From the Division of Molecular Cardiology,
Cardiovascular Research Institute, The Texas A&M University System
Health Science Center, College of Medicine, Temple, Texas 76504 and the
§ Department of Experimental Therapeutics, University Health
Network and Department of Medical Biophysics, University of
Toronto, Toronto, Ontario M5G 2M9, Canada
Received for publication, November 13, 2002
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ABSTRACT |
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Mixed lineage kinase 3 (MLK3) is a
mitogen-activated protein kinase kinase kinase (MAPKKK) that activates
c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By
contrast, the activation of phosphatidylinositol 3-kinase
and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis.
Here, we report a functional interaction between MLK3 and AKT1/PKB The cellular decision to undergo either cell death or cell
survival is determined by the integration of multiple survival and
death signals. Mixed lineage kinase 3 (MLK3)1 is a member of a
growing family of mixed lineage kinases (1). Recently, it has been
shown that overexpression of MLK3 or NGF withdrawal leads to neuronal
cell death, which can be prevented by treatment with a small molecule
inhibitor of MLKs, CEP-1347 (2). Similarly, CEP-11004, an analog of
CEP-1347, has also been shown to prevent neuronal cell death upon NGF
withdrawal (3). These results indicate a significant and direct
involvement of MLKs in regulating cell death; however, the detailed
mechanism by which MLKs are regulated is still unknown.
The c-jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK)
is stimulated by proinflammatory cytokines, oxidative stress, heat
shock, UV, In this study, we demonstrate that MLK3 is a direct substrate of AKT1
and their interaction is regulated by insulin. This regulatory event
has measurable consequences for MLK3 downstream signaling including
inhibition of MKK7, JNK activities, and apoptosis by MLK3. These
findings suggest that MLK3 is a physiological target of AKT and raises
the intriguing possibility that cell death rendered by MLK3 in a
cell-specific context could be regulated through this mechanism.
Cell Culture, Transfection, and Treatments--
Human embryonic
kidney 293 (HEK 293) and human cervical carcinoma (HeLa) cells were
maintained as described previously (1). Human hepatoma HepG2 cells were
maintained in minimum essential medium supplemented with 10% fetal
bovine serum, sodium pyruvate, and non-essential amino acids.
HEK293 and HeLa cells were transfected with the appropriate expression
vectors using LipofectAMINE (Invitrogen). All of the treatments
including those of LY294002, wortmannin, and insulin (Calbiochem) were
preceded by 12-h starvation in medium containing 0.2% fetal
bovine serum and then harvested for further experiments.
Immunoblot Analysis--
For immunoblotting, the equal protein
content of cell extracts or the immunoprecipitated protein samples was
taken. The proteins were separated on denaturing SDS-PAGE and
transferred onto polyvinylidene difluoride membrane and blotted with
antibodies as indicated. Antibodies used were JNK and MKK7 (Santa Cruz
Biotechnology, Santa Cruz, CA), AKT (Cell Signaling Technology, Inc.,
Beverly, MA), anti-GST (Upstate Biotechnology, Lake Placid, NY), HA tag
(BAbCo, Richmond, CA), and anti phospho-JNK (Promega, Madison,
WI). Antibody for immunoprecipitating endogenous JNK was provided by
Dr. Joseph Avruch (Massachusetts General Hospital, Boston, MA). For
immunoprecipitating endogenous MLK3, the antibody against the
C-terminal peptide-(CRAQTKDMGAQAPWVPE) of the protein was developed in
our laboratory.
Immunoprecipitation, Kinase Assay, and Metabolic Labeling with
32P--
HEK 293 or HepG2 cells were lysed in lysis buffer
as described previously (1). The cell extracts were clarified by
centrifugation at 15,000 × g for 5 min, and protein
contents were measured using the Bradford method. Immunoprecipitation
and glutathione S-transferase (GST) pull-down assays were
performed either by specific antibodies or glutathione-Sepharose beads,
respectively. After thorough washing of the immunoprecipitates, they
were either processed for immunoblotting or kinase assay as described
previously (1). For in vitro kinase assay, both eukaryotic
and prokaryotic recombinant proteins were expressed and purified as
described previously (1). Transfected HEK 293 cells were labeled for
3 h with [32P]orthophosphate (100 µCi/ml)
(PerkinElmer Life Sciences) as described previously (9).
Site-directed Mutagenesis--
Site-directed mutagenesis of
human MLK3 was performed with a QuickChangeTM kit
(Stratagene). The MLK3 (S674A) mutant cDNA was generated with the
oligonucleotide 5'-CGCGAGCGCGGGGAGaCCCCGACAACACCCCCC-3' (mismatch with
the wild-type MLK3 template is indicated by lowercase letter).
Apoptotic Cell Death--
HeLa cells were transfected with pEGFP
along with the vectors encoding the proteins as indicated. 36 h
post-transfection, the cells were fixed with 4% paraformaldehyde and
permeabilized with phosphate-buffered saline containing 0.1% Triton
X-100 and stained with 4,6-diamidino-2-phenylindole dihydrochloride
(DAPI) (Vector Laboratories). The DAPI-stained nuclei in GFP-positive cells were analyzed for apoptotic morphology by fluorescence
microscopy. The percentage of apoptotic cells was calculated as the
number of GFP-positive cells with apoptotic nuclei divided by the total number of GFP-positive cells.
PI3K We first examined whether stress-induced JNK activation is regulated by
pretreatment with insulin. Pretreatment of HepG2 cells with insulin
significantly attenuated the effect of anisomycin on JNK activation.
The inhibitory effect of insulin was more profound on JNK2 compared
with JNK1 (Fig. 1, A and
B). The pretreatment of HepG2 cells with the PI3K
inhibitors, LY294002 and wortmannin, prevented the inhibitory effect of
insulin on JNK, indicating that the JNK pathway could be down-regulated
by PI3K.
Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction
is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a
putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7,
and MLK3 kinase activities in HepG2 cells are significantly attenuated
by insulin treatment, whereas the phosphatidylinositol 3-kinase
inhibitors LY294002 and wortmannin reversed the effect. Finally,
MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates
MLK3 on serine 674 both in vitro and in vivo.
Furthermore, the expression of activated AKT1 inhibits MLK3-mediated
cell death in a manner dependent on serine 674 phosphorylation. Thus,
these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-irradiation, and by other cellular stresses (4, 5). The
signals in stress-activated JNK pathway are transmitted through three
core modules: MAP3Ks such as members of the mixed lineage kinases or
MEKK members, a MAP2K such as SEK1/MKK4 or MKK7, and MAPK such as JNK
family members (4, 5). The activated MAP3K phosphorylates and activates
MKK7 or SEK1, which in turn phosphorylates and activates JNK. JNKs
phosphorylate several nuclear transcription factors that include ELK1,
c-Jun, and ATF2 (4, 5). In several cell types, the
activation of JNKs is directly linked to cell death (6-8). Therefore,
one mechanism of cell survival could be to block JNK pathway induction.
The activation of phosphatidylinositol 3-kinase (PI3K)
correlates with increased cell survival, and this effect is largely
mediated through the activation of a serine/threonine kinase, AKT (also known as PKB). PI3K agonists such as insulin and insulin-like growth
factor-1 (IGF-1) have been shown to inhibit anisomycin and tumor
necrosis factor-
(TNF-
)-induced JNK activation (9, 10). Earlier,
we have shown that MLK3 is a potent activator of JNK pathway and that
the activation of JNK by MLK3 is through direct phosphorylation and
activation of SEK1/MKK4 (1, 11). MLK3 has also been shown to activate
MKK7 (12), another MAPKK member in the JNK pathway.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
AKT pathway, which is induced by insulin or IGF-1,
has been reported to act as a cell survival pathway in many cell types (13, 14). It has also been shown that JNK pathway is down-regulated by
insulin or IGF-1 treatments (9, 10), which results in greater cell
survival (9). Therefore, we attempted to identify the molecule(s) in
the JNK pathway that could be regulated by PI3K
AKT signaling.
AKT pathway.
View larger version (45K):
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Fig. 1.
Insulin suppresses the JNK signaling pathway
through PI3K-AKT signaling. HepG2 cells were sequentially
treated as indicated with anisomycin (10 µg/ml) for 20 min, insulin
(100 nM) for 30 min, and LY294002 (50 µM) and
wortmannin (100 nM) for 2 h. The effect of insulin,
LY294002, and wortmannin pretreatment on anisomycin-induced JNK1
(A) and JNK2 (B) activation was measured by
immunoblotting with phospho-JNK antibody. C, MKK7 was
immunoprecipitated, and kinase assay was performed using JNK (Lys-Ala)
as the substrate. Lanes 1 and 6 are controls
without any treatment and substrate alone. D, MLK3 was
immunoprecipitated with a specific antibody raised against the
C-terminal region of MLK3, and the kinase activity was assayed using
MKK7 (Ala) protein as the substrate. Lanes 1 and
6 are controls without any treatment and substrate alone.
Cell lysates in the above experiments were also immunoblotted for JNK,
MKK7, and MLK3 protein expression. The graphs above the blots are the
average ± S.E. of four similar experiments. The blots shown here are
representative of four independent experiments with comparable
results.
MKK7 and SEK1/MKK4 are two distinct MAPKK members that can directly phosphorylate and activate downstream target JNK (5). Recently, it has been reported that SEK1 activity is inhibited by insulin, and this leads to the inhibition of JNK activity (9). We have also observed that SEK1 kinase activity is inhibited by insulin (data not shown). Because MKK7 is a specific activator of JNK pathway, unlike SEK1 in which SEK1 can activate the p38 MAPK as well as JNK pathways (5), we examined the effect of insulin on anisomycin-induced MKK7 activity. Endogenous MKK7 from HepG2 cells was also inhibited by insulin, and pretreatment with PI3K inhibitors, LY294002 and wortmannin, were able to block the effect of insulin on MKK7 kinase activity (Fig. 1C).
We have reported earlier that MLK3 activates JNK pathway through direct phosphorylation and activation of SEK1 (1, 11), and MLK3 has also been demonstrated to phosphorylate MKK7 (12). MLK3 kinase activity, estimated by using MKK7 (Ala) as substrate, was similarly inhibited by pretreatment with insulin, and the PI3K inhibitors, wortmannin and LY294002, blocked the inhibitory effect of insulin on MLK3 kinase activity as well as the MLK3 autophosphorylation (Fig. 1D).
The inhibitory effect of insulin on MKK7 and MLK3 was modest but quite consistent and was not as profound as on anisomycin-induced JNK activity, indicating that there could be other targets of insulin regulating JNK activity. Recently, it has been shown that SEK1, an immediate upstream kinase to JNK, is also down-regulated by insulin through PI3K-AKT pathways (9), whereas the JNK activator ASK1 is similarly inhibited by AKT (15). Thus, the modest but consistent inhibition of JNK upstream activator (i.e. MKK7 and MLK3) was not unexpected; rather, the potent inhibition of JNK by insulin points to the fact that the inhibitory effect of insulin on JNK is an additive effect of insulin targets within the JNK pathway.
AKT recognizes its substrate proteins at the conserved consensus
sequence of RXRXX(S/T)X (16,
17). A putative consensus AKT phosphorylation sequence (RERGESP) is
present at amino acids 669-675 of human MLK3. The putative AKT
consensus phosphorylation sequence is not present on either MLK1 or
MLK2; however, two AKT consensus phosphorylation motifs are present on
dual leucine zipper-bearing kinase (DLK). Because insulin
inhibited JNK activity and MLK3 contains a potential AKT
phosphorylation site, we postulated that the inhibitory effect of the
PI3KAKT pathway on JNK pathway is partly mediated proximally
through MLK3. To test this hypothesis, we probed for endogenous
association of MLK3 with AKT in HepG2 cells. MLK3 co-precipitated with
AKT under basal conditions, but this association was attenuated upon
insulin treatment. The treatment of cells with LY294002 and wortmannin
prevented the insulin-induced decrease in association between AKT and
MLK3 (Fig. 2A). Whether DLK
having the putative AKT phosphorylation sites associates with AKT and
is regulated by insulin in a manner similar to MLK3 is an area yet to
be investigated.
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The potential AKT phosphorylation site on MLK3 is located in the C-terminal half of the protein. To determine whether the interaction between MLK3 and AKT is mediated through this region on MLK3, the N-terminal half (amino acids 1-386) or the C-terminal half (amino acids 511-847) of MLK3 was each transfected into HEK 293 cells. Both full-length and the C-terminal portion of MLK3 were found to interact with AKT, but the N-terminal half of MLK3 failed to bind (Fig. 2B). Since we observed that insulin regulates endogenous interaction between AKT and MLK3 in HepG2 cells and that the full-length as well as C-terminal domain of MLK3 interacted with AKT in HEK293 cells, we examined the effect of insulin on the interaction between C-terminal half of MLK3 with AKT. Whereas recombinant full-length MLK3 interacted with AKT in a manner similar to the endogenous MLK3, interaction of the C-terminal truncated mutant with AKT was not regulated by insulin, suggesting that the truncation mutant lacks a domain necessary for dissociation from AKT (Fig. 2C).
We next examined the effect of PI3K-AKT pathway activation on MLK3-mediated JNK activation. In HEK 293 cells, overexpression of AKT inhibited JNK activation through MLK3, whereas insulin treatment further inhibited the JNK activation (Fig. 2D). In the same experiment, we measured the kinase activity of the MLK3 and found it to be inhibited by insulin in presence of AKT, and this effect was reversed by LY294002 (Fig. 2E).
Since a potential AKT consensus phosphorylation site in MLK3 was
present at serine 674 (Fig.
3A), we examined whether
serine 674 in MLK3 is targeted by AKT. We mutated this residue to
alanine in full-length MLK3 and also within the C-terminal regulatory tail and performed in vitro phosphorylation in the presence
of recombinant AKT. Although a low level of phosphorylation of
MLK3-(511-847) S674A was observed, the full-length kinase inactive
MLK3 and MLK3 truncation mutant (amino acids 511-847) were both
phosphorylated to higher degree by AKT (Fig. 3B). The
significant decrease in the phosphorylation of S674A mutant suggests
that serine 674 represents an important phosphorylation site. In
vivo, metabolic 32P-labeling experiments in HEK 293 cells showed that active AKT was able to phosphorylate kinase-inactive
MLK3, whereas phosphorylation of the MLK3 (K144A) S674A mutant
was very low, in agreement with the in vitro phosphorylation
data (Fig. 3C). In transfection experiments, AKT and insulin
treatment could not regulate the activity of MLK3 S674A mutant (Fig.
3D), and consequently, no alteration in the JNK activation
was observed under similar conditions (Fig. 3E), proving
that serine 674 in the MLK3 is the target of AKT.
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It has been shown that cell death caused by overexpression of MLK1, MLK2, MLK3, and DLK is rescued by overexpressing active AKT in neuronal cells (18). Because MLK1 and MLK2 do not contain putative AKT phosphorylation sites, the one possible way by which AKT can exert its cell survival effect could be through phosphorylating and inactivating SEK1, since SEK1 has been shown to be down-regulated by AKT (9). However, in the case of MLK3-mediated cell death, the effect of AKT could be the synergistic outcome through inactivating MLK3 as well as SEK1. The DLK contains two putative AKT phosphorylation sites, one at serine 551 and another at threonine 626. It is possible that DLK could be regulated in a manner similar to MLK3 by AKT, which needs to be determined.
Since MLK3 has been implicated in neuronal cell death (2), we examined whether MLK3 overexpression causes cell death in HeLa cells similar to neuronal cells and whether the cell death induced by MLK3 can be regulated by AKT. Transfection of MLK3 into HeLa cells resulted in an increase in apoptotic cell death, and this effect was inhibited by co-expressing a constitutively active AKT (Myr-AKT). The co-expression of a mutant of MLK3 (lacking the putative AKT phosphorylation site) with AKT induced apoptosis, suggesting that the AKT-mediated blockage of MLK3 induced death was dependent upon phosphorylation of serine 674 (Fig. 3F). It is possible that AKT in some cellular contexts may keep MLK3 kinase activity suppressed under normal conditions. For example, in PC-12 cells and sympathetic neurons, which die in a MLK-dependent manner upon NGF withdrawal (18), NGF-dependent survival may in part reflect AKT inhibition of MLK3. This hypothesis is further substantiated by a recent finding (19) in which cerebellar granule neurons maintained under high potassium and low serum, the condition under which PI3K-AKT pathway is active, showing profound JNK activation and rapid cell death upon LY294002 treatment. However, this JNK activation and cell death are prevented if cells are treated with CEP-1347 along with LY294002, indicating that PI3K-AKT pathway down-regulates the MLK-mediated JNK activation and cell death process.
IGF-1 and IGF-2 inhibits TNF--induced JNK activation in HEK 293 (10)
and CG4 cell lines, respectively (20). Recently, we have shown that
TNF-
also induces MLK3 activity in Jurkat T Cells (22) and that
TNF-
-induced cell death was inhibited by CEP-11004, a specific mixed
lineage kinase inhibitor in PC-12 cells (21). These observations along
with our present results suggest that insulin mediated inhibition of
TNF-
-induced JNK pathway may be partly mediated through inhibition
of MLK3 activity by AKT.
Taken together, our results identify AKT as the first reported negative
regulator of MLK3 kinase activity. Further studies may help to unravel
the mechanism leading to neuronal apoptosis as well as other cell death
pathways and also help in the development of targeted pharmacological
interventions in neurodegenerative disorders involving neuronal
apoptosis such as in idiopathic Parkinson's disease and Alzheimer's disease.
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ACKNOWLEDGEMENTS |
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We thank Drs. Joseph Avruch, R. J. Davis, and L. I. Zon for providing anti-JNK antibody, MKK7, and SEK1 cDNAs, respectively. We are also grateful to Dr. Jing Jin for help in generating PKB/AKT cDNA constructs.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM55853 (to A. R.).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: Division of Molecular Cardiology, Cardiovascular Research Institute, 1901 South 1st St., Bldg.162, Temple, TX 76504. Tel.: 254-778-4811 (ext. 1200); Fax: 254-899-6165; E-mail.arana@medicine.tamu.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211598200
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ABBREVIATIONS |
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The abbreviations used are: MLK, mixed lineage kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; SEK1, SAPK/ERK Kinase-1; MKK7, MAPK kinase 7; HA, hemagglutinin; GST, glutathione S-transferase; HEK 293 cells, human embryonic kidney 293 cells; HepG2 cells, hepatocellular carcinoma cells, NGF, nerve growth factor; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; PI3K, phosphatidylinositol 3-kinase; IGF, insulin-like growth factor; TNF, tumor necrosis factor; MAPKK, MAPK kinase kinase; GFP, green fluorescent protein; PKB, protein kinase B; DLK, dual leucine zipper-bearing kinase.
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