Negative Regulation of Mixed Lineage Kinase 3 by Protein Kinase B/AKT Leads to Cell Survival*

Manoj K. BarthwalDagger , Pradeep SathyanarayanaDagger , Chanakya N. KunduDagger , Basabi RanaDagger , Anamika PradeepDagger , Chandan SharmaDagger , James R. Woodgett§, and Ajay RanaDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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/PKBalpha . 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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, gamma -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-alpha (TNF-alpha )-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.

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.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

PI3K-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.

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-AKT pathway.


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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 PI3K-AKT 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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Fig. 2.   Endogenous and recombinant MLK3 interact with AKT and mediate JNK inactivation. A, HepG2 cells were treated sequentially as indicated with insulin (100 nM) for 30 min and with LY294002 (50 µM) and wortmannin (100 nM) for 2 h. The endogenous AKT was immunoprecipitated (IP) and immunoblotted with affinity-purified MLK3 antibody. Cell lysates were also immunoblotted with anti-AKT and anti-MLK3 antibody. B, different GST-MLK3 constructs, as indicated, were co-transfected with wild type AKT. Cell lysates were subjected to GST pull-down assay, and immunoblotting was performed by anti-HA antibody to detect the AKT interaction. Lane 1 is mock IP. C, GST-MLK3(W/T), GST-MLK3-(511-847), and HA-AKT(W/T) were transfected in HEK 293 cells and then subsequently treated with insulin alone (100 nM for 30 min) or in combination with LY294002 (50 µM, 2 h). Cell lysates were subjected to GST pull-down assay and immunoblotted with anti-HA antibody. Cell lysates were also immunoblotted with antibodies against HA and GST. D, GST-MLK3(W/T) and HA-AKT(W/T) were transfected in HEK 293 cells and sequentially treated with LY294002 (50 µM, 2 h) and insulin (100 nM, 30min) as indicated. Total JNK activity was measured after immunoprecipitating endogenous JNK, and kinase assay was performed using GST-c-Jun as the substrate. Lanes 1 and 7 are controls without any treatment and substrate alone. E, GST-MLK3 was pulled down on glutathione-Sepharose beads from cell extracts as used in D, and kinase assay was performed using MKK7 (Ala) as the substrate. Lanes 1 and 6 are controls without any treatment and substrate alone. Cell lysates were also immunoblotted with anti-HA, anti-GST, and anti-JNK antibodies to monitor the expression of recombinant and endogenous proteins. The graphs above the blots are the average ± S.E. of three similar experiments. The blots shown here are representative of three independent experiments with comparable results.

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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Fig. 3.   MLK3-induced cell death is prevented by AKT through protein phosphorylation. A, a putative AKT consensus phosphorylation motif is present in MLK3. Several known AKT substrate sequences are also shown. B, different GST-MLK3 proteins expressed in bacterial or mammalian systems were isolated, and in vitro kinase assays were performed with recombinant GST-AKT protein in presence of [gamma -32P]ATP. Immunoblotting with anti-GST antibody was done to check the expression of various constructs used in the assay. First, four lanes are substrate alone (MLK3 proteins), and the subsequent four are with AKT. Data represent one of three similar experiments. C, HEK293 cells were transfected either with GST-MLK3 (Lys-Ala) or GST-MLK3 (Lys-Ala) S674A along with myristoylated M2-AKT. Post-transfection, cells were labeled with [32P]orthophosphate, and the expressed GST-MLK3 constructs were pulled down using glutathione-Sepharose beads and subjected to SDS-PAGE. Phosphorylation of MLK3 was detected by autoradiography. Simultaneously, anti-GST and anti-M2 immunoblot (IB) analyses were carried out to monitor the expression of MLK3 and AKT, respectively. Data represent one of two similar experiments. D, HEK 293 cells were transfected with MLK3 S674A alone or along with AKT as indicated and treated with insulin and LY294002, and the MLK3 kinase activity was measured as described in Fig. 2. The recombinant MLK3 S674A and AKT were blotted for their expression as indicated. E, cell extracts as described in D were immunoprecipitated for endogenous JNK, and kinase assay was performed as described in Fig. 2. The recombinant MLK3, AKT, and endogenous JNK were blotted as indicated. D and E, the graphs above the blots are the average ± S.E. of three similar experiments. The blots shown are representative of three independent experiments with comparable results. F, HeLa cells were transfected with a vector (pEGFP) expressing green fluorescence protein along with the expression vectors as indicated, encoding MLK3(W/T), MLK3(W/T) S674A, and Myr-AKT. After transfection, cells were treated with vehicle or anisomycin (10 µg/ml) for 20 min as indicated and were fixed and stained with DAPI. GFP-positive cells were analyzed for the presence of apoptotic nuclei with fluorescence microscope. The graph shows the average ± S.E. of three similar experiments.

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-alpha -induced JNK activation in HEK 293 (10) and CG4 cell lines, respectively (20). Recently, we have shown that TNF-alpha also induces MLK3 activity in Jurkat T Cells (22) and that TNF-alpha -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-alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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