From Cephalon Inc.,
West Chester, Pennsylvania 19380 and the ¶ Department of
Pathology and Center for Neurobiology and Behavior, College of
Physicians and Surgeons, Columbia University,
New York, New York 10032
Received for publication, December 22, 2000, and in revised form, March 16, 2001
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ABSTRACT |
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CEP-1347 (KT7515) promotes neuronal survival at
dosages that inhibit activation of the c-Jun amino-terminal kinases
(JNKs) in primary embryonic cultures and differentiated PC12 cells
after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of
JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347
could modulate JNK1 activation. CEP-1347 blocked JNK1 activation
induced by members of the mixed lineage kinase (MLK) family (MLK3,
MLK2, MLK1, dual leucine zipper kinase, and leucine zipper
kinase). The response was selective because CEP-1347 did not inhibit
JNK1 activation in cells induced by kinases independent of the MLK
cascade. CEP-1347 inhibition of recombinant MLK members in
vitro was competitive with ATP, resulting in IC50
values ranging from 23 to 51 nM, comparable to inhibitory
potencies observed in intact cells. In addition, overexpression of MLK3
led to death in Chinese hamster ovary cells, and CEP-1347 blocked this
death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.
The stress-activated protein kinases are members of the
mitogen-activated protein kinase
(MAPK)1 superfamily and
include the c-Jun amino-terminal kinases (JNKs) and p38 kinases. These
kinases are key regulators involved in cellular responses to
environmental stress, cytokines, and initiators of cell death (1, 2).
In particular, the JNKs and a substrate of the JNKs, c-Jun, have been
implicated in the progression of neuronal cell death (3). In
differentiated PC12 cells and primary sympathetic, hippocampal, and
cerebellar granule neurons, activation of c-Jun leads to cell death
(4-7). Conversely, a dominant negative mutant of c-Jun can block death
induced by trophic factor withdrawal or potassium depolarization. In
addition, death of hippocampal neurons induced by kainic acid is
prevented in mice lacking the jnk3 gene or in mice
expressing mutations in c-Jun at its JNK phosphorylation sites (8, 9).
Agents that can block the JNK signaling cascade may have therapeutic
impact in preventing neuronal cell death that has been observed in a
variety of neurodegenerative diseases, including Parkinson's,
Huntington's, and Alzheimer's (10-12).
The JNKs are activated by the dual specificity MAP kinases (MAPKKs)
MEK4/MKK4 and MEK7/MKK7 (13-21). Upstream of the MAPKKs, there
are multiple MAPKK kinase (MAPKKK) families that regulate JNK activity,
including the mixed lineage kinase (MLK) and MEKK family, as well as
Tpl-2, a member of the Raf family (2). Overexpression of constitutively
active MEKK1 or MEKK5/ASK1 causes death of sympathetic neurons, whereas
inhibition of JNK activation by dominant negative variants can block
cell death caused by trophic factor withdrawal (22, 23). Many of the
MAPKKKs are regulated by small GTPases via a Cdc42/Rac-interactive
binding motif. Similar to overexpression of the MEKKs, expression of
constitutively active Cdc42 leads to death of sympathetic neurons and
of PC12 cells (24). Therefore, kinases and small GTPases located
upstream of JNK/c-Jun may be participants in neuronal cell death processes.
CEP-1347 (KT7515), a derivative of the natural product K-252a found in
broths of Narcodiopsis bacterium,
demonstrates neuroprotective properties (25). CEP-1347 promotes
survival of a variety of primary neurons in cell culture, including
dorsal root, sympathetic and motor neurons, and PC12 cells after either
trophic factor withdrawal, DNA damage, or oxidative stress (26-28).
Moreover, CEP-1347 effectively maintains survival of motor neurons in
several in vivo models of programmed cell death, such as
developmental cell death of postnatal rat motor neurons of the spinal
nucleus of the bulbocavernosis or of chick lumbar motor neurons
in ovo and of adult rat hypoglossal neurons subjected to
axotomy (29). Furthermore, neurons are spared in the presence of
CEP-1347 after mice are exposed to toxins such as ibotenic acid or
1-methyl-4-phenyl tetrahydropyridine (30, 31).
Investigating the mechanism of action of CEP-1347, it was first
discovered that concentrations of CEP-1347 needed to promote survival
after trophic withdrawal of differentiated PC12 cells, primary
embryonic motor neurons, and sympathetic neurons or after treating mice
with 1-methyl-4-phenyl tetrahydropyridine were the same as those needed
to inhibit JNK activation, suggesting that these two activities were
related (27, 28, 32). In an effort to elucidate molecular targets of
CEP-1347 in the JNK signaling cascade, we demonstrated that
CEP-1347 inhibits the MLKs in vitro and in intact cells at
concentrations that block MLK3-induced cell death.
Reagents--
Dulbecco's modified Eagle's medium
(catalog no. 11995-040) was purchased from Life Technologies,
Inc. HA antibody was purchased from Babco (Richmond, CA). MKK4
monoclonal antibody (catalog no. 65731a) was purchased from BD
PharMingen (Bedford, MA). Phospho-MKK4 polyclonal antibody (catalog no.
9151) was purchased from New England Biolabs (Beverly, MA). Goat
anti-rabbit alkaline phosphatase (catalog no. 4050-04) and goat
anti-mouse IgG (catalog no. 1070-01) were purchased from Southern
Biotech (Birmingham, AL). FluoroNunc Maxisorp (catalog no. 437796)
96-well plates were purchased from Nunc (Naperville, IL), and 96-well
Multiscreen-DP plates were obtained from Millipore (Bedford, MA).
Superblock (catalog no. 37545) was purchased from Pierce. AP-1
(c-Jun) substrate was purchased from Promega (Madison, WI),
and the NH2 terminus of mouse c-Jun (residues 1-79)
was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein and purified by standard
glutathione affinity chromatography (33). Myelin basic protein
was obtained from Upstate Biotechnology (Lake Placid, NY).
[ Compound--
CEP-1347 was solubilized in
Me2SO at a concentration of 4 mM and
stored at Cell Culture--
Green monkey kidney Cos-7 (ATCC catalog no.
CRL 1651) and Chinese hamster ovary (CHO-K1; ATCC catalog no. CCL-61)
cells were obtained from American Type Culture Collection (Manassas,
VA). The SH-SY5Y human neuroblastoma cell line was kindly provided by
Dr. June Beidler (Memorial Sloan-Kettering Cancer Center, Rye, NY).
Cos-7, CHO, and SH-SY5Y cells were grown at 37 °C in a humidified atmosphere of 10% CO2:90% air in complete Dulbecco's
modified Eagle's medium containing 2 mM glutamine, 1 mM pyruvate, 50 units/ml penicillin/streptomycin, and
either 10% bovine serum (Cos-7 cells) or 10% fetal bovine serum
(SH-SY5Y and CHO cells). All cell lines were detached for passaging by
adding 0.25% trypsin. Spodoptera frugiperda cells
(Sf21) (34) were grown in suspension as previously described
(35). Expressions were performed with Sf21 cells grown in
suspension in serum-free Ex-Cell 420 medium (JRH Biosciences, Lenexa, KS).
cDNA Constructs in Mammalian Expression Vectors--
The
cDNAs for MKK4b and MKK7 Cos-7 Cell Transfection--
Cos-7 cells were plated to 80%
confluency in 60-mm dishes and transfected with 2 µg of test cDNA
and 2 µg of hemagglutinin (HA)-tagged JNK1 constructs using
LipofectAMINE as recommended by the provider (Life Technologies, Inc.).
Two days after transfection, the cells were treated with either
0.0125% Me2SO or 500 nM CEP-1347 for 2 h,
rinsed with ice-cold phosphate buffered saline followed by lysis in 0.4 ml of Triton buffer (1% Triton X-100, 50 mM sodium chloride, 10 mM Tris (pH 7.6), 0.1% bovine serum albumin,
30 µM sodium pyrophosphate, 50 mM sodium
fluoride, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 1 mM sodium vanadate).
Immunoprecipitation and Kinase Assay--
Immunoprecipitation
and kinase assays were performed as previously described (44). Lysates
from Cos-7 cells were normalized to protein concentration. The
HA-tagged JNK1 was immunoprecipitated with the HA-antibody.
Immunoprecipitates were rinsed three times with Triton buffer followed
by a final wash in kinase buffer (20 mM Hepes, pH 7.4, 20 mM MgCl2, 2 mM dithiothreitol, 0.1 mM sodium vanadate). Reactions were incubated in kinase
buffer containing 1 µM ATP, 5 µCi of
[ c-Jun Luciferase Reporter Assay--
A c-Jun luciferase reporter
system called PathDetectTM (Stratagene, La Jolla, CA) was
transfected into 150-mm dishes of Cos-7 cells at 40-50% confluency
with LipofectAMINE (Life Technologies, Inc.), as recommended by the
manufacturer, along with other cDNA constructs as indicated. The
composition of the transfected cDNA consisted of 45% pFA-Luc, 5%
pFA-Jun, and 50% pcDNA3 vector containing either MLK3 or pFC-MEKK1
(Stratagene). After 24 h, the cells were detached with trypsin and
seeded to a 24-well plate at 2 × 105 cells/well.
After the cells were attached, they were washed with serum-free medium
containing 0.5% fetal bovine serum and treated with either 0.01%
Me2SO (control) or 500 nM CEP-1347 in the same medium overnight. At the end of the incubation, the cells were lysed in
buffer provided from the Promega luciferase kit, and luciferase
activity was measured as recommended by the manufacturer. Luciferase
activity was normalized to protein concentration as measured by the
Bradford assay (Bio-Rad). Luciferase activity is expressed as pg of
luciferase by comparison to a standard curve constructed with firefly
luciferase (Sigma, catalog no. L1759).
Recombinant Protein--
Portions of the MLK family members were
expressed with an NH2-terminal GST fusion partner in insect
cells. The region was chosen based on the in vitro kinase
activity of the fusion protein, with the NH2 terminus
beginning with the last 5 amino acids of the Src homology 3 domain
through the end of either the kinase domain (MLK1 and MLK3) or the dual
leucine zipper (MLK2). Recombinant viruses were prepared using the
Bac-to-Bac (Life Technologies, Inc.) baculovirus system following the
manufacturer's protocol. Expressed GST fusion proteins were purified
using glutathione affinity chromatography (33).
In Vitro Kinase Assays--
Kinase assays were performed using
the Millipore Multiscreen trichloroacetic acid "in-plate" format as
described for protein kinase C (45). Each 50-µl assay mixture
contained 20 mM Hepes, pH 7.2, 5 mM EGTA, 15 mM MgCl2, 1 mM dithiothreitol, 25 mM
The kinase inhibitory activity of CEP-1347 was evaluated at various
concentrations (0.001-10 µM) in order to determine
IC50 values. Data were analyzed using the sigmoidal
dose-response (variable slope) equation in GraphPad (San Diego,
CA) Prism. Double reciprocal plots of CEP-1347 versus
ATP were generated in the MLK1 reaction under assay conditions
described above. Kinetic data were fitted to equations for competitive,
noncompetitive, or uncompetitive inhibition using the BASIC versions of
the computer programs of Cleland (46).
MLK Cell-based Assay for Determining IC50
Values--
CHO cells were plated at 6.9 × 105
cells/100-mm dish. After 24 h, the cells were transfected using
LipofectAMINE Plus (Life Technologies, Inc., catalog no. 10964-013),
with 25% MLK-1, 5% MLK-2, or 4% MLK-3, where the remainder of total
cDNA was composed of dominant negative (dn) MKK4 cDNA
substrate. The ratio of MLKs to dnMKK4 was predetermined to be in the
linear range with respect to MLK expression and phosphorylation of the
dnMKK4 substrate. Four h after the transfection, complete growth medium
was added. On the following day, cells were replated at 2 × 104cells/well in 96-well plates. The next day cells were
treated with CEP-1347 for 1 h, washed in cold PBS, and lysed in
Triton buffer on ice for 10 min. Lysates were then assayed for
phospho-MKK4 activity in a phospho-MKK4 enzyme-linked immunosorbent assay.
Phospho-MKK4 Enzyme-linked Immunosorbent
Assay--
FluoroNunc Maxisorp 96-well plates were precoated with 100 µl/well goat anti-mouse IgG at 330 ng/well in 0.1 M
sodium bicarbonate. After a 1-h incubation at 37 °C, the wells were
washed five times with TTBS (0.05% Tween in Tris-buffered saline).
Each well was coated with MKK4 monoclonal capture antibody at 100 ng/well in Superblock for 1 h at 37 °C. Wells were washed again
five times with TTBS and blocked with Superblock for another 1 h,
followed by repeated washing with TTBS. Lysate samples, normalized to
protein, were diluted 50% in Superblock, added to the wells, and then
incubated overnight at 4 °C. Further washing as described above was
followed by incubation with the phospho-MKK4 antibody at a 1:500
dilution in Superblock for 1 h at 37 °C. After repeated washing
(five times), wells were treated with goat anti-rabbit alkaline
phosphatase at 1:2000 in 2% bovine serum albumin/TBS for 1 h at
37 °C. This incubation was followed by three washes with TTBS and
three washes with TBS. 4-Methyl-umbelliferyl phosphate substrate (0.02 mg/ml) was dispensed in diethanolamine/MgCl2 buffer (1 M diethanolamine, pH 9.6, 1 mM
MgCl2) and allowed to incubate for 20 min at 37 °C. Fluorescence was read on the CytoFluor (PerSeptive Biosystems, Foster
City, CA) (excitation filter, 360 nm; emission filter, 460 nm). Each
data point represents the average of triplicate samples. Phospho-MKK4
in treated wells was expressed as a percentage of Me2SO
control treatment. IC50 values were determined using GraphPad Prism software, with fixed upper and lower limits of 100 and
0%, respectively.
Cell Death Assay--
CHO cells were plated to 80% confluency
in 24-well dishes and transfected with either pCMV-EGFP
(CLONTECH, Palo Alto, CA) alone or with
pCDNA3.0-MLK3. For each well, 0.5 µg of DNA and 2 µl of
Lipofectin (Life Technologies, Inc.) were used. CEP-1347 was added at
the indicated concentrations immediately following the transfection.
Cells were washed, and the medium was changed to Dulbecco's modified
Eagle's medium containing 0.5% fetal bovine serum 9 h after the
transfection in the presence of CEP-1347. Two days later, cells were
fixed with 3.7% formaldehyde and stained with Hoechst dye to visualize
nuclei. Within an experiment, the nuclei of at least 100 transfected
cells (as judged by EGFP fluorescence) were scored for apoptotic
appearance (condensed and fragmented chromatin) per well in duplicate
for each condition. Data given are the percentage of apoptotic
nuclei relative to total number of cells for each condition after
subtracting background cell death due to the transfection alone.
MLK3 Mobility Shifts--
SH-SY5Y cells were preincubated with
either 0.1% Me2SO or 500 nM CEP-1347 for
1 h prior to UV irradiation for 45 s in a UV2400 Stratolinker
(Stratagene). The cells were then returned to the incubator for another
3 h followed by lysis in Triton buffer. Lysate from Cos-7 cells
overexpressing MLK3 or from SH-SY5Y cells treated with UV irradiation
were run on a Nupage 3-8% Tris acetate gel, transferred to Millipore
polyvinylidene difluoride membrane and probed with an MLK3 antibody
(Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. 536) as
previously described (28). For the phospho-JNK immunoblots, lysates
were run on a 10% gel and the membranes were probed with a phospho-JNK
antibody (New England Biolabs catalog no. 9251). Blots were
stripped by washing twice for 30 min at 70 °C in buffer containing
2% SDS, 62.5 mM Tris (pH 7.5), and 100 mM
2-mercaptoethanol. The blots were reprobed with JNK antibody from
PharMingen. Secondary antibodies conjugated to alkaline phosphatase
or horseradish peroxidase were used at a 1:20,000 dilution. Blots were
analyzed using ECL reagent or enhanced chemifluorescence reagent
as described by the manufacturer (Amersham, Piscataway, NJ). The
enhanced chemiflorescense blots were quantified on a Molecular Dynamics PhosphorImager.
Effect of CEP-1347 on the Activation of JNK1 in Cos-7 Cells
Overexpressing Upstream Regulators of JNK1 Activity--
Many proteins
leading to JNK activation have been delineated in the JNK cascade
through overexpression studies in Cos-7 cells, because these cells are
highly amenable to efficient transfection. To define the molecular
target(s) of CEP-1347 in the JNK signaling cascade, co-transfection
experiments were performed with HA-JNK1 and upstream regulators of the
JNK signaling cascade in the presence or absence of CEP-1347.
Initially, to define broadly the level at which CEP-1347 acts, Cos-7
cells were co-transfected with HA-JNK1 and activated forms of Cdc42 or
Rac (47), to determine whether the site of inhibition was
upstream or downstream of these small GTPases. At 48 h after
transfection, JNK1 activation was 58- and 24-fold higher in Cos-7 cells
co-transfected with HA-JNK1 and activated Rac or Cdc42, respectively,
compared with its activation in Cos-7 cells transfected with HA-JNK1
alone (Table I). Addition of CEP-1347
(500 nM) inhibited JNK1 activation induced by activated Rac
and Cdc42 by 86 and 95%, respectively (Table I). These data indicated
that potential targets of CEP-1347 in the JNK signaling cascade were
either the small GTPases directly or proteins downstream of the small
GTPases and upstream of HA-JNK1. No specific binding of
[3H]CEP-1347 to these small GTPases was detected (data
not shown), indicating that the targets of CEP-1347 were downstream of
the GTPases Rac and Cdc42.
Below the GTPases, there are three tiers of kinases that link the
GTPases to JNK activation that are generally referred to as MAPKKK
kinases, MAPKKKs, and MAPKKs (Fig. 1). To
date, there are two MAPKKK kinase families (GCK and PAK) three MAPKKK
families (MEKK, MLK, and Raf), and one MAPKK family (MKK) that
lead to JNK activation (48). At least one member from each family was examined in the overexpression system to determine whether HA-JNK1 activation was sensitive to CEP-1347. The results are summarized in
Table I. No significant inhibitory activity by CEP-1347 was observed
when select members of the MEKK, raf, or MKK family were overexpressed.
ASK1 was not considered to be a target for CEP-1347 because inhibition
was not consistently observed over a range of ASK1 concentrations at
which the level of JNK activation was directly dependent on the levels
of ASK1 protein (data not shown). The regulation of JNK by PAKs is
controversial (47). One member of the PAK family, PAK3, did not
activate the JNK pathway when overexpressed in Cos-7 cells (data not
shown) and was, therefore, eliminated as a possible candidate. In
contrast, CEP-1347 inhibited to varying degrees the JNK activation
induced by overexpressing all five reported members of the MLK family
(MLK1, MLK2, MLK3, DLK, and LZK) and members of the GCK family (GCK,
kinase homologous to STE20, and NIK) (Table I). These data
delineated the potential molecular target(s) of CEP-1347 to either the
GCK and/or MLK family directly or undefined species downstream of these
kinases, with the exclusion of MKK4/MKK7, and upstream of JNK.
A Dominant Negative Mutant of MLK3 Blocks JNK Activation Induced By
GCK--
It has been previously reported that HPK1, a member of the
GCK family, activates JNK via both MEKK1 and MLK3 (49, 50). To
determine whether other members of the GCK family could also activate
JNK via MLK3, JNK1 activity was assessed in cells overexpressing GCK,
HA-JNK1, and a dn mutant of MLK3. As reported for HPK1, co-transfection of dnMLK3 and GCK cDNAs at a ratio of 2:1 completely blocked
GCK-induced JNK1 activation (Fig. 2).
Altogether, these data indicated that MLK3 is downstream of at least
two members in the GCK family leading to JNK activation. Therefore, the
inhibitory effect of CEP-1347 on JNK activation induced by both GCK and
the MLKs could potentially be accounted for via direct inhibition of
the MLKs.
CEP-1347 Does Not Directly Inhibit JNK in CHO
Cells--
The overexpression cell-based approach indicated that
CEP-1347 inhibited upstream activators of JNKs but did not exclude the possibility of direct interactions with JNKs because CEP-1347 is a
reversible inhibitor that likely could wash out during the immunoprecipitation process. Therefore, to address whether CEP-1347 could directly inhibit endogenous JNK in cells, a c-Jun luciferase reporter construct driven by c-Jun phosphorylation was co-transfected with MEKK1 or MLK3. Luciferase activity driven by either MLK3 or MEKK1
was 15- and 7-fold above vector control, respectively (Fig.
3). Because it was previously established
that CEP-1347 could block MLK3 activation of JNK1 (Table I) it was not
unexpected that treatment with 500 nM CEP-1347 inhibited
MLK3 driven c-Jun luciferase activity. However, CEP-1347 had no effect
on the luciferase activity driven by MEKK1 (Fig. 3). These data
corroborate the previous results in Table I, demonstrating that
CEP-1347 does not inhibit the MEKK1 to JNK pathway, and further
indicates that CEP-1347 does not directly inhibit the endogenous JNK
expressed in CHO cells.
CEP-1347 Directly Inhibits the Kinase Activities of the MLK Family
Members--
To determine whether CEP-1347 could directly inhibit the
kinase activities of the MLK family, GST-tagged truncated kinase-active forms of three MLK family members (MLK1, MLK2, and MLK3) were expressed
in and purified from lysate of insect cells infected with baculovirus
constructs expressing these proteins. Kinase assays were established
using myelin basic protein as a substrate via a radioactive Multiscreen
format. As a negative control, purified baculoviral GST was used in
place of the GST-tagged MLK; no formation of phosphorylated myelin
basic protein was detected (data not shown). The observed Michaelis
constants for the substrates in the reactions catalyzed by the
baculovirus-expressed MLKs ranged from 2-14 µM for
myelin basic protein and 58-111 µM for ATP (Table II). Evaluation of CEP-1347 indicated
potent inhibition of the three MLKs in vitro with
IC50 values of 38, 51, and 23 nM for MLK1,
MLK2, and MLK3, respectively (Table II). These values were in the same
range as the IC50 values obtained using cells co-expressing full-length MLK family members and a substrate, dnMKK4 (Table II).
In order to understand the mechanism by which CEP-1347
inhibits members of the MLK family, the mode of inhibition of MLK1 with
respect to ATP was investigated. Double reciprocal inhibition plots
(Fig. 4) were consistent with competitive
type kinetics for CEP-1347 versus ATP and yielded a
calculated Ki value of 17 ± 2 nM.
Effect of CEP-1347 on Cell Death Induced by Overexpression of
MLK3--
It has recently been reported that microinjection or
overexpression of MLK2 can lead to cell death in fibroblasts and a rat hippocampal cell line, respectively, where the cells display
morphological changes consistent with apoptosis (51, 52). To determine
whether CEP-1347 could block MLK-induced cell death, cDNA plasmids
containing a green fluorescent probe alone or with MLK3 were
transfected into CHO cells in the presence of various concentrations of
CEP-1347. At 48 h following transfection, ~7-10% of cells
expressing MLK3 displayed condensed chromatin as determined by
staining with Hoechst dye (Fig.
5A). CEP-1347 had no effect on
death in cells transfected with vector alone. In contrast, CEP-1347
suppressed MLK3-driven apoptotic death at concentrations at which it
inhibits MLK3 kinase activity (Fig. 5).
CEP-1347 Blocks Endogenous Activation of MLK3 in SH-SY5Y
Cells--
It has recently been reported that activated MLK3 has a
reduced electrophoretic mobility compared with the nonactivated protein (53). This slower migrating form of MLK3 presumably reflects autophosphorylation (54-56). In order to determine whether CEP-1347 could modulate endogenous MLK3 activity, the effect of CEP-1347 on
electrophoretic mobility of MLK3 was examined in CHO cells overexpressing MLK3. As expected from results identifying MLK3 as a
target of CEP-1347 (Table II), a faster migrating MLK3 protein was
observed from lysates of MLK3-overexpressing cells treated with 500 nM CEP-1347 as compared with sister cultures not treated with the compound (Fig. 6A).
To determine whether CEP-1347 could modulate endogenous MLK3 activated
by a cellular stress, lysates from SH-SY5Y human neuroblastoma cells
previously exposed to UV irradiation in the absence or presence of
CEP-1347 were compared. Although we previously reported that
differentiated PC12 cells are protected from UV-induced death in the
presence of CEP-1347 (28), SH-SY5Y cells were chosen for these studies
because the MLK3 antibody readily recognizes human but not rodent MLK3.
As in PC12 cells and sympathetic ganglion neurons, CEP-1347 partially rescued SH-SY5Y cells from cell death induced by UV irradiation (data
not shown). Migration of MLK3 detected from SH-SY5Y lysates of
UV-irradiated cells was retarded on gels compared with control and
CEP-1347 prevented this upward mobility shift (Fig. 6A). In addition, CEP-1347 also blocked the UV-induced downstream
phosphorylation of JNK (Fig. 6B). These data demonstrate
that CEP-1347 can modulate endogenous MLK3 protein, as determined by
mobility analysis.
CEP-1347 is a potent inhibitor of neuronal cell death induced by a
variety of stress stimuli both in vitro and in
vivo. In search of the biochemical mechanism by which CEP-1347 is
neuroprotective, we first discovered that within the MAPK signaling
cascades, CEP-1347 was a potent and selective inhibitor of the JNK but
not the p38 or the extracellular signal-regulated kinase signaling
cascades in primary embryonic motor neurons subjected to trophic
withdrawal (27). The correlation between inhibition of the JNK
signaling cascade and neuroprotection by CEP-1347 was extended to
differentiated PC12 cells exposed to a variety of cellular stresses,
including trophic factor withdrawal, oxidative stress, and DNA damage
(28). Furthermore, CEP-1347 blocked phosphorylation of MKK4 and JNK at
doses that prevented 1-methyl-4-phenyl tetrahydropyridine-induced toxicity in mice (32). In this report, it is demonstrated that a
cellular target of CEP-1347 within the JNK signaling cascade is the MLK
family (Tables I and II).
The five known members of the MLK family (MLK1, MLK2/MST,
MLK3/PTK1/SPRK, DLK/ZPK/MUK, and LZK) are so named because they share
sequence homology to both tyrosine and serine/threonine kinases,
although functionally they are serine/threonine kinases (39-43,
57-61). CEP-1347 inhibits the kinase activities of MLK family members
in vitro with similar potency in living cells (Table II). Downstream of MLK activation, several reports suggest that the MLK
family prefer MKK7 as a substrate (38, 66, 67). However, activation of
MKK4 has also been shown (38, 66, 67). In the overexpression system
utilized to obtain cellular IC50 values, all of the MLKs
reported here could utilize dnMKK4 as a substrate (Table II). CEP-1347
provides another example in a growing list of ATP competitive kinase
inhibitors (62-65) that display the same relative inhibitory potency
in vitro and in intact cells, reflecting the cellular
accessibility of these molecules.
CEP-1347 displayed minimal inhibitory activity against multiple
alternate kinases, beyond the MLK family, in the JNK signaling pathway
when these kinases were overexpressed in Cos-7 cells. However, CEP-1347
did block JNK activation driven by overexpression of members of the GCK
family, indicating that CEP-1347 may also directly inhibit members of
the GCK family. Alternatively, inhibition may be indirect via the MLKs
because GCK-induced JNK activation was blocked by overexpression of
dnMLK3, suggesting that MLKs are functionally downstream of the GCK
family (Fig. 2). In addition, CEP-1347 did not directly inhibit GCK
activity from immunoprecipitates of CHO cells overexpressing GCK
protein (data not shown). However, these latter results are complicated
by potential contaminating kinase activity coprecipitated with GCK and
insensitive to CEP-1347 that could mask detection of GCK inhibition and
does not address the effect of the compound on the other family
members. Further experimentation is necessary to define the status of
GCK and the GCK family as targets for CEP-1347. Nevertheless, the MLK
family represents the most downstream JNK pathway component inhibited by CEP-1347 in cells and is an important target responsible for the
regulation of JNK activation by the compound.
The conserved structural elements within the MLK family
provide insight into the regulation of MLK activity. All of the MLK family members contain a dual leucine zipper motif, a basic protein domain, and a carboxyl-terminal proline/serine-rich domain. However, MLK1, MLK2, and MLK3 are distinct in that they also contain Src homology-3 and Cdc42/Rac-interactive binding domains. It has recently been demonstrated that a monomeric form of MLK3 can bind via the Cdc42/Rac-interactive binding domain to the small GTPase, Cdc42, and
has increased autophosphorylating kinase activity yet fails to activate
the JNK pathway (68). Indeed, dimerization via the leucine zipper
domain along with association to the small GTPases represents at least
one means by which MLKs are regulated so that the downstream signal can
be transmitted to JNK (54-56). Activation of MLK3 leads to a change in
the mobility of the protein on gels, presumably reflecting
autophosphorylation (53). CEP-1347 blocks the UV-induced activation of
JNK in human neuroblastoma SH-SY5Y cells concomitant with preventing
the UV-induced change in electrophoretic mobility of MLK3 (Fig. 6).
These data demonstrate that CEP-1347 can affect endogenous MLK3, as
indicated by this measure of activation.
Although the mechanisms of MLK regulation are becoming
clearer, comparatively little is known regarding the biological end point of endogenous MLK activation. Based upon the protective activity
observed with CEP-1347 under various stresses and the discovery of the
MLKs as molecular targets of CEP-1347, our data suggested that the MLK
family could play a critical role in certain cell death models. Indeed,
overexpression of MLK3 in CHO cells leads to cell death, as measured by
chromatin condensation (Fig. 5). In addition, we failed to establish
stable MLK-overexpressing clones in a CHO or Cos-7 background, further
suggesting that long term activation of the MLKs is incompatible with
cell survival. This is consistent with reduced colony formation in
NIH3T3 cells overexpressing the human homologue of DLK called ZPK (74).
It has also been recently reported that either microinjection or transfection of MLK2 into a fibroblastic or rat hippocampal cell line,
respectively, results in apoptotic cell death (51, 52). In contrast,
stable lines of MLK3 transformed NIH3T3 cells have been reported (75).
It is unclear why overexpression of MLK3 in NIH3T3 cells leads to
transformation rather than cell death as observed in other cell lines,
but perhaps this may indicate that the level of MLK3 activity and the
cellular environment may alter the consequences of its activation.
Our results demonstrate clear correlations between MLK activation, JNK
activation, and cell death. However, MLK activation may affect
additional molecular targets and signaling pathways. Multiple proteins,
such as In summary, we find that the MLK family represents molecular targets of
CEP-1347 in the JNK pathway. Concentrations of CEP-1347 necessary for
inhibition of MLK activities are consistent with concentrations
necessary for inhibition of JNK activity and neuronal protection
previously reported (27, 28). The inhibitory activity of CEP-1347
against the MLK family provides an opportunity to further explore the
role of the MLK family in neuronal cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (6000 Ci/mmol) was purchased from Amersham
Pharmacia Biotech.
20 °C in amber glass vials (25). Further dilutions were
made in appropriate solutions containing 0.05% bovine serum albumin
(fraction V, protease-free, Roche Molecular Biochemicals).
1 were polymerase chain
reaction-amplified from human placenta and brain, respectively,
sequence-verified, and inserted into pcDNA3.1. The cDNA
encoding a dominant negative (K113A) mutation of MKK4b was generated by
polymerase chain reaction-mediated site-directed mutagenesis (36). A
truncated cDNA of mouse MEKK1 (encoding amino acids 817-1493 of
full-length MEKK1) and cDNAs encoding full-length human c-Jun
NH2-terminal kinase (JNK1
1), as well as constitutively
activated Rac 1 (QL) and Cdc42 (QL), kindly provided by J. Silvio Gutkind (National Institutes of Health, Bethesda, MD), were
subcloned into the pcDNA3 vector (Invitrogen, San Diego, CA). The
following constructs were generously provided as indicated: human
ASK1/MEKK5 from Hidenori Ichijo (37) (Tokyo Medical and Dental
University, Tokyo, Japan), and mouse MKK7
and dual leucine zipper
kinase (DLK) from Lawrence Holzman (38, 39) (University of Michigan
Medical School, Ann Arbor, MI). Experiments involving JNK activation
were performed with human MLK2 and partial MLK1 from Donna Dorow
(40, 41) (Peter MacCallum Cancer Center, Melbourne, Australia); leucine
zipper kinase (LZK) from Toshisuke Kawasaki (42) (Kyoto University,
Kyoto, Japan); and kinase homologous to STE20, GCK,
NIK, PAK3, tpl-2, and MLK3 from Jonathan Chernoff (Fox Chase Cancer
Center, Philadelphia, PA). Subsequent studies with full-length human
MLKs were performed with the following constructs: MLK3, kindly
provided by Richard Spritz (43) (University of Wisconsin, Madison, WI),
and MLK1 and MLK2 cloned from a human brain cDNA library. The
cDNA encoding the kinase-inactive K144R mutation of MLK3 was
generated by polymerase chain reaction-mediated site-directed
mutagenesis (36). MLK2 and MLK3 were subcloned into pcDNA-3.1,
whereas MLK1 was expressed with an NH2-terminal
His6-XpressTM epitope fusion partner in the
vector pcDNA4/HisMax (Invitrogen).
-32P]ATP, and 1 µg/sample of AP-1 substrate for 15 min at 30 °C. The kinase reaction was stopped by addition of sample
buffer. Samples were heated to 80 °C for 5 min, and proteins were
separated by electrophoresis on 10% SDS-polyacrylamide gels.
Quantitation of results was performed on a Molecular Dynamics
PhosphorImager (Sunnyvale, CA).
-glycerophosphate, Km of ATP (60 µM for MLK1 or 100 µM for MLK2 and MLK3), 0.25 µCi of [
-32P]ATP, 0.1% bovine serum albumin,
2% Me2SO, and 500 µg/ml myelin basic protein. Reaction
was initiated by adding purified recombinant kinases (50, 150, and 100 ng of GST-MLK1KD, GST-MLK2KD/LZ, and GST-MLK3KD, respectively). Samples were incubated for 15 min at 37 °C. The reaction was stopped by adding ice-cold 50%
trichloroacetic acid, and the proteins were allowed to precipitate for
30 min at 4 °C. The plates were then washed with ice-cold 25%
trichloroacetic acid. Supermix scintillation mixture was added, and the
plates were allowed to equilibrate for 1-2 h prior to counting using the Wallac MicroBeta 1450 PLUS scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of the effect of CEP-1347 on HA-JNK1 activation induced by
upstream regulators of JNK1 activity
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Fig. 1.
JNK signaling cascade. Various members
from each of the kinase families (noted in boldface)
upstream of JNK and downstream of the small GTPases (Rac and Cdc42)
were co-expressed in Cos-7 cells with HA-JNK1 and assayed for JNK
activity in the absence or presence of CEP-1347. KHS, kinase
homologous to STE20; Tpl-2, tumor progression locus 2.
View larger version (13K):
[in a new window]
Fig. 2.
Effect of dominant negative MLK3 on JNK1
activation induced by the GCK family. Cos-7 cells were grown to
80% confluency and transfected with HA-tagged JNK1 and GCK along with
vector or dnMLK3 at a 1:1:2 ratio of cDNA. Two days after
transfection, the cells were lysed and assayed for HA-JNK1 activity as
described under "Experimental Procedures." Activity is expressed as
fold change relative to vector control, which was set at 1. Data are
average ± range of duplicate samples.
View larger version (14K):
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Fig. 3.
c-Jun luciferase reporter activity driven by
MLK3 or MEKK1 in the absence or presence of CEP-1347. Cos-7 cells
were transfected with the Jun-Luc reporter constructs and a construct
coding for either MLK3, constitutively active (NH2-terminal
deleted) MEKK1, or vector control as described under "Experimental
Procedures." One day after transfection the cells were treated with
500 nM CEP-1347 or 0.01% Me2SO carrier
control. Two days after transfection, the cells were lysed and assayed
for luciferase activity. The specific activity of luciferase,
normalized to protein concentration, is reported as pg/mg protein. The
data shown are typical of three independent experiments. Data are
average ± range of duplicate samples.
Inhibitory activity of CEP-1347 towards the MLK family in living cells
and in in vitro kinase assays
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Fig. 4.
Kinetics of inhibition of MLK1 by
CEP-1347. The activity of GST-MLK1KD was determined by
varying the concentration of ATP (18-200 µM) at
different fixed concentrations of CEP-1347 (0 ( ), 15 (
), 30 (
), 45 (
), and 60 (
) nM). The plot shown here is
representative of three separate determinations.
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Fig. 5.
Overexpression of MLK3 in CHO cells leads to
cell death. CHO cells were transfected with either
pCMV-EGFP alone or together with pCDNA3.0-MLK3 as described under
"Experimental Procedures." A, cells were incubated with
various amounts of CEP-1347 following transfection. Two days later,
cells were fixed with 3.7% formaldehyde and stained with Hoechst dye
to visualize nuclei. The nuclei of at least 100 transfected cells per
well in duplicate (as judged by EGFP fluorescence) were assessed for
apoptotic appearance (condensed and fragmented chromatin). Data
presented are the percent of apoptotic nuclei relative to total number
of cells for each condition after subtracting background cell death due
to the transfection alone (1.6% of total cells in vector control). The
experiment was performed three times and results from a representative
experiment are shown. B, morphology of CHO cells transfected
with pCMV-EGFP alone (a, b, and c) or together
with pcDNA3.0-MLK3 (d-h) 48 h after transfection
in the absence (a-f) or presence (g and
h) of 200 nM CEP-1347. Cells were visualized for
EGFP fluorescence (a, d, and g), Hoechst dye
(b, e, and h), and phase contrast (c
and f).
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Fig. 6.
CEP-1347 prevents retarded mobility of
activated MLK-3. A, CHO cells overexpressing MLK-3
protein or UV-irradiated SH-SY5Y cells were incubated in the presence
of carrier Me2SO or 500 nM CEP-1347 for the
indicated times followed by lysis. Equal amounts of total protein (20 µg) for each sample were run on a polyacrylamide gel. The gel was
transferred to a polyvinylidene difluoride membrane and probed with an
MLK3 antibody as described under "Experimental Procedures."
B, total lysate from UV-irradiated SH-SY5Y cells were
collected as in A, and immunoblots were probed with a
phospho-JNK antibody (top) and developed with ECF
reagents. The phospho-JNK blot was stripped and reprobed with a JNK
antibody (bottom) and developed with ECL reagents.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin, prohibitin, dynamin, and members of the KIP3
family, have been found to be associated with MLKs, but the nature of
these associations remains unknown (51, 69, 70). Similar to MEKK1 and
NIK, MLK-3 has been implicated in T cell receptor-mediated responses
via phosphorylation of the I
B kinases, leading to
up-regulation of NF
B activity (53, 71-73).
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ACKNOWLEDGEMENTS |
---|
We thank Jonathan Chernoff, Fox Chase Institute, for reagents and helpful discussions. We also acknowledge Beverly Holskin, Chrysanthe Spais, Joan Nuss Abrams, and Frank Maslanka for contributing to the recombinant MLK effort; Kevin Walton for providing molecular reagents; and Nicola T. Neff for helpful discussions.
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FOOTNOTES |
---|
* 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: Cephalon Inc., 145 Brandywine Pkwy., West Chester, PA 19380. Tel.: 610-738-6227; Fax: 610-344-0065; E-mail: amaroney@cephalon.com.
These authors were supported by the National Institutes of
Health, NINDS, and by the Blanchette Rockefeller Foundation.
Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.M011601200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; DLK, dual leucine zipper kinase; dn, dominant negative; EGFP, enhanced green fluorescent protein; GCK, germinal center kinase; GST, glutathione S-transferase; HA, hemagglutinin; JNK, c-Jun amino-terminal kinase; LZK, leucine zipper kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MEK, MAPK/extracellular signal-regulated kinase; MEKK, MEK kinase; MKK, mitogen-activated kinase kinase; MLK, mixed lineage kinase; NIK, Nck-interacting kinase; PAK, p21-activated protein kinase.
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