Activated JNK Phosphorylates the C-terminal Domain of MLK2 That Is Required for MLK2-induced Apoptosis*

David R. PhelanDagger §, Gareth PriceDagger §, Ya Fang Liu, and Donna S. DorowDagger ||

From the Dagger  Trescowthick Research Centre, Peter MacCallum Cancer Institute, Melbourne 8006, Victoria, Australia and  Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115

Received for publication, September 8, 2000, and in revised form, December 7, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MAP kinase signaling pathways are important mediators of cellular responses to a wide variety of stimuli. Signals pass along these pathways via kinase cascades in which three protein kinases are sequentially phosphorylated and activated, initiating a range of cellular programs including cellular proliferation, immune and inflammatory responses, and apoptosis. One such cascade involves the mixed lineage kinase, MLK2, signaling through MAP kinase kinase 4 and/or MAP kinase kinase 7 to the SAPK/JNK, resulting in phosphorylation of transcription factors including the oncogene, c-jun. Recently we showed that MLK2 causes apoptosis in cultured neuronal cells and that this effect is dependent on activation of the JNK pathway (Liu, Y. F., Dorow, D. S., and Marshall, J. (2000) J. Biol. Chem. 275, 19035-19040). Furthermore, dominant-negative MLK2 blocked apoptosis induced by polyglutamine-expanded huntingtin protein, the product of the mutant Huntington's disease gene. Here we show that as well as activating the stress-signaling pathway, MLK2 is a target for phosphorylation by activated JNK. Phosphopeptide mapping of MLK2 proteins revealed that activated JNK2 phosphorylates multiple sites mainly within the noncatalytic C-terminal region of MLK2 including the C-terminal 100 amino acid peptide. In addition, MLK2 is phosphorylated in vivo within several of the same C-terminal peptides phosphorylated by JNK2 in vitro, and this phosphorylation is increased by cotransfection of JNK2 and treatment with the JNK activator, anisomycin. Cotransfection of dominant-negative JNK kinase inhibits phosphorylation of kinase-negative MLK2 by anisomycin-activated JNK. Furthermore, we show that the N-terminal region of MLK2 is sufficient to activate JNK but that removal of the C-terminal domain abrogates the apoptotic response. Taken together, these data indicate that the apoptotic activity of MLK2 is dependent on the C-terminal domain that is the main target for MLK2 phosphorylation by activated JNK.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

One mechanism by which cells respond to extracellular stimuli is the recruitment of signaling networks that regulate changes in the internal environment and control gene transcription (reviewed in Refs. 1 and 2). A group of signaling pathways that are highly conserved through evolution are collectively known as mitogen-activated protein kinase (MAPK)1 pathways (3, 4). The classical MAPK pathway is triggered by growth factors binding to cell surface receptors leading to the activation of the GTPase, Ras. This pathway results in activation of the extracellular signal-regulated kinases (ERKs) leading to gene transcription and cellular proliferation. A parallel MAPK pathway triggered by stress factors such as osmotic shock, UV irradiation, cytotoxic drugs, and inflammatory cytokines results in activation of the stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs) (2). A second stress-activated pathway leads to activation of p38 MAPK. Effects of stress activation vary from proliferation, differentiation, and gene transcription to cell cycle arrest and apoptosis depending on cell type and stimulus (2).

All MAPK pathways utilize a "three-kinase cascade" mechanism to modulate incoming signals. Thus, each MAPK has upstream-activating kinases (MAPKKs) that are in turn regulated by MAPKK kinases (MAPKKKs) (reviewed in Ref. 5). Some of the upstream kinases appear to be highly specific for a particular cascade, whereas others link to more than one MAPK. The MAPKKs for the ERKs are the MAP/ERK kinases (MEKs) (6-8), whereas activators for JNKs are the SAPK/ERK kinase/JNK kinase (SEK1/JNKK), also known as MAPK kinase 4 (MKK4) (9-11), and the more recently discovered MKK7 (12-14). In the case of p38, the main upstream-activating kinases are MKK3 and MKK6 (11, 15, 16), but p38 can also be activated by JNKK/MKK4 (11), providing crossover between the JNK and the p38 pathways. MAPKKKs are a larger group of kinases that have overlapping specificities for MAPKKs and are themselves activated by an array of interactions in response to extracellular and internal stimuli (reviewed in Ref. 17). As more upstream activators of the MAPKs are identified, the picture of how these three parallel pathways are regulated becomes more complex.

Recently it has become clear that MAPKs not only control downstream signals to transcription factors but also exert effects upstream on their activators. In budding yeast, the three components of the pheromone-induced MAPK cascade are Ste11 Right-arrow  Ste7 Right-arrow  Fus3/KSS (18). Errede and co-workers (19) coined the term "feedback phosphorylation" to describe upstream phosphorylation of Ste7 by Fus3. In mammalian cells, Xu and Cobb (20) report that JNK binds to and phosphorylates MEKK1, a major MAPKKK for the JNK pathway (21, 22). In addition, cotransfection of the noncatalytic domain of MEKK1 potentiates JNK activation

The mixed lineage kinases (MLKs) (23, 24) are MAPKKKs that act upstream of JNKK/MKK4 and MKK7 in the SAPK/JNK pathway (25, 26). MLK family members MLK2, MLK3, and DLK activate the SAPK/JNKs, and to a lesser extent p38 and ERKs, and MLK2 directly phosphorylates JNKK/MKK4 in vitro (26-30). Within their N-terminal regions, MLKs 1-3 contain several domains associated with protein interaction including an Src homology (SH3) domain and two leucine zipper motifs (24). In addition, each has a large C-terminal domain of unknown function that is rich in serine, threonine, and proline amino acids. Recently we reported that overexpression of active full-length MLK2 causes apoptosis in neuronal cells, and this is dependent on activation of JNK (31). Transfection of kinase-negative MLK2 inhibits neuronal cell apoptosis caused by expression of polyglutamine-expanded huntingtin protein, the gene product associated with neuronal pathology in Huntington's disease (32, 33). In addition, overexpression of the N-terminal region of normal huntingtin protein partially rescues this neuronal toxicity. A proline-rich peptide near the polyglutamine insertion site in the N-terminal region of normal huntingtin protein binds to the SH3 domain of MLK2, and polyglutamine expansion interferes with this interaction. As MLK2 is expressed at high levels in the brain, these results suggest a role for MLK2 in the neuronal pathology associated with polyglutamine expansion of huntingtin protein.

In the present report, we investigated signaling of MLK2 in the JNK pathway and found that as well as being an activator of the SAPK/JNKs, MLK2 is a substrate of activated MAPKs. JNK phosphorylation occurs at a number of specific sites mainly within the C-terminal region of the MLK2 protein, including the C-terminal 100 amino acid peptide. Furthermore, the MLK2 C-terminal domain is phosphorylated in vivo, and this phosphorylation increases after activation of JNK by anisomycin treatment. In studies of MLK2 expression in epithelial cells, wild-type MLK2 induced apoptosis, and this is dependent on both its catalytic activity and the presence of the noncatalytic C-terminal domain. Taken together, these studies demonstrate that the MLK2 C-terminal domain is a target for regulation by activated JNK and is vital for MLK2-induced apoptosis.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Dulbecco's modified Eagle's medium (DMEM), leupeptin, pepstatin, and aprotinin were purchased from ICN, Sydney, Australia. Fetal calf serum (FCS) was from the Commonwealth Serum Laboratories, Parkville, Victoria, Australia, and gentamicin was from David Bull Laboratories, Melbourne, Australia. Rabbit anti-HA antibodies were provided by Dr. D. Bowtell and monoclonal mouse anti-HA (12CA5) by Dr. R. Pearson, both from the Peter MacCallum Cancer Institute, Melbourne, Australia. Rabbit anti-JNK1 antibody (C-17) from Santa Cruz Biotechnology and biotinylated sheep anti-mouse and goat anti-rabbit immunoglobulins, streptavidin-biotin-conjugated alkaline phosphatase, and Hybond C Super nitrocellulose membrane were from Amersham Pharmacia Biotech. Alkaline phosphatase substrate reagent, 5-bromo-4-chloro-3-indolylphosphate and FuGENETM 6 transfection reagent were from Roche Molecular Biochemicals, and nitro blue tetrazolium was from Sigma. Protein A-Sepharose was from Amersham Pharmacia Biotech, and [gamma -32P]ATP (3000 Ci/mmol) and [32P]orthophosphoric acid (9000 Ci/mmol) were from PerkinElmer Life Sciences. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (248 units/mg protein) was from Worthington, and pre-coated thin layer cellulose plates were from Merck. VectashieldTM mounting medium was from Vector Laboratories (Burlingame, CA).

Plasmids-- cDNAs encoding the complete human MLK2 polypeptide (24), the N-terminal region (nucleotides 289-1777), the C-terminal region (nucleotides 1777-3454, including a stop codon beginning at nucleotide 3151), and the C-terminal 300 nucleotides (nucleotides 2851-3153) were each cloned into the vector pKH3, immediately 3' to 3 copies of an oligonucleotide encoding the hemagglutinin (HA) tag peptide. The pKH3 vector for mammalian expression from the cytomegalovirus promoter was a kind gift of Dr. Ian Macara, Department of Pathology, University of Vermont. FLAG®-tagged versions of MLK2 cDNAs were made by replacing the 3× HA tag coding sequence in pKH3 with a polymerase chain reaction product encoding the FLAG® peptide epitope. GFP-tagged MLK2 cDNAs were constructed by subcloning MLK2 cDNAs into the vector pEGFP-C1 (CLONTECH) that includes a gene for neomycin resistance. Expression vectors for human JNK1 (34), JNK2 (35), and JNKK (10) were HA-tagged full-length cDNAs in the vector pSRalpha (10). The JNK vectors and a cDNA encoding c-Jun fused to glutathione transferase (GST-c-Jun) in pGEX were kind gifts from Dr. M. Karin, University of California, San Diego.

Site-directed Mutagenesis-- The MLK2-K125A (KN-MLK2) mutant was made using the Altered Sites® II in vitro mutagenesis kit (Promega) essentially according to manufacturer's instructions, with the following modifications. Taq polymerase (8 units/30 µl) was substituted for T4 polymerase in the second strand synthesis reaction, and mutant plasmid DNA was transfected into Escherichia coli ES1301 mutS by electroporation using a Bio-Rad Gene Pulser. The mutagenesis template for KN-MLK2, a BamHI fragment containing the first 1488 base pairs of the coding region of human MLK2 cDNA (24), was subcloned into the pALTER-1 vector (Promega). The oligonucleotide used to create the MLK2-K125A mutation was 5'-146AGGAGGTGGCAGTCGCGGCCGCCCGGCTGG176 (altered bases underlined) based on the MLK2 nucleotide sequence. The entire nucleotide sequence of the mutated insert was confirmed by automated nucleotide sequence analysis on an ABI Prism model 373 DNA Sequencer (Applied Biosystems) before re-inserting into HA-pKH3-HA-MLK2 vector.

Cell Culture, Transfection, and Treatment-- COS7 cells were maintained in DMEM supplemented with 10% FCS and 20 µg/ml (20 IU) gentamicin at 37 °C in 5% CO2 in air. Cells (0.5-1 × 106) were transiently transfected with 2-5 µg plasmid DNA using FuGENETM 6 transfection reagent, electroporation, or CaPO4. For phosphopeptide mapping experiments, 4 × 106 COS7 cells were transfected by electroporation with 5 µg of HA-MLK2 pKH3, HA-KN-MLK2 pKH3, HA-C-MLK2 pKH3, and/or 3 µg of HA-JNK2 pSR3alpha and incubated in complete DMEM at 37 °C in 5% CO2 in air for 24-36 h. For anisomycin treatment, JNK2-expressing cells were washed after 12 h with serum-free DMEM and incubated in serum-free conditions for 24 h. In some cultures, JNK2 was activated by addition of 10 µg/ml anisomycin to serum-starved cells for 30 min before harvest. For stable transfectants, HEK 293 cells were electroporated with 5 µg of pEGFPC1 mutant MLK2 cDNA, and the cells were grown in the presence of 400 µg/ml G418 antibiotic. Clones of resistant cells were isolated and assayed by microscopy for expression of the GFP fusion protein and by immunoblotting with anti-GFP antibodies to confirm the size of the expressed fusion protein.

Western Blotting and Immune Complex Kinase Assays-- Cells were lysed in 1 ml of ice-cold lysis buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, and 1.5 mM MgCl2) containing proteinase inhibitors (1 µg/ml each of aprotinin, leupeptin, and pepstatin and 1 mM phenylmethylsulfonyl fluoride) and 50 mM NaPO4. Lysates were incubated on ice for 10 min and then clarified by centrifugation in a microcentrifuge at 4 °C. Lysate protein concentrations were determined using the bicinchoninic acid assay (36). For Western blotting, aliquots of cellular lysate containing 30 µg of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis and proteins transferred to nitrocellulose. For immune complex kinase assays (IP kinase assays), HA-tagged proteins were immunoprecipitated from cell lysate volumes adjusted to contain equal amounts of protein with anti-HA antibodies bound to protein A-Sepharose beads. Beads were resuspended in 30 µl of assay buffer (25 mM HEPES, pH 7.2, 100 mM NaCl, 10 mM MgCl2, 5 mM MnCl2, 100 µM NaVO4, and 10% glycerol) containing 60 µM ATP and 20 µCi of [gamma -32P]ATP. Reactions were allowed to proceed for 30 min at room temperature before proteins were separated on 10% SDS-polyacrylamide gel electrophoresis, and incorporated radioactivity was analyzed using a Molecular Dynamics PhosphorImager (Sunnyvale, CA). Assays were repeated in triplicate, and raw data were corrected for protein expression levels as determined by anti-HA immunoblotting of an aliquot of each cell lysate.

Phosphopeptide Mapping-- MLK2 tryptic phosphopeptides were separated on two-dimensional thin layer cellulose essentially by the method of Hunter and co-workers (37). Briefly, phosphorylated proteins were excised from polyacrylamide gels and the gel slices suspended in 200 µl of 20 mM NH4HCO3. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (10 µg) was added, and the digest was allowed to proceed for 18 h at 37 °C. A total of 4 × 103 cpm/digest were dried and resuspended in electrophoresis buffer, loaded onto thin layer cellulose plates, and peptides resolved in two dimensions by electrophoresis followed by ascending chromatography. The electrophoresis buffer was 2% formic acid and 7.8% acetic acid in H2O, pH 1.9, and chromatography was in 37.5% n-butyl alcohol, 25% pyridine, and 7.5% acetic acid in H2O. Labeled peptides were visualized by PhosphorImage analysis of the thin layer plates.

In Vivo Labeling-- COS7 cells were transfected with pKH3-HA-KN-MLK2 together with either pSRalpha -HA-JNK2 or empty vector and incubated for 24 h in complete DMEM. For cells transfected with JNK2, complete DMEM was replaced with serum-free DMEM, and cells were incubated for a further 18 h. The medium of all cells was then replaced with phosphate-free DMEM with or without 10% dialyzed FCS for 2 h. Cells were then incubated with [32P]orthophosphoric acid (200 µCi per ml of medium) alone or together with 10 µg/ml anisomycin for 30 min. Cells were harvested and proteins immunoprecipitated with anti HA-antibodies. Following electrophoresis, the KN-MLK2 protein was subjected to two-dimensional phosphopeptide mapping as described above.

Phosphoamino Acid Analysis-- Following tryptic digestion of phosphorylated MLK2 protein, extracted peptides were incubated with constant boiling HCl at 95 °C for 120 min. After drying, amino acids were dissolved in 7.5% acetic acid, 25% pyridine, 37.5% n-butyl alcohol in H2O, resolved on a thin layer cellulose plate at 1500 V for 2 h, and visualized by PhosphorImage analysis.

Staining of Cultured Cells for Microscopy-- Cells grown on glass coverslips were incubated for 10 min in fixing solution (4% paraformaldehyde in 100 mM Pipes, pH 6.9, 5 mM MgSO4, 10 mM EGTA, and 2 mM dithiothreitol), permeabilized in 0.1% Triton X-100 and 0.5% bovine serum albumin in phosphate-buffered saline for 5 min, and treated with RNase (400 µg/ml) for 30 min. Cells were stained with propidium iodide (1 µg/ml, 20 min) before mounting coverslips onto glass slides. Confocal images were captured using a Bio-Rad MRC-1000 Confocal Imaging System.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MLK2 Activates JNKs 1 and 2-- Within MAPK cascades, preferences for activation of particular components or downstream targets have been reported in various cell types or activation conditions. In the case of the JNK cascade, differential involvement of SAPK/JNK isoforms in growth regulation and apoptosis induction has been demonstrated in several different cell types (38, 39). To determine whether MLK2 activation of the SAPK pathway leads to preferential activation of JNK isoforms, COS7 cells were cotransfected with expression plasmids for HA-MLK2 (Fig. 1) together with either HA-JNK1 or HA-JNK2. Lysates of the transfected cells were assayed for JNK activation in IP kinase assays using GST-c-Jun as a substrate. As a control for these experiments, a kinase-negative mutant in which alanine replaces lysine at position 125 in the MLK2 kinase catalytic domain (KN-MLK2) was prepared. Lysine 125 of MLK2 corresponds to lysine 72 of cyclic AMP-dependent kinase that is directly involved in the phospho-transfer reaction (40) during catalysis. As can be seen in Fig. 1B, there is very efficient phosphorylation of c-Jun by both JNK1 and JNK2 when isolated from cells expressing wild-type MLK2 (wt-MLK2), whereas both JNKs were essentially inactive toward c-Jun when coexpressed with KN-MLK2. Although JNK1 appears to be more highly activated than JNK2 in this assay, JNK1 has a higher basal activity and thus the degree of activation by MLK2 is about 8-fold for both JNKs (Fig. 1C).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   MLK2 activates both JNKs 1 and 2. A, schematic representation of MLK2 structural domains (24) and constructs used for expression of epitope-tagged MLK2 proteins. HA, 3 copies of the HA tag peptide; SH3, Src homology 3 domain; kinase, catalytic domain; LeuZip, dual leucine zipper domain; Basic, basic motif; CRIB, cdc42/rac interactive binding motif (41); K125A, catalytic domain inactivating mutation. B, upper panels, IP kinase assays. COS7 cells were cotransfected with 5 µg of plasmid, pSRalpha -HA-JNK1 (lanes 1-3), or pSRalpha -HA-JNK2 (lanes 5-7) together with either pKH3 empty vector (lanes 1 and 5), pKH3-MLK2 (lanes 2 and 6), or pKH3-KN-MLK2 (lanes 3 and 7), or pKH3-MLK2 with pKH3 empty vector (lane 4) as indicated. After 24 h of incubation, cells were lysed and anti-HA IP kinase assays performed with GST-c-Jun added as a substrate (as described under "Experimental Procedures"). 32P incorporation was visualized by PhosphorImage analysis of the kinase assay gel. Lower panels, anti-HA immunoblots of cell lysate proteins before immunoprecipitation. C, graphical representation of 32P incorporation (PhosphorImager units) into GST-c-Jun by JNK1 or JNK2, when coexpressed with HA tag (control), HA-MLK2, or HA-KN-MLK2, as indicated. Each bar represents an average of data from three separate experiments.

JNK Phosphorylates Its Upstream Activator MLK2-- In the course of analyzing SAPK/JNK signaling, COS7 cells expressing KN-MLK2 were treated with the protein synthesis inhibitor anisomycin, a potent activator of JNK (2). In anti-HA immunoblots of recombinant MLK2 proteins from these cells, broadening of KN-MLK2 bands was evident (data not shown). This suggested that MLK2 is phosphorylated in vivo in response to anisomycin. However, due to the strong constitutive autophosphorylation activity of overexpressed wt-MLK2, this effect is only observable with the KN-MLK2 mutant. To determine whether activated JNK has ability to phosphorylate MLK2, HA-JNK2 was transfected into COS7 cells and immunoprecipitated after either serum starvation or anisomycin treatment of the cells. Immune complex kinase assays were performed with HA-KN-MLK2 that had been immunoprecipitated from separately transfected untreated COS7 cells as the added substrate (Fig. 2A). This revealed low level phosphorylation of added KN-MLK2 by JNK2 isolated from serum-starved cells, and JNK2 isolated from anisomycin-treated cells phosphorylated KN-MLK2 efficiently. Anisomycin activation of JNK in the COS7 cells was confirmed by immunoblotting cellular lysates with anti-JNK antibodies where a clear bandshift in JNK from anisomycin-treated cells was observed (Fig. 2A, lower panel).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   Kinase-negative MLK2 is phosphorylated by activated JNK2. A, COS7 cells transfected with 5 µg of pSRalpha -HA-JNK2 were incubated for 24 h and then serum-starved for 18 h before treatment with either medium alone or 10 µg/ml anisomycin in medium, for 30 min, as indicated. Anti-HA kinase assays were performed as described in the legend to Fig. 1, with HA-KN-MLK2 added as substrate. Upper panels, in vitro kinase assay; lower panels, anti-HA and anti-JNK immunoblots of expressed proteins before immunoprecipitation. B, COS7 cells were transfected with 5 µg of pKH3-FLAG-MLK2, pKH3-FLAG-KN-MLK2, and/or pSRalpha -HA-JNK2, as indicated. Cells were lysed, and HA-tagged proteins were immunoprecipitated. Upper panels, IP kinase assays performed as in A, lower panel, anti-HA immunoblot of expressed proteins. C, diagram of the phosphorylation cascade from MLK2 to JNK and the upstream phosphorylation of MLK2 by JNK. D, COS7 cells transfected with 3 µg of pSRalpha -HA-JNK2 together with 5 µg of either pSRalpha -HA-wt-JNKK or pSRalpha -HA-KN-JNKK were incubated for 24 h and then serum-starved for 18 h before treatment with either medium alone or 10 µg/ml anisomycin in medium, for 30 min, as indicated. Upper panels, IP kinase assays performed as in A, lower panel, anti-HA immunoblot of expressed proteins.

As coexpression of active MLK2 has a significant effect on JNK activity, we measured the ability of JNK2 to phosphorylate KN-MLK2 after activation by wt-MLK2. In this case, HA-JNK2 was coexpressed with FLAG-tagged versions of either wt-MLK2 or KN-MLK2 and the JNK2 selectively immunoprecipitated with anti-HA antibodies. HA-KN-MLK2 was separately transfected into COS7 cells and immunoprecipitated. HA-KN-MLK2 immunoprecipitate was then added to the HA-JNK2 for a kinase reaction. As can be seen in Fig. 2B, HA-JNK2 that had been coexpressed with active FLAG-MLK2 readily phosphorylated the added KN-MLK2. Where JNK2 was expressed either alone or coexpressed with FLAG-KN-MLK2, however, phosphorylation activity toward KN-MLK2 was reduced, and no phosphorylation of KN-MLK2 was detected in anti-HA immunoprecipitates from cells expressing FLAG-MLK2 alone (Fig. 2B, lane 3). This confirms that no active FLAG-MLK2 was present in the anti-HA immunoprecipitates and rules out the possibility that another kinase coimmunoprecipitating with KN-MLK2 may be responsible for the observed phosphorylation. Taken together, these data demonstrate that JNK2 activated either by coexpression of wt-MLK2 or the classical JNK activator, anisomycin, readily phosphorylates KN-MLK2 in vitro. The signaling pathway from MLK2 to JNK leading to JNK phosphorylation of MLK2 is depicted in the diagram in Fig. 2C. To demonstrate the involvement of the JNK cascade further, we examined the requirement for JNKK activity in the ability of activated JNK to phosphorylate KN-MLK2 in vitro (Fig. 2D). For this, HA-JNK2 was coexpressed with either wild-type or kinase-negative JNKK, and the cells were activated with anisomycin. The HA-JNK was immunoprecipitated and tested for the ability to phosphorylate added HA-KN-MLK2 immunoprecipitated from separately transfected cells. In this assay, phosphorylation of KN-MLK2 increased by 10-fold after anisomycin activation of the JNK pathway, and coexpression of kinase-negative JNKK inhibited this activation 50%.

It has been shown previously that JNK both binds to and phosphorylates another MAPKKK, MEKK1 (20). To test the possibility that JNK may bind directly to MLK2, HA-JNK2 was coexpressed with GFP-tagged wt-MLK2, and anti-GFP immunoprecipitates were assayed for the presence of HA-JNK2 by anti-HA immunoblotting. In addition, anti-GFP-MLK2 immune precipitates were tested for c-Jun phosphorylation activity in an IP kinase assay. In both cases, although GFP-wt-MLK2 was fully active, no HA-JNK2 band or phosphorylation of c-Jun was detected (data not shown). Thus, by two criteria, JNK was not detectable in the anti-GFP-MLK2 immune precipitates.

The C-terminal Region of MLK2 Is a Target for MAPK Phosphorylation-- The 496-amino acid N-terminal region of the MLK2 protein (Fig. 1A) contains structural domains associated with protein-protein interaction (24). The C-terminal 458-amino acid region contains no known structural domains but has many potential sites for proline-directed MAPK phosphorylation. To determine whether sequences within the C-terminal region are necessary for MLK2 signaling activity, HA-JNK2 was coexpressed with HA-MLK2, HA-KN-MLK2, HA-N-MLK2, or HA-C-MLK2, and activation of JNK2 was measured in IP kinase assays with GST-c-Jun as a substrate (Fig. 3A). This experiment revealed that JNK2 immunoprecipitated from cells cotransfected with either MLK2 or N-MLK2 (lanes 2 and 4) phosphorylated c-Jun efficiently, whereas JNK2 coexpressed with KN-MLK2 or C-MLK2 was essentially inactive toward c-Jun.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.   The N-terminal 495 amino acids of MLK2 are sufficient to activate JNK2. A, COS7 cells were transfected with 5 µg of pSRalpha -HA-JNK2 together with 5 µg of pKH3 empty vector, pHK3-HA-MLK2, pKH3-HA-KN-MLK2, pKH3-HA-N-MLK2, or pKH3-HA-C-MLK2, as indicated, and cells were lysed 24-36 h later. Upper panels, anti-HA IP kinase assays with added GST-c-Jun substrate. Lower panels, anti-HA immunoblots of cell lysates before immunoprecipitation.

It is also clear in this assay that there is a low level of incorporation of phosphate into both KN-MLK2 and C-MLK2 (Fig. 3A, lanes 3 and 5) when they are coexpressed and coimmunoprecipitated with JNK2. Although C-MLK2 appears to be more highly phosphorylated than KN-MLK2, the expression level of C-MLK2 is consistently higher than that of full-length MLK2, as is evident in anti-HA immunoblots of cell lysates from the assay (Fig. 3A, lower panels). In addition, it is noteworthy that the C-MLK2 band recognized by anti-HA antibodies in the immunoblot is somewhat broad, and the in vitro incorporation of radioactive phosphate is concentrated in the lower portion of the band (Fig. 3A, upper panel, lane 5). As the bandshift is associated with phosphorylation, this indicates that some phosphate is already incorporated into C-MLK2 in vivo. The C-MLK2 protein generally runs as a broad band in immunoblots, and its bandwidth is increased in lysates where JNK2 is coexpressed. The opposite situation is observed for N-MLK2, which has a narrow band in the immunoblot but shows a substantial shift after the in vitro kinase assay (Fig. 3A, lane 4) indicating that much of the phosphorylation has taken place in vitro.

To examine the effect of JNK activation on phosphorylation of HA-KN-MLK2 and HA-C-MLK2, each was expressed in COS7 cells, immunoprecipitated, and used as substrates for JNK2 immunoprecipitated from either serum-starved or anisomycin-activated cells. IP kinase assays (Fig. 4A) confirm that both KN-MLK2 and C-MLK2 are phosphorylated by JNK2 and that there is a substantial increase in their phosphorylation (KN-MLK2 -3.5-fold and C-MLK2 -5-fold) by JNK2 following anisomycin treatment (Fig. 4A, upper panel, lanes 2 and 4).



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 4.   JNK2 phosphorylates KN-MLK2 and C-MLK2. Upper panel, HA-JNK2 was transfected into 2 plates of COS7 cells, and after 24 h incubation, cells were serum-starved and incubated with or without anisomycin (as described under "Experimental Procedures"). Anti-HA-JNK2 IP kinase assays performed as in the legend to Fig. 1 with substrates HA-CMLK2 (lanes 1 and 2) or HA-KN-MLK2 (lanes 3 and 4) added as substrates. Lower panel, anti-HA immunoblots of expressed proteins in cell lysates before immunoprecipitation. SS, serum-starved cells; A, anisomycin treated cells.

It has been reported that expression of MLK2 activates both p38-MAPK and ERK (30, 42). JNK, ERK, and p38 all phosphorylate serine or threonine residues followed by proline (5, 43), and such dipeptides are prevalent in the MLK2 C-terminal domain (see Table I). To test the possibility that phosphorylation of MLK2 may be mediated by members of the ERK and p38 MAPK families, COS7 cells were transfected with HA-C-MLK2 together with HA-MLK2, HA-JNK2, HA-p38, or HA-ERK2, and phosphorylation of C-MLK2 was measured in IP kinase assays (Fig. 5A). In this assay, C-MLK2 was most efficiently phosphorylated by full-length MLK2 itself (Fig. 5A, lane 4) and by JNK2 (Fig. 5A, lane 2). Phosphorylation of C-MLK2 by ERK2 (Fig. 5B) was observed; however, this was weak in comparison to that by JNK2, whereas phosphorylation by p38 was barely detectable.


                              
View this table:
[in this window]
[in a new window]
 
Table I
MLK2 tryptic peptides containing serine or threonine



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   P38, ERK2, and wt-MLK2 phosphorylate the MLK2 C-terminal domain. COS7 cells were transfected with 5 µg of plasmid pKH3-HA-C-MLK2 together with either 5 µg of pKH3 empty vector (lane 1), pKH3-HA-MLK2, pSRalpha -HA-JNK2, or pSRalpha -HA-p38 (A) as indicated or pSRalpha -HA-JNK2 or pSRalpha -HA-ERK2 (B). Cells were lysed 24-36 h later for anti-HA IP kinase assays (upper panels) and anti-HA immunoblotting (lower panels).

MLK2 Is Phosphorylated at Multiple Sites by Autophosphorylation and by JNK2-- The catalytic domain of MLK2 has sequence motifs associated with both the serine/threonine- and the tyrosine-specific kinase families (24). To confirm the specificity of MLK2 autophosphorylation, the phosphorylated MLK2 band from an anti-HA IP kinase reaction gel was excised and subjected to phosphoamino acid analysis (Fig. 6A). The migration pattern of phosphorylated amino acids was compared with that of standard phosphoserine, phosphothreonine, and phosphotyrosine. This analysis revealed that MLK2 autophosphorylates on both serine and threonine but not tyrosine.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Activated JNK2 phosphorylates MLK2 at multiple sites. Two-dimensional phosphopeptide maps of MLK2 proteins phosphorylated in vitro. COS7 cells were electroporated with either 3 µg of pSRalpha -HA-JNK2 or 5 µg of pKH3-HA-MLK2, pKH3-HA-KN-MLK2, or pKH3-HA-C-MLK2. JNK2-expressing cells were serum-starved and then incubated with or without anisomycin (as described under "Experimental Procedures"). Proteins were immunoprecipitated, and anti-HA IP kinase assays wer performed as described in the legend to Fig. 1. Phosphorylated MLK2 proteins were subjected to either phosphoamino acid analysis or two-dimensional phosphopeptide mapping by electrophoresis (E) followed by ascending chromatography (C) on thin layer cellulose as described by Boyle et al. (37). A, autophosphorylated HA-MLK2 and phosphoamino acid analysis (left panel) and phosphopeptide map (right panel). Maps of phosphopeptides from HA-KN-MLK2 phosphorylated by JNK2 from serum-starved (B) or anisomycin-activated cells (C). Maps of phosphopeptides from HA-C-MLK2 phosphorylated by JNK2 from serum-starved (D) or anisomycin-activated cells (E). Numbers 1, 4, 5, and 7 in E refer to phosphopeptides that appear in the map of autophosphorylated WT-MLK2 (A) and numbers J1-J7 refer to peptides that are specific to KN-MLK2 phosphorylated by JNK2 in vitro. Circles show positions of phosphopeptides from the map of KN-MLK2 phosphorylated by activated JNK2 (C).

Due to the constitutive activity of overexpressed wt-MLK2, JNK2 phosphorylation of MLK2 can only be detected using KN-MLK2 as a substrate, and this precludes measurement of MLK2 activation in response to JNK phosphorylation. Therefore, we used phosphopeptide mapping to demonstrate that JNK2 phosphorylation of KN-MLK2 targets sites distinct form that of MLK2 autophosphorylation. For this analysis, phosphorylated HA-MLK2 was isolated from an IP kinase reaction gel, cleaved with trypsin, and resulting peptides resolved on thin layer cellulose by electrophoresis in the first dimension followed by ascending chromatography (37). The phosphopeptide mapping experiment was repeated several times to ensure that the pattern was reproducible and represented as complete a digest as possible. The final map (Fig. 6A) shows that 8 tryptic peptides are targets for MLK2 autophosphorylation with peptides 1, 4, and 5 being most highly phosphorylated. In a time course in which MLK2 in vitro autophosphorylated peptides were mapped at 2, 5, 10, and 20 min, all of the same peptides were present, and only the intensity appeared to change over time (data not shown).

Next, JNK2 immunoprecipitated from either serum-starved or anisomycin-treated COS7 cells was used to phosphorylate HA-KN-MLK2 in vitro. Phosphopeptide mapping of the phosphorylated HA-KN-MLK2 (Fig. 6B) revealed that JNK2 from serum-starved cells targets a single MLK2 tryptic peptide. This peptide corresponds to a major phosphopeptide present in the autophosphorylated wt-MLK2 map (Fig. 6A, peptide 1). When activated JNK2 from anisomycin-treated cells was used, a number of tryptic phosphopeptides appear on the KN-MLK2 map (Fig. 6C), and four of these (peptides 1, 4, 5, and 7) run at similar positions to phosphopeptides in the wt-MLK2 autophosphorylation profile (Fig. 6A). Although it is possible that distinct phosphopeptides may run at overlapping positions in the two maps, the pattern created by these four phosphopeptides is strikingly similar in both maps, and it would be surprising to find four sets of distinct MLK2 phosphopeptides that comigrate in such a manner. It is possible that another kinase coimmunoprecipitating with KN-MLK2 could be responsible for the phosphorylation; however, when KN-MLK2 expressed alone was subjected to an IP kinase assay no in vitro phosphorylation was detected (see Fig. 1).

In addition to the four peptides from the wt-MLK2 profile, there are seven JNK2-specific phosphopeptides in the map of KN-MLK2 phosphorylated by activated JNK2 (Fig. 6C, peptides J1-J7). Within the MLK2 protein, there are 40 predicted serine- and or threonine-containing tryptic peptides (Table I) fairly evenly distributed throughout the polypeptide chain. However, there are but two hydroxy-amino acid-proline dipeptides in the N-terminal 496 amino acids (peptides 1 and 20). In contrast, the 457-amino acid C-MLK2 polypeptide contains a total of 77 serine and threonine residues of which 20 constitute potential sites for proline-directed phosphorylation. These 20 sites are located within 12 predicted tryptic peptides.

To determine which JNK2 phosphorylation sites are located within the C-terminal region of the MLK2 molecule, the JNK2 phosphorylation assay was repeated with HA-C-MLK2 as the added substrate. Maps of HA-C-MLK2 tryptic peptides phosphorylated in vitro by JNK2 from either serum-starved or anisomycin-treated cells are shown in Fig. 6, D and E, with positions of phosphopeptides from the KN-MLK2 map circled. These maps confirm that most of the MLK2 sites phosphorylated by JNK2 are indeed located within the C-MLK2 protein. Only phosphopeptide J5, which is a prominent phosphopeptide in the KN-MLK2 map, does not appear on the C-MLK2 map. However, signals for several phosphopeptides, including numbers 4, J1, and J3, are somewhat reduced in the C-MLK2 map compared with that of KN-MLK2, and two new phosphopeptides (Fig. 6E, peptides C1 and C2) that do not appear in the KN-MLK2 map are present in the C-MLK2 map. Taken together, the phosphopeptide maps clearly demonstrate specific phosphorylation of multiple MLK2 C-terminal domain sites by activated JNK2.

JNK2 Phosphorylates the C-terminal 100-Amino Acid Peptide of MLK2-- As can be seen in Table I, several regions of the MLK2 C-terminal domain sequence are rich in potential proline-directed phosphorylation sites. Peptide 38 in particular contains three Ser- or Thr-Pro pairs as well as three other serine and four threonine residues. The C-terminal 100 amino acids of MLK2 includes peptide 38 as well as 2 other tryptic peptides containing hydroxy-amino acids (Table I, peptides 36 and 37). To begin to identify specific regions of the MLK2 C-terminal domain that may be targets for JNK phosphorylation, we cloned a cDNA fragment encoding the C-terminal 100 amino acids of MLK2 (C100-MLK2) into a vector for mammalian expression with an HA tag. The HA-C100-MLK2 vector was transfected into COS7 cells together with either HA-MLK2 or HA-MLK2 plus HA-JNK2, and phosphorylation of C100-MLK2 was determined by IP kinase assay (Fig. 7). In this assay, HA-C100-MLK2 was phosphorylated by activated JNK2, while no phosphorylation of this peptide by active MLK2 was detected. Phosphorylation of C100-MLK2 by JNK2 but not MLK2 is consistent with data from the two-dimensional phosphopeptide mapping experiments (Fig. 6) in which a number of C-MLK2 peptides are phosphorylated by JNK2 that are not present in the MLK2 autophosphorylation map.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   JNK2 phosphorylates the C-terminal 100-amino acid peptide of MLK2. COS7 cells were transfected with 3 µg of plasmid pKH3-C-100-MLK2 together with 3 µg of pKH3-HA-MLK2 and pSRalpha -HA-JNK2 (lane 1) or pKH3-HA-MLK2 and pKH3 empty vector (lane 2) and lysed 24 h later. Left panel, anti-HA IP kinase assays; right panel, anti-HA immunoblots of cell lysates before immunoprecipitation.

JNK2 Phosphorylates MLK2 in Vivo-- To examine MLK2 phosphorylation in vivo, COS7 cells were transfected with HA-KN-MLK2 together with either empty vector or HA-JNK2 and labeled with [32P]orthophosphoric acid. Cells coexpressing JNK2 were first serum-starved and then incubated with or without anisomycin. After immunoprecipitation and electrophoresis, phosphorylated KN-MLK2 was excised from the gel and subjected to two-dimensional tryptic phosphopeptide mapping (Fig. 8). It is evident from this analysis (Fig. 8A) that KN-MLK2 is phosphorylated in vivo on six of the same peptides phosphorylated by JNK2 in vitro. Furthermore, most of the in vivo labeled peptides are also represented in the C-MLK2 map (Fig. 6E). In addition, a new phosphopeptide (V), that is poorly labeled during in vitro JNK2 phosphorylation of KN-MLK2 and C-MLK2 (Fig. 6, C and E) appears in the in vivo maps. Surprisingly, however, peptide 1 that is present in all of the in vitro phosphopeptide maps is absent from the in vivo maps, suggesting that phosphorylation of this peptide is an artifact of the in vitro system.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 8.   JNK2 phosphorylates MLK2 in vivo. COS7 cells were cotransfected with 5 µg of pKH3-KN-MLK2 and either 3 µg of pKH3 empty vector (A) or 3 µg of pSRalpha -HA-JNK2 (B and C). Cells were labeled with [32P]orthophosphoric acid (as described under "Experimental Procedures") before immunoprecipitation and two-dimensional phosphopeptide mapping. A, maps of KN-MLK2 peptides phosphorylated in vivo (phosphopeptides are numbered according to their positions in Fig. 6 and arrowheads refer to peptides absent from the maps of MLK2 proteins phosphorylated in vitro); B, KN-MLK2 phosphorylated in serum-starved cells coexpressing JNK2. C, KN-MLK2 phosphorylated in anisomycin-treated cells coexpressing JNK2 (arrows indicate peptides with increased signal, and circles indicate peptides with decreased signal, following anisomycin treatment). D, autoradiograph of immunoprecipitated JNK2 proteins from serum-starved (SS) or anisomycin (Aniso)-treated cells.

When KN-MLK2 is coexpressed with JNK2 (Fig. 8B), the in vivo labeled phosphopeptide pattern is almost identical to that of KN-MLK2 expressed alone. Interestingly, however, two phosphopeptides that are not present in the in vitro maps appear very faintly when JNK2 is coexpressed with KN-MLK2 (arrowheads in Fig. 8B). When the cells are treated with anisomycin (Fig. 8C), phosphorylation of these two new peptides increases dramatically as does that of 5 other phosphopeptides (2, 4, J2, J5, and V), indicating that stress activation is involved in phosphorylation at these sites. Interestingly, however, the signals for two peptides (J4 and J6, circled in Fig. 8C) decrease when the cells are treated with anisomycin. It is not clear why these peptides, which are prominent phosphopeptides in maps of KN-MLK2 phosphorylated in vitro, decrease in signal while anisomycin causes an ~3.5-fold increase in KN-MLK2 phosphorylation (Fig. 4). The fact that the position of peptides in the overall map does not change, however, shows that the cleavage and migration conditions have not varied significantly.

Expression of wt-MLK2 causes apoptosis in neuronal cells, and this is dependent on JNK activation (31). MLK2 expression also leads to apoptosis in other cell types including melanoma and breast tumor lines (data not shown) and Swiss 3T3 fibroblasts (42). To examine the roles of the C-terminal domain and catalytic activity in MLK2-induced apoptosis, we expressed GFP-tagged wt-MLK2, KN-MLK2, N-MLK2, and C-MLK2 into HEK 293 cells (Fig. 9). In transient transfection experiments, all of the GFP-wt-MLK2-expressing cells displayed classic hallmarks of apoptosis including cytoplasmic shrinkage, membrane blebbing, and nuclear distortion or fragmentation (Fig. 9A) within 24-48 h of transfection. No such characteristics were observed with the expression of the kinase-negative or truncated MLK2 proteins (Fig. 9, B-D), and cells with stable expression of GFP-KN-MLK2, GFP-C-MLK2, or GFP-N-MLK2 were selected. After these cells were expanded into stable cell lines, the catalytic activity of the GFP-N-MLK2 protein was compared with that of GFP-wt-MLK2 (Fig. 9E), and correct sizes of expressed GFP-tagged MLK2, N-MLK2, and C-MLK2 proteins were confirmed by anti-GFP immunoblotting. Wt-MLK2 and N-MLK2 are both fully competent to signal to the JNK pathway as shown by immunoblotting of coexpressed HA-JNKK, where a clear bandshift of phosphorylated JNKK was evident (Fig. 9E, bottom panel). Although there are morphological differences among the cell lines,2 each has been retrieved after storage in liquid nitrogen and cultured for periods of several weeks without appreciable loss of viability or protein expression. Furthermore, all of these cell lines appear to cycle normally with only small differences in doubling times (data not shown). These experiments clearly show that both kinase catalytic activity and the C-terminal domain of MLK2 are required to induce apoptosis.



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 9.   Catalytic activity and the presence of the C-terminal domain are required for MLK2 induced apoptosis. A-D, confocal fluorescence micrographs of HEK 293 cells. Transiently transfected with pEGFPC1-MLK2 (A) or cell lines stably expressing GFP-KN-MLK2 (B); GFP-C-MLK2 (C); or GFP-N-MLK2 (D). Cells cultured on glass coverslips were fixed and stained with propidium iodide to highlight nuclei (A-C) or left unstained (D). E, analysis of signaling activity and sizes of expressed GFP-tagged MLK2 proteins. HEK 293 cells were transiently transfected with 5 µg of pEGFPC1-MLK2 and 3 µg of pSRalpha -HA-JNKK or stable cell lines expressing GFP, GFP-N-MLK2, or GFP-C-MLK2 were transiently transfected with 3 µg of pSRalpha -JNKK. Upper panels, IP kinase assays; lower panels, anti-GFP or anti-HA immunoblots of cell lysates.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we have shown that MLK2 activates both JNK1 and JNK2 and that all of the information required for JNK activation is contained within the N-terminal 496 amino acids of the MLK2 protein (Fig. 3). Furthermore, we show that MLK2 is phosphorylated by JNK mainly within the C-terminal domain of the MLK2 protein. When JNK2 is activated by either anisomycin treatment or coexpression of wt-MLK2 incorporation of phosphate into the KN-MLK2 protein increases, and this increase is significantly inhibited by coexpression of dominant-negative JNKK. Furthermore, MLK2 is also phosphorylated by both p38 and ERK2 but somewhat weakly compared with JNK. In previous studies, MLK2 was shown to activate p38 and ERK (30, 42); however, JNK is activated to a much more significant extent than either of the other MAPKs. In addition, MLK2 phosphorylates and activates JNKK and MKK7, the direct upstream activators of JNK (25, 26), but is unable to phosphorylate MEK, the MAPKK for ERK (30). Thus, in MAPK signaling pathways, MLK2 activates MAPKs in a hierarchical manner, and this is mirrored by the extent to which each MAPK reciprocally phosphorylates MLK2 (Fig. 5).

Phosphopeptide mapping revealed that four MLK2 tryptic peptides are phosphorylated by both autophosphorylation and by activated JNK2 (Fig. 6C, peptides 1, 4, 5, and 7), whereas seven MLK2 peptides are phosphorylated specifically by activated JNK. All but one of the JNK-specific peptides are located within the MLK2 C-terminal domain (Fig. 6, C and E). In cotransfection analysis, the C-terminal 100-amino acid peptide of MLK2 is phosphorylated specifically by JNK2 but not by MLK2 (Fig. 7). As all potential proline-directed sites within this peptide are located within a 12-amino acid stretch near the C terminus of MLK2 (Table I, tryptic peptide 38), this region of the MLK2 protein may be important for regulation by MAPK phosphorylation.

Mapping of in vivo labeled KN-MLK2 phosphopeptides revealed that all but one of the MLK2 peptides phosphorylated in vivo have counterparts in the maps of both KN-MLK2 and C-MLK2 phosphorylated by JNK2 in vitro. This suggests that endogenous JNK, or another kinase with very similar specificity, is responsible for a large proportion of the in vivo phosphorylation of KN-MLK2 and that the MLK2 C-terminal domain is the main target for in vivo phosphorylation. Furthermore, three KN-MLK2 peptides that are not present in the in vitro maps (Fig. 8) are labeled in vivo. It is possible that phosphorylation of these three peptides within the cell requires the presence of other factors or specific interactions that are missing in the in vitro kinase reaction. It is also possible that kinases other than JNK2 are involved. However, when JNK2 is coexpressed with KN-MLK2 and the cells treated with anisomycin, incorporation of radiolabel into these three peptides increases (Fig. 8C). In addition, anisomycin treatment increases phosphorylation of four peptides that are also present in the in vitro KN-MLK2 map. Thus, when MLK2 is phosphorylated in vivo in conditions that activate JNK signaling, sites are targeted that are also specifically phosphorylated by activated JNK2 in vitro. Taken together, these results strongly support the conclusion that activated JNK is responsible for the majority of phosphorylation in the in vivo labeling of KN-MLK2.

Unexpectedly, two peptides observed during in vivo phosphate labeling (Fig. 8, J5 and J6) decrease in signal after anisomycin treatment. Although it is possible that phosphatases activated by anisomycin treatment may be responsible for the reduction in signal at specific MLK2 sites, to our knowledge, activation of phosphatases by anisomycin has not been reported. Therefore, it is likely that activation of the JNK pathway by anisomycin influences either MLK2 conformation or interactions within the cell such that certain sites become unavailable or unfavorable for phosphorylation. Recently, it has been shown that several MAPK pathways are regulated by interactions of pathway components with scaffolding proteins (19, 44-49). These proteins may play roles in channeling MAPK signals to maintain specificity or in localizing MAPK cascade components at specific sites of activity within the cell. In budding yeast, a scaffolding protein, Ste5, binds the three components of the pheromone MAPK cascade, Ste11 (MAPKKK), Ste7 (MAPKK), and Fus3/KSS (MAPK), and directs their specificity (44). Fus3 phosphorylates both Ste7 (19) and Ste5 (45), and this "feedback phosphorylation" is thought to play a role in complex formation among these proteins (19).

In the mammalian SAPK/JNK pathway, JNK binds to and phosphorylates MEKK1 (20). As MEKK1 also binds JNKK (20), MEKK1 may act as a scaffolding protein to regulate specificity of this JNK cascade (20). In the present study, no direct binding of MLK2 to JNK was detected. However, a pair of recently discovered mammalian scaffolding proteins, JNK-interacting proteins 1 and 2, complex with MLKs as well as MKK7 and JNK (46-49) and enhance JNK signaling in response to coexpression of active MLK (49). Thus, JNK-interacting proteins play similar roles to Ste5 for the kinase cascade from MLK to JNK in mammalian cells (47, 49). Therefore, phosphorylation of MLK2 by JNK may regulate such a complex by a feedback mechanism, as has been suggested for Fus3 phosphorylation of Ste7 (19).

Expression of wt-MLK2 causes apoptosis in neuronal cells, and this is dependent on its catalytic activity (Fig. 9) and on JNK activation (31). However, the viability of cells stably expressing active N-MLK2, which is fully competent to activate JNK, clearly demonstrates that MLK2 signaling activity in itself is not sufficient to induce apoptosis. Thus, sequences within the C-terminal region are required to trigger a cell death response. As the C-terminal domain is a target for phosphorylation by activated JNK, this phosphorylation has the potential to regulate MLK2 interactions with proteins that may be required for the apoptotic activity. It has previously been reported that the MLK2 C-terminal domain binds to the signaling adapter 14.3.3 epsilon  (42). 14.3.3 proteins are involved in both MAPK signaling and regulation of apoptosis (50, 51), and they bind their targets through motifs containing phosphorylated serine. The MLK2 C-terminal domain 14.3.3 motif is Arg719-Gly-Leu-Ser-Pro-Pro724 (42) in which the serine is a proline-directed MAPK phosphorylation site. Thus, the 14.3.3/MLK2 interaction is a prime example of a complex in which regulation of MLK2 activity and apoptosis may be influenced through phosphorylation by activated JNK. Studies are now underway to identify the specific sites of JNK phosphorylation of the MLK2 C-terminal domain and to determine the role of phosphorylation at these sites in MLK2 induced apoptosis.


    ACKNOWLEDGEMENTS

We thank Dr. Michael Karin, University of California, San Diego, for providing plasmids. We are also grateful to Dr. Richard Simpson for advice on the manuscript and Drs. Richard Pearson and Nelly Marmy-Conus, Peter MacCallum Cancer Institute, for valuable discussion and assistance with phosphopeptide mapping.


    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.

§ Both authors contributed equally to this work.

|| To whom correspondence should be addressed: Trescowthick Research Centre, Peter MacCallum Cancer Institute, Locked Bag #1 A'Beckett St., Melbourne 8006, Victoria, Australia. Tel.: 61-3-9656-1249; Fax: 61-3-9656-1411; E-mail: d.dorow@pmci.unimelb.edu.au.

Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M008237200

2 G. Price, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MLK, mixed lineage kinase; KN-MLK2, kinase-negative MLK2 mutant; C-MLK2, C-terminal 457 amino acids of MLK2; C100-MLK2, C-terminal 100 amino acids of MLK2; MEK, MAP/ERK kinases, MKK4 or MKK7, MAPK kinase 4 or 7; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; IP, immunoprecipitate; Pipes, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Avruch, J. (1998) Mol. Cell. Biochem. 182, 31-48[CrossRef][Medline] [Order article via Infotrieve]
2. Karin, M. (1998) Ann. N. Y. Acad. Sci. 851, 139-146[Free Full Text]
3. Gupta, S., Barrett, T., Whitmarsh, A. J., Cavanagh, J., Sluss, H. K., Derijard, B., and Davis, R. J. (1996) EMBO J. 15, 2760-2770[Abstract]
4. Madhani, H. D., and Fink, G. R. (1998) Trends Genet. 14, 151-155[CrossRef][Medline] [Order article via Infotrieve]
5. Brunet, A., and Pouyssegur, J. (1997) Essays Biochem. 32, 1-16[Medline] [Order article via Infotrieve]
6. Kyriakis, J. M., App, H., Zhang, X. F., Banerjee, P., Brautigan, D. L., Rapp, U. R., and Avruch, J. (1992) Nature 358, 417-421[CrossRef][Medline] [Order article via Infotrieve]
7. Crews, C. M., and Erikson, R. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8205-8209[Abstract]
8. Zheng, C. F., and Guan, K. L. (1993) J. Biol. Chem. 268, 11435-11439[Abstract/Free Full Text]
9. Sánchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794-798[Medline] [Order article via Infotrieve]
10. Lin, A., Minden, A., Martinetto, H., Claret, F.-X., Lange-Carter, C., Mercurio, F., Johnson, G., and Karin, M. (1995) Science 268, 286-290[Medline] [Order article via Infotrieve]
11. Dérijard, B., Raingeaud, J., Barrett, T., Wu, I., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) Science 267, 682-685[Medline] [Order article via Infotrieve]
12. Holland, P. M., Suzanne, M., Campbell, J. S., Noselli, S., and Cooper, J. A. (1997) J. Biol. Chem. 272, 24994-24998[Abstract/Free Full Text]
13. Tournier, C., Whitmarsh, A. J., Cavanagh, J., Barrett, T., and Davis, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7337-7342[Abstract/Free Full Text]
14. Moriguchi, T., Toyoshima, F., Masuyama, N., Hanafusa, H., Gotoh, Y., and Nishida, E. (1997) EMBO J. 16, 7045-7053[Abstract/Free Full Text]
15. Han, J., Lee, J.-D., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R. J. (1996) J. Biol. Chem. 271, 2886-2891[Abstract/Free Full Text]
16. Stein, B., Brady, H., Yang, M. X., Young, D. B., and Barbosa, M. S. (1996) J. Biol. Chem. 271, 11427-11433[Abstract/Free Full Text]
17. Fanger, G. R., Gerwins, P., Widmann, C., Jarpe, M. B., and Johnson, G. L. (1997) Curr. Opin. Genet. & Dev. 7, 67-74[CrossRef][Medline] [Order article via Infotrieve]
18. Neiman, A. M., and Herskowitz, I. (1994) Proc. Natl. Acad. Sci., U. S. A. 91, 3398-3394[Abstract]
19. Errede, B., and Ge, Q. Y. (1996) Philos. Trans. R. Soc. Lond-Biol. Sci. 351, 143-148[Medline] [Order article via Infotrieve]
20. Xu, S., and Cobb, M. H. (1998) J. Biol. Chem. 272, 32056-32060[Abstract/Free Full Text]
21. Lange-Carter, C. A., Pleiman, C. M., Gardner, A. M., Blumer, K. J., and Johnson, G. L. (1993) Science 260, 315-319[Medline] [Order article via Infotrieve]
22. Xu, S., Robbins, D. J., Christerson, L. B., English, J. M., Vanderbilt, C. A., and Cobb, M. H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5291-5295[Abstract/Free Full Text]
23. Dorow, D. S., Devereux, D., Dietzsch, E., and deKretser, T. A. (1993) Eur. J. Biochem. 213, 701-710[Abstract]
24. Dorow, D. S., Devereux, L., Tu, G.-F., Price, G., Nicholl, J. K., Sutherland, G. R., and Simpson, R. J. (1995) Eur. J. Biochem. 234, 492-500[Abstract]
25. Cuenda, A. I., and Dorow, D. S. (1998) Biochem. J. 333, 11-15[Medline] [Order article via Infotrieve]
26. Hirai, S., Noda, K., Moriguchi, T., Nishida, E., Yamashita, A., Deyama, T., Fukuyama, K., and Ohno, S. (1998) J. Biol. Chem. 273, 7406-7412[Abstract/Free Full Text]
27. Rana, A., Gallo, K., Godowski, P., Hirai, S., Ohno, S., Zon, L., Kyriakis, J. M., and Avruch, J. (1996) J. Biol. Chem. 271, 19025-19028[Abstract/Free Full Text]
28. Hirai, S., Izawa, M., Osada, S., Spyrou, G., and Ohno, S. (1996) Oncogene 12, 641-650[Medline] [Order article via Infotrieve]
29. Fan, G., Merritt, S. E., Kortenjann, M., Shaw, P. E., and Holzman, L. B. (1996) J. Biol. Chem. 271, 24788-24793[Abstract/Free Full Text]
30. Hirai, S., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I., Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167-15173[Abstract/Free Full Text]
31. Liu, Y. F., Dorow, D. S., and Marshall, J. (2000) J. Biol. Chem. 275, 19035-19040[Abstract/Free Full Text]
32. MacDonald, M. E., and Gusella, J. F. (1996) Curr. Opin. Neurobiol. 5, 638-643
33. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W., and Bates, G. P. (1996) Cell 87, 493-506[Medline] [Order article via Infotrieve]
34. Dérijard, B., Hibi, M., Wu, I.-H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037[Medline] [Order article via Infotrieve]
35. Kallunki, T., Su, B., Tsigelny, I., Sluss, H. K., Derijard, B., Moore, G., Davis, R., and Karin, M. (1994) Genes Dev. 8, 2996-3007[Abstract]
36. Smith, P. K., Krohn, R. L., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fugimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[Medline] [Order article via Infotrieve]
37. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110-149[Medline] [Order article via Infotrieve]
38. Potapova, O., Gorospe, M., Dougherty, R. H., Dean, N. M., Gaarde, W. A., and Holbrook, N. J. (2000) Mol. Cell. Biol. 20, 1713-1722[Abstract/Free Full Text]
39. Cerezo, A., Martinez-A, C., Gonzalez, A., Gomez, J., and Rebollo, A. (1999) Cell Death Differ. 6, 87-94[CrossRef][Medline] [Order article via Infotrieve]
40. Zheng, J., Knighton, D. R., Ten Eyck, L. F., Karlsson, R., Xuong, N.-H., Taylor, S. S., and Sowadski, J. M. (1993) Biochemistry 32, 2154-2161[Medline] [Order article via Infotrieve]
41. Burbelo, P. D., Drechsel, D., and Hall, A. (1995) J. Biol. Chem. 270, 29071-29074[Abstract/Free Full Text]
42. Nagata, K.-I., Puls, A., Futter, C., Aspenstrom, P., Schaefer, E., Nakata, T., Hirokawa, H., and Hall, A. (1998) EMBO J. 17, 149-158[Abstract/Free Full Text]
43. Mukhopadhyay, N. K., Price, D. J., Kyriakis, J. M., Pelech, S., Sanghera, J., and Avruch, J. (1992) J. Biol. Chem. 267, 3325-3335[Abstract/Free Full Text]
44. Choi, K. Y., Satterberg, B., Lyons, D. M., and Elion, E. A. (1994) Cell 78, 499-512[Medline] [Order article via Infotrieve]
45. Krantz, J. E., Satterberg, B., and Elion, E. A. (1994) Genes Dev. 8, 313-327[Abstract]
46. Dickens, M., Rogers, J. S., Cavanagh, J., Raitano, A., Xia, Z., Halpern, J. R., Greenberg, M. E., Sawyers, C. L., and Davis, R. J. (1997) Science 277, 693-696[Abstract/Free Full Text]
47. Whitmarsh, A. J., Cavanagh, J., Tourneir, C., Yasuda, J., and Davis, R. J. (1998) Science 281, 1671-1674[Abstract/Free Full Text]
48. Yasuda, J., Whitmarsh, A. J., Cavanagh, J., Sharma, M., and Davis, R. J. (1999) Mol. Cell. Biol. 19, 7245-7254[Abstract/Free Full Text]
49. Schaeffer, H. J., Catling, A. D., Eblen, S. T., Collier, L. S., Krauss, A., and Weber, M. J. (1998) Science 281, 1668-1671[Abstract/Free Full Text]
50. Xing, H., Zhang, S., Weinheimer, C., Kovacs, A., and Muslin, A. J. (2000) EMBO J. 19, 349-358[Abstract/Free Full Text]
51. Zhang, L., Chen, J., and Fu, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8511-8515[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.