CEP-1347 (KT7515), a Semisynthetic Inhibitor of the Mixed Lineage Kinase Family*

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 IC50values 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)(14)(15)(16)(17)(18)(19)(20)(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 re-lated (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.
Compound-CEP-1347 was solubilized in Me 2 SO at a concentration of 4 mM and stored at Ϫ20°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).
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 HAtagged 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 MgCl 2 , 2 mM dithiothreitol, 0.1 mM sodium vanadate). Reactions were incubated in kinase buffer containing 1 M ATP, 5 Ci of [␥-32 P]ATP, and 1 g/ 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% SDSpolyacrylamide gels. Quantitation of results was performed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA).
c-Jun Luciferase Reporter Assay-A c-Jun luciferase reporter system called PathDetect TM (Stratagene, La Jolla, CA) was transfected into 150-mm dishes of Cos-7 cells at 40 -50% confluency with Lipo-fectAMINE (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 ϫ 10 5 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% Me 2 SO (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 NH 2 -terminal GST fusion partner in insect cells. The region was chosen based on the in vitro kinase activity of the fusion protein, with the NH 2 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-l assay mixture contained 20 mM Hepes, pH 7.2, 5 mM EGTA, 15 mM MgCl 2 , 1 mM dithiothreitol, 25 mM ␤-glycerophosphate, K m of ATP (60 M for MLK1 or 100 M for MLK2 and MLK3), 0.25 Ci of [␥-32 P]ATP, 0.1% bovine serum albumin, 2% Me 2 SO, and 500 g/ml myelin basic protein. Reaction was initiated by adding purified recombinant kinases (50,150, and 100 ng of GST-MLK1 KD, GST-MLK2 KD/LZ , and GST-MLK3 KD , 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.
The kinase inhibitory activity of CEP-1347 was evaluated at various concentrations (0.001-10 M) in order to determine IC 50 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 IC 50 Values-CHO cells were plated at 6.9 ϫ 10 5 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 ϫ 10 4 cells/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 l/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/MgCl 2 buffer (1 M diethanolamine, pH 9.6, 1 mM MgCl 2 ) 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 Me 2 SO control treatment. IC 50 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 24well dishes and transfected with either pCMV-EGFP (CLONTECH, Palo Alto, CA) alone or with pCDNA3.0-MLK3. For each well, 0.5 g of DNA and 2 l 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% Me 2 SO 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 Phar-Mingen. 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 [ 3 H]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 controver- by upstream regulators of JNK1 activity Cos-7 cells were grown to 80% confluency and transfected with HA-JNK1 alone or with various constructs at a 1:1 ratio as indicated in the left column. Two days after the transfection, cells were incubated with carrier (0.01% Me 2 SO) or 500 nM CEP-1347 for 2 h followed by lysis in Triton buffer. Lysates were normalized to protein and immunoprecipitated with the HA antibody. The immunoprecipitates were assayed for kinase activity using c-Jun as substrate. Experiments were performed at least two times, and results from a representative experiment are shown. All values were derived from duplicate samples where the range was less than 10%. Activations of JNK1 by the constructs noted in the left column were expressed as fold increase relative to Me 2 SO-treated vector/HA-JNK-transfected cells. The effect of CEP-1347 on JNK1 activation is given as the percentage of inhibition relative to total activity. sial (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, GSTtagged 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 M for myelin basic protein and 58 -111 M for ATP (Table II). Evaluation of CEP-1347 indicated potent inhibition of the three MLKs in vitro with IC 50 values of 38, 51, and 23 nM for MLK1, MLK2, and MLK3, respectively (Table II). These values were in the same range as the IC 50 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  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 Ϯ range of duplicate samples.

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 (NH 2 -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% Me 2 SO 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 Ϯ range of duplicate samples. kinetics for CEP-1347 versus ATP and yielded a calculated K i value of 17 Ϯ 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, ϳ7-10% 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 UVinduced 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 irradia-  tion (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. DISCUSSION 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 IC 50 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 MLKoverexpressing 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 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 Me 2 SO or 500 nM CEP-1347 for the indicated times followed by lysis. Equal amounts of total protein (20 g) 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. activation, JNK activation, and cell death. However, MLK activation may affect additional molecular targets and signaling pathways. Multiple proteins, such as ␤-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 IB kinases, leading to up-regulation of NFB activity (53,(71)(72)(73).
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.