inc., Champagne, IL) using a 3-mm
cuvette with a magnetic stirrer. Coumarin-labeled proteins were excited
at 350 nm and scanned from 380-480 nm. To determine the binding
affinities for protein-protein interactions, the energy transfer data
were analyzed using Sigma Plot.
Data Analysis--
All immunoblot and SDS-polyacrylamide gel
electrophoresis gels were scanned using a UMAX Powerbook III flatbed
with Adobe Photoshop software. Scanned images were quantified by NIH
image program. Prism software was used for data analysis.
Purification of JNK Proteins--
Full-length JNK1 was
soluble and purified directly by Ni2+
chromatography. Both truncated forms were insoluble in aqueous buffer and were initially solubilized in detergent followed by slow
dialysis against renaturing buffers. The renatured proteins were then
similarly purified by Ni2+ chromatography. The purity of
these proteins was confirmed by single band staining on
SDS-polyacrylamide gel electrophoresis (data not shown).
Circular dichroism of these purified proteins was used to monitor the
refolding process. Fig. 1 shows the
proportional secondary structure of the truncated fragments derived
from the circular dichroism spectra. These data were analyzed further
using a Kohonen neural network with two-dimensional output layer using
K2D software (14, 15). JNK1 shows a >95% identity to JNK3, a protein
which has been crystallized (19). The C-terminal of JNK3 is comprised primarily of FRET Analysis of GSTP1-1 Association with JNK--
In
vitro association kinetics between the purified JNK proteins and
GSTP1-1 was analyzed using (FRET) and is shown in Fig. 2. GSTP1-1 was labeled with the succinyl
ester of 7-methoxycoumarin. Full-length and truncated JNK1 was labeled
with the non-fluorescence energy transfer acceptor,
4-(dimethylamino) phenylazophenyl-4-sulfonyl chloride succinyl ester.
Saturation binding experiments were used to measure the specific
binding at equilibrium at various concentrations of the fluorescent
acceptor protein to determine acceptor affinity. Fig. 2A
shows that for the full-length JNK1 a loss in 7-methoxycoumarin fluorescence was observed as the protein interacted with GSTP1-1. A
similar result was found for the C-terminal fragment (Fig.
2B), while neither the N-terminal fragment nor buffer
conditions produced any change in fluorescence. The calculated
Kd for GSTP1-1 association with full-length JNK1
was 188 ± 38 nM and for the C-terminal fragment,
217 ± 72 nM. These data provide quantitative evidence
for the direct protein-protein interaction between GSTP1-1 and
JNK1.
Immunoprecipitation Analysis of GSTP1-1-C-JNK
Association--
These results were extended to a cellular system by
using a tet-inducible construct of HA-tagged C-JNK. To measure the
in vivo association of C-JNK and GSTP1-1,
immunoprecipitation of WCE from cells expressing HA-tagged C-JNK was
performed. Induction of C-JNK expression following removal of
tetracycline resulted in an enhanced level of GSTP1-1
co-immunoprecipitation when an HA-tag antibody was used (Fig.
3).
These data are consistent with the conclusion that C-JNK and GSTP1-1
interact in a cellular milieu. Similarly, the intracellular expression
of the C-terminal truncated form of JNK1 leads to an increase in JNK1
kinase activity as a consequence of competing for the GSTP1-1 binding
site and liberating catalytically active JNK1 (see Fig. 5).
C-JNK Blocks the Interaction between GSTP1-1 and Full-length JNK
and Increases JNK Enzymatic Activity--
For confirmation that the
C-terminal portion of JNK1 is critical to the JNK1-GSTP1-1
interaction, a tetracycline-inducible expression system was used to
examine whether the increased expression of the C-terminal truncated
JNK1 could have a dominant-negative effect upon GSTP1-1 inhibition
of JNK1 activity. Fig. 4 shows the
time-dependent increase in expression of the C-terminal
truncated JNK1 fragment following removal of tetracycline. Induction of the C-terminal fragment was detected by 1 h and persisted for at
least 48 h. In these same cells, full-length JNK1 expression was
constitutive and unaltered throughout this period (Fig. 4). JNK1
activity was measured by the phosphorylation of the downstream c-Jun
substrate (Fig. 5). The
time-dependent increase in phosphorylated c-Jun was
coincident with the enhanced expression of the truncated C-terminal
fragment. If tetracycline was not removed from the media, there was no
enhanced production of phosphorylated c-Jun product (Fig. 5). Overall,
these results support the conclusion that the C-JNK fragment through
competitive binding with GSTP1-1 can act in a dominant-negative
fashion and increase the catalytic activity of JNK1.
Glutathione S-transferases are a multigene family of
isozymes that primarily have been linked functionally with phase II
metabolism or xenobiotics. In particular, GSTP1-1 is frequently
overexpressed in solid tumors and in cells that have acquired
resistance to anticancer drugs even when the selected drug is not a GST
substrate (11). The precise reason for the high levels of expression in tumor cells has not been explained adequately. Recently, we have carried out a series of studies to show that GSTP1-1 can act as a
ligand-binding protein and an endogenous switch for the control of the
catalytic activity of JNK1 (1, 10). This has led to other studies
implicating that GSTP1-1 is in control of apoptosis (20). Maintenance
of cellular kinases that influence cell growth and apoptosis must be
finely tuned. In particular, external stimuli such as reactive oxygen
species may influence signaling kinase activities, influencing pathways
in stress response and cell survival (21). It now seems reasonable to
conclude that GSTP1-1 can elicit protection against apoptosis induced
by reactive oxygen species by controlling the balance of kinase
activity elicited by JNK1 versus other cellular kinases such
as extracellular signal regulated kinase, I JNK1 shares a high level of amino acid identity with JNK3 (Fig.
6). By extrapolation, we were able to
establish that the folding pattern of the C-terminal truncated JNK1 was
similar to the structure of JNK3 established by crystallography data
(19). Both the in vitro and cellular data confirmed that the
truncated C-terminal JNK1 contained amino acid residues that can be
implicated in the binding to GSTP1-1. A recent study (22) has used
computation of average structures to reveal that residues 194-201 of
GSTP1-1 may be involved in the interaction with JNK. This sequence
(SSPEHVNR) contains residues that are positively charged or contain
polar groups in their side chains. Sequence analysis of the C terminus of JNK1 shows both a loop region and an
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
pathways (8, 9). c-Jun, a component of the transcription factor AP-1
complex is the most characterized downstream target of JNK, where
regulation is achieved by phosphorylation at Ser-63 or Ser-73 (3).
Extracellular signals such as growth factors, transforming
oncoproteins, and UV irradiation stimulate phosphorylation of c-Jun at
ser-63/73 and activate c-Jun-dependent transcription. The
binding of JNK to the N-terminal region of c-Jun permits substrate
phosphorylation. The JNK pathway has also been shown to be important in
the control of cell survival and death pathways, and interference with
the JNK pathway suppresses the induction of apoptosis by a variety of
agents (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet, and random structure for the
protein. K2D also estimates the probable error in the estimation based
on the training procedure results.
3 vector. Cell clones that stably
express both constructs were selected in 600 µg/ml geneticin in the
presence of hygromycin (100 µg/ml) 24 h after
N,N,N-trimethyl 1-2-3-bis
(1-oxo-9-octa-decenyl)oxy-(Z,Z)-1-propanaminium methyl sulfate transfection with the
pTet-C-JNK-UHD-10
3 construct. Cells
expressing C-terminal JNK were maintained in Dulbecco's modified
Eagle's medium containing fetal bovine serum (10%), hygromycin (100 µg/ml), and geneticin (400 µg/ml). To maintain suppression of
C-terminal JNK expression, tetracycline was added to the medium every 3 days (to a final concentration of 1 µg/ml). Analysis of JNK1 activity
was achieved by measurement of phosphorylation of c-Jun as a substrate
(1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices, whereas the N-terminal is predominantly comprised of
-sheet. Such structures are in agreement with the data
shown in Table I, confirming that
accurate folding in solution has occurred.
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Fig. 1.
Circular dichroic spectra of
(A) C-terminal JNK1 and (B)
N-terminal JNK1 solubilized in 20 mM phosphate buffer (pH
7.2). Data were collected at room temperature. Spectra were fit to
secondary structural elements that are listed under "Experimental
Procedures" and analyzed to yield the secondary structures listed in
Table I.
K2D software analysis of circular dichroism results
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Fig. 2.
Binding GSTP1-1 to full-length or truncated
JNK1. The FRET assay was carried out as described under
"Experimental Procedures" at room temperature. GSTP1-1 labeled
with 7-methoxy coumarin (20 nM) titrated with either N- or
C-terminal JNK and full-length JNK labeled with dabsyl
(DAB). The reaction buffer contained 20 mM Hepes
and 150 mM NaCl. A, the normalized decrease in
the fluorescence intensity obtained by dividing the zero-point
fluorescence intensity with background subtraction of 2 nM
methoxycoumarin labeled GSTP1-1 titrated with full-length JNK1 ( ),
N-terminal-JNK1 (
), or buffer only (
). B, the
association of GSTP1-1 with C-terminal JNK1 under the same
experimental procedures as Fig. 4A; C-terminal JNK
(
).
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Fig. 3.
Co-immunoprecipitation of GSTP1-1 with
C-JNK. HA antibody was added to WCE. Protein G beads were added to
the mixture to pull down HA-tagged C-JNK followed by immunoblotting
with GSTP1-1 polyclonal antibodies. Endogenous GSTP1-1 expression
levels stayed the same throughout the 48 h induction after removal
of tetracycline. The histogram quantified (mean ± S.D.
of three experiments) that the GST association with C-JNK increased as
more C-JNK was expressed.
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Fig. 4.
A), induction of protein expression of
C-terminal JNK in NIH3T3 cells by removal of tetracycline. HA-tagged
C-JNK (residues 200-424) was used to establish NIH3T3 cells that are
tet-regulatable. NIH3T3 cells transfected with
ptet-C-JNK-UHD-10 3 were harvested at
different time points for protein isolation after tetracycline removal.
WCE were analyzed on 12% polyacrylamide gels. Immunoblot analysis
revealed the expression of a 30-kDa protein detected with
polyclonal antibodies to C-JNK1. B, expression of
full-length JNK1 under the same conditions. The full-length JNK protein
was used to monitor equal loading for each lane. Immunoblots
were scanned and quantified by NIH image software. The final results
were plotted as histograms and are mean ± S.D. of
three experiments.
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Fig. 5.
JNK kinase activity in the presence of
truncated C-terminal JNK expression. Cells were harvested at the
indicated time points (hours) after removal of tetracycline.
c-Jun fusion protein beads were added to the WCE. Equal amounts of
total cell lysate (~200 µg of protein) were electrophoresed on 12%
polyacrylamide gels. Immunoblot detection was performed using the ECL
system with rabbit monoclonal antibodies to phosphorylated c-Jun
(top panel) for the detection of JNK kinase catalytic
activity or c-Jun (bottom panel). Absorption was quantified
and plotted in arbitrary units in the presence ( ) or absence (
)
of tetracycline.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B kinase, and p38 (1,
10). Our earlier experiments showing interactions between GSTP1-1 and
JNK1 relied primarily on immunoprecipitation technology. In this
report, we combine a number of technical approaches to provide evidence
for direct protein-protein interaction between GSTP1-1 and JNK1. These
data also establish quantitative binding kinetics for these two
proteins with Kd values in the nanomolar range. The
specificity of the C terminus of JNK1 was confirmed by a number of
controls that included the N-terminal and other unlabeled proteins, all of which failed to bind. The affinity of the truncated C-JNK fragment for GSTP1-1 was similar to the full-length protein.
-helix rich in negatively charged residues, particularly glutamic and aspartic acid (Fig. 6).
These residues are either in the flexible loop structure
(Asp-377, Asp-381, Glu-382, Glu-384) or the
-helix (Glu-388,
Glu-389, Glu-392, Glu-397, Asp-400, Glu-402, Glu-403) of the protein
and can form a negatively charged binding face capable of interacting
with GSTP1-1. This, in turn, is on the surface of JNK1 and therefore in a location conducive to protein-protein interaction with GSTP1-1. Fig. 6 shows the sequence alignments for the C-terminal components of
JNK family members (19). The potential interaction site implicated in
the molecular dynamic analysis (22) is distal to the GST subunit
dimerization domain (involving Cys-47 and Cys-101) (23) suggesting that
JNK may interact in vivo with homodimeric GSTP1-1. In
addition, because the catalytic kinase domain is localized in the
N-terminal of JNK1, our results suggest that the capacity of GSTP1-1
to suppress JNK enzyme activity will be through an allosteric
inhibition mechanism.
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Fig. 6.
Multiple sequence alignment of
JNK proteins using the program MAC showing conserved sequence
homology among JNK proteins. A cluster of acidic residues (marked
in light gray) are possible sites interacting with the basic
residues of GSTP1-1 identified by Monaco et al. (22).
This information may be facile in the design of potential small
molecule inhibitors of the ligand-binding properties of these two
proteins. Targeting this area on JNK1 may impact kinase expression and,
by extrapolation, influence apoptosis (10). We have previously shown
that a peptidomimetic inhibitor of GSTP1-1 (24) causes a dissociation
of the GSTP1-1-JNK1 complex resulting in increased JNK catalytic
activity (1). This drug has significant effects on proliferative
pathways in bone marrow
cells.2 It is plausible that
pharmacological manipulation with such agents could achieve
control of the coordinated regulation of stress kinases with a
significant impact in the therapy of cancer.
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ACKNOWLEDGEMENTS |
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We thank Zhiming Yin for providing the partial JNK constructs, Ma Jinguo for the NIH3T3 tet-off cell system, and Dr. Suzanne Scarlata for use of ISIS instrumentation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants CA06927 and RR05539, National Institutes of Health Grant CA85660 (to KDT), National Institutes of Health Grant CA77389 (to ZR), and by an appropriation from the Commonwealth of Pennsylvania.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: Dept. of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3147; Fax: 215-728-4333; E-mail: kd_tew@fccc.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M101355200
2 Ruscoe, J. E., Rosario, L. A., Wang, T., Gate, L., Arifoglu, P., Wolf, C. R., Henderson, C. J., Ronai, Z., and Tew, K. D. (2001) J. Pharmocol. Exp. Ther., in press.
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ABBREVIATIONS |
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The abbreviations used are: JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferases; ASK, apoptosis-signaling kinase; WCE, whole cell extract; FRET, fluorescence resonance energy transfer; HA, hemagglutinin; ECL, enhanced chemiluminescence.
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