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J. Biol. Chem., Vol. 275, Issue 42, 32482-32490, October 20, 2000

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The Mixed Lineage Kinase DLK Is Oligomerized by Tissue Transglutaminase during Apoptosis^*

Sébastien S. Hébert, Alex Daviau, Gilles Grondin, Mathieu Latreille, Rémy A. Aubin^§, and Richard Blouin^¶

From the  Centre de Recherche sur les Mécanismes d'Expression Génétique, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec J1K 2R1 and ^§ Santé Canada, Programme des Produits Thérapeutiques/Bureau des Produits Biologiques et Radiopharmaectiques/Division des Services de Recherche-Biotechnologie, Centre de Recherche Sir F. G. Banting, Parc Tunney, Ottawa, Ontario K1A OL2, Canada

Received for publication, July 21, 2000, and in revised form, August 1, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A variety of extracellular "stress" stimuli responsible for the induction of growth arrest and apoptosis in multicellular organisms are transduced from the cell membrane to the nucleus via a phosphorylation cascade involving members of the c-Jun NH₂-terminal kinase (JNK)¹ subgroup of mitogen-activated protein kinases (MAPKs) (1-3). Once activated, these signal-transducing kinases transit from their primary subcellular location to the nucleus where they phosphorylate specific Ser-Pro and Thr-Pro motifs on transcription factors, which in turn regulate the expression of prescribed sets of downstream effector genes. Phosphorylation of transcription factors by members of the JNK family is an integrative and dynamic process, capable of eliciting an up- or down-regulation of gene expression, depending on the transcription factor(s) targeted (1-3). Like the extracellular signal-regulated kinase (ERK) and p38 subfamilies of MAPKs, the activation of JNKs requires the phosphorylation of two conserved threonine and tyrosine residues in subdomain VIII of their catalytic domains: a process catalyzed by the dual specificity MAPK kinases (MKKs) MKK4 (4) and MKK7 (5, 6). The latter are themselves substrates for activation by upstream MAPK kinase kinases (MKKKs). Examples of JNK MKKKs identified in mammalian cells include the MAPK/ERK kinase kinases (MEKKs), transforming growth factor--activated kinase, tumor progression locus-2, apoptosis signal-regulating kinase 1, and the mixed lineage kinases (MLKs) (7).

The MLK family of MKKKs is composed of five distinct members, designated MLK1, MLK2, MLK3, DLK (also known as MUK and ZPK), and LZK, which share several unique structural features. These include a "hybrid" catalytic domain bearing amino acid and structural motifs found in serine/threonine and tyrosine kinases and two leucine/isoleucine zipper motifs and a proline-rich carboxyl-terminal domain surmised to be intimately involved in facilitating protein/protein interactions (8-17). Other motifs important for protein binding have also been identified in the MLK family members. For example, MLK2 and MLK3 both contain a Src homology 3 domain in their respective amino-terminal regions. These domains bind the GTPase dynamin and the Ste20-related protein kinase HPK1, respectively (18, 19). Both kinases also possess functional Cdc42/Rac interactive binding motifs that mediate association with Cdc42 and Rac1 in a GTP-dependent manner (20, 21). Examples of other proteins capable of interacting with the carboxyl-terminal region of MLK2 have recently been identified using the yeast two-hybrid system and include the Ca²⁺-binding protein hippocalcin, 14-3-3 protein, and several members of the kinesin family of microtubule motor proteins (20). The biological significance of these associations, however, remains to be determined.

Despite our increasing knowledge regarding the involvement of MLKs in the JNK signaling cascade, relatively little is known about their physiological roles in mammalian cells or about the mechanisms responsible for their regulation in response to various stimuli. In this regard, recent studies from our laboratory have provided evidence in support of a role for the MLK family member DLK in regulating cell proliferation and differentiation, particularly during the process of tissue remodeling. This working model was originally formulated following the observation that DLK expression is not ubiquitously distributed during mouse embryogenesis, but is highest and apparently restricted to tissues where terminal cell differentiation is ongoing or where cell proliferation has strongly declined (22). Subsequent reports have further revealed a strong correlation between DLK expression and tissue regeneration (23, 24). In more recent studies, transfection and overexpression of DLK was shown to induce dramatic growth inhibition in several cell types, including fibroblasts (25) and primary cultured human keratinocytes (26). Interestingly, in the latter case, the ectopic expression of DLK led to the induction of terminal differentiation (26). Although DLK, like most other signaling molecules, might carry out different functions depending on cellular context and microenvironment, these data, when considered in sum, strongly support a role for this kinase during such pivotal biological processes as cell growth, cell differentiation and/or cell survival.

In the present report, we investigated this working notion further by studying the regulation of DLK activity during apoptosis. To this end, cultures of NIH 3T3 and DLK-transfected COS-1 cells were exposed to calphostin C, a potent and selective inhibitor of protein kinase C (PKC) (27). Calphostin C exposure concurrently leads to activation of JNK (28) and induces apoptotic cell death in various cellular backgrounds (29, 30). We demonstrate that calphostin C-mediated apoptosis is characterized, in both cellular backgrounds, by a rapid and dramatic down-regulation of DLK during the early phases of cell death, that this event is paralleled by the accumulation of high molecular weight DLK polymers, and that the formation of DLK oligomers is almost completely antagonized by monodansylcadaverine, a specific inhibitor of the Ca²⁺-dependent cross-linking enzyme tissue transglutaminase (tTG) (31, 32). We also show that DLK oligomers can be generated in vitro in the presence of purified tTG and that transglutamination results in altered kinase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Antibodies-- Calphostin C, bisindolylmaleimide I, staurosporine, MG-132, EST, calpeptin, DEVD-CHO, and Z-VAD-FMK, were purchased from Calbiochem-Novabiochem Co. (San Diego, CA). Guinea pig liver tTG, monodansylcadaverine, protease inhibitors, and all other common reagents were obtained from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). Lactacystin was kindly provided by Dr. D. LeBel (Université de Sherbrooke, Québec, Canada). The rabbit polyclonal antiserum to mouse DLK was described previously (33). Rabbit polyclonal antibodies raised against cleaved caspase-3, phospho-JNK, and JNK were purchased from New England Biolabs Inc. (Beverly, MA). The rabbit polyclonal antibody against  actin was obtained from Sigma-Aldrich Ltd. Rabbit polyclonal antibodies for the detection of phosphoserine, phosphothreonine, phosphotyrosine, and the FLAG epitope tag were purchased from Zymed Laboratories Inc. (South San Francisco, CA).

Cell Culture-- NIH 3T3, Neuro-2a, AtT-20, and COS-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin B. Stock cultures were maintained at 37 °C in a humidified atmosphere consisting of 5% CO₂, 95% air. Exposure to calphostin C was routinely carried out on late log (i.e. 80% confluent) cultures in the continuous presence of fluorescent light. Where indicated (see "Results"), cells were pre-incubated with selected inhibitors for 1 h before the addition of calphostin C. The final concentrations of diluent/vehicle (Me₂SO or ethanol) never exceeded 0.1% (v/v).

Immunoelectron Microscopy-- NIH 3T3 cells were seeded in 35-mm culture dishes and allowed to reach 70% confluence. Cells were rinsed twice with phosphate-buffered saline (PBS) and fixed in paraformaldehyde-lysine-periodate fixative (34) for 4 h at room temperature. The cells were then permeabilized with saponin and incubated, in sequence, with DLK antiserum (33), followed by goat anti-rabbit antibodies coupled to 1.4-nm Nanogold^® particles (Nanoprobes, Inc., Yaphank, NY). The samples were further fixed in 1.3% glutaraldehyde, subjected to silver enhancement (HQ Silver Enhancement Kit, Nanoprobes Inc.), and postfixed in 1% osmium tetroxide, 1% potassium ferrocyanide. Cells were then embedded in situ with Poly/Bed^® 812 epoxy resin (Polysciences, Inc., Warrington, PA). Sections were cut to 70 nm, post-stained with uranyl acetate and lead citrate, and examined with a Philips model 201 electron microscope.

Cytochemical Staining of Apoptotic Cells-- Nuclear morphology and chromatin condensation in response to calphostin C treatment were monitored by staining with the DNA-intercalating dye acridine orange. Briefly, cells were fixed in methanol/acetone, washed with PBS, and stained with acridine orange (1 mg/ml) for 1 min. Following a post-stain rinse in PBS, cells were mounted onto glass slides, viewed, and photographed by epifluorescence microscopy.

Plasmids and Transfection-- A BamHI fragment of the mouse DLK cDNA (amino acids 30-662) (35) was inserted in frame with a hexahistidine (His) tag sequence in the bacterial expression vector pET-30b (Novagen Inc., Madison, WI). The complete coding region of rat JNK cDNA was amplified by polymerase chain reaction (PCR) using pMT2 p54 SAPK-HA (a gift of Dr. J. Woodgett, University of Toronto) as template for subsequent in-frame insertion into the BamHI-XhoI sites of the bacterial expression vector pET-30c (Novagen Inc.). The prokaryotic expression vectors for glutathione S-transferase (GST)-MKK7 and GST-c-Jun were kindly provided by Dr. R. J. Davis (University of Massachusetts, Worcester, MA) and Dr. N. Rivard (Université de Sherbrooke, Québec, Canada), respectively. Recombinant proteins were expressed in Escherichia coli strain BL21 pLys and purified by affinity chromatography through columns packed with Ni²⁺-NTA-agarose or glutathione-Sepharose beads according to the supplier's recommended procedure. Three different DLK eukaryotic expression constructs were used. The pcDNA3 (Invitrogen Corp., Carlsbad, CA) expression plasmid carrying the entire coding region of mouse DLK cDNA has been described elsewhere (33). A COOH-terminal truncated construct encoding amino acid residues 1-719 of DLK was generated by PCR using Pwo polymerase and subcloned into the mammalian expression vector pLXSN (CLONTECH Laboratories Inc., Palo Alto, CA). The downstream primer was designed to introduce an artificial stop codon in the amplified cDNA fragment. A NH₂- and COOH-terminal truncated DLK construct (amino acid 160-511) tagged with the FLAG epitope sequence (DYKDDDDK) was generated similarly by PCR and inserted into pcDNA3 (Invitrogen Corp.). The upstream and downstream primers used for this amplification were designed to introduce artificial start and stop codons.

In preparation for gene transfer experiments, COS-1 cells in the exponential phase of growth were seeded at 3 × 10⁶ viable cells/60-mm dish and allowed to recover for 24 h. Thereafter, cells were transfected with 5 µg of the various DLK expression vectors using Superfect^TM (Qiagen Inc., Mississauga, Ontario, Canada) according to the manufacturer's protocol. Cells were harvested and processed for immunoblot analyses 48 h after transfection.

Preparation of Cell Lysates and Immunoblotting-- NIH 3T3, Neuro-2a, AtT-20, parental COS-1, and DLK transfected COS-1 cells were lysed for 30 min at 4 °C in lysis buffer A (15 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM -mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 1 µg/ml aprotinin). Lysates were clarified by centrifugation (12,000 × g for 15 min at 4 °C), and the concentration of total protein in the supernatant fraction was quantified by the modified Bradford protein assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada). For immunoblotting, equal amounts of proteins (50 µg/lane) were fractionated on 7% reducing SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Roche Diagnostics, Laval, Québec, Canada) using a semidry transfer apparatus (Amersham Pharmacia Biotech, Inc., Baie d'Urfé, Québec, Canada). Membranes were "blocked" overnight at 4 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20 containing 5% skim milk powder before addition of the primary antibody (1 h at room temperature). Immunoreactive bands were detected by enhanced chemiluminescence using a horseradish peroxidase-conjugated anti-rabbit (ECL Plus Western blotting kit, Amersham Pharmacia Biotech).

Subcellular Fractionation-- NIH 3T3 cells were disrupted in homogenization buffer (250 mM sucrose, 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 1 µg/ml aprotinin) with a Dounce homogenizer. The homogenates were centrifuged at 600 × g for 3 min at 4 °C to yield a nuclear pellet and a postnuclear supernatant. Nuclei were resuspended in 20 mM Hepes, pH 7.9, 400 mM NaCl, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.2 mM EDTA, 20% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 1 µg/ml aprotinin, incubated at 4 °C for 1 h with constant rotation, and then centrifuged at 13,000 × g for 20 min to obtain a supernatant fraction enriched with nuclear proteins. The postnuclear supernatant was centrifuged at 100,000 × g for 1 h at 4 °C in a Beckman SW50.1 rotor. The supernatant containing the cytosolic components was removed and saved, and the pellet consisting of the crude membrane fraction was resuspended in homogenization buffer supplemented with 1% (v/v) Triton X-100. Following incubation at 4 °C for 1 h with constant rotation, the membrane fraction was centrifuged (13,000 × g for 30 min) to yield a supernatant fraction enriched with membrane proteins. Equivalent amounts of nuclear, cytosolic, and membrane proteins (40 µg/lane) were fractionated on SDS-PAGE and processed for immunoblot analysis with DLK antiserum as described above.

Immunoprecipitation-- NIH 3T3 cells incubated in the presence or absence of calphostin C were lysed for 30 min at 4 °C in lysis buffer B (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 0.2 mM Na₃VO₄, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin). Lysates were clarified by centrifugation (12,000 × g for 15 min at 4 °C), and the concentration of total protein in the supernatant fraction was quantified using the modified Bradford protein assay (Bio-Rad). Typically, 1 mg of protein extract were immunoprecipitated for 2 h at 4 °C with constant rotation using preimmune serum, DLK antiserum (1:2000 dilution), or a combination of anti-phosphoserine/anti-phosphothreonine/anti-phosphotyrosine antibodies (5 µg each) and protein A-Sepharose beads. Immune complexes were collected by centrifugation (12,000 × g for 30 s) and washed three times with lysis buffer B. The resulting pellet was resuspended in 1× SDS-PAGE sample buffer, boiled for 5 min, fractionated on SDS-PAGE, and processed for immunoblot analysis with the DLK antiserum as described above.

In Vitro Transglutamination of DLK-- NIH 3T3 cells were harvested and lysed for 30 min at 4 °C in PBS containing 1% Triton X-100, 5 mM CaCl₂, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 1 µg/ml aprotinin (PBS-T). Cell lysates were clarified by centrifugation (13,000 × g for 15 min at 4 °C). Equal amounts of protein (typically 100 µg) recovered from the supernatant were incubated for 1 h at 37 °C in the presence of bovine serum albumin or purified guinea pig liver tTG. The samples were then subjected to SDS-PAGE electrophoresis and Western blot analysis with the DLK antiserum. In parallel experiments, purified mouse recombinant DLK expressed as a His-tagged fusion protein in E. coli was incubated in PBS-T supplemented with tTG for 1 h at 37 °C. Following incubation, the reaction was stopped by addition of an appropriate volume of 6× sample buffer and boiling for 3 min. The samples were fractionated on SDS-PAGE, and transglutaminated DLK oligomers were visualized by staining the gels with Coomassie Brilliant Blue R-250.

The functional consequences resulting from tTG-mediated oligomerization of DLK were measured by in vitro sequential kinase assays, essentially as described previously (36). Briefly, purified mouse recombinant His-tagged DLK was precipitated for 2 h at 4 °C in PBS-T containing DLK antiserum and protein A-Sepharose beads. Immunoprecipitates were then washed three times in PBS-T before incubation in the presence of purified tTG for 1 h at 37 °C in PBS-T. At the end of the incubation, the immunocomplexes were washed three times with PBS-T and three times with kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 mM Na₃VO_4, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Immunocomplex kinase assays were performed by incubating the immune complexes in 40 µl of kinase buffer containing 2.5 µCi of [-³²P]ATP (Amersham Pharmacia Biotech), 25 µM ATP, 1 µg of GST-MKK7, 1 µg of His-JNK, and 1 µg of GST-c-Jun as substrates. Following a 20-min incubation at 30 °C, the reaction was stopped by adding an appropriate volume of 6× SDS-PAGE sample buffer and boiling for 3 min. Phosphorylated proteins were visualized by autoradiography after fractionation on SDS-PAGE. Immune complexes incubated in the presence of tTG were also subjected to electrophoresis and Western blot analysis with the DLK antiserum to monitor the formation of transglutaminated oligomers.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Subcellular Distribution of DLK in Mouse NIH 3T3 Fibroblasts-- Since recent studies have indicated that DLK is subject to cell type-dependent variations in its subcellular distribution (33, 36, 37), we began by monitoring the expression and defining the localization of native (i.e. host) DLK in NIH 3T3 cells. In agreement with our previous report (33), Western blot analysis of total proteins isolated from NIH 3T3 cells using an antiserum specific for DLK revealed the presence of a major immunoreactive band at approximately 150 kDa (Fig. 1A). The DLK antiserum also detected two smaller and less prominent fragments with molecular masses of 85 and 57 kDa. These appear to be similar to those detected by Reddy et al. (37) with an antibody raised against the human homologue of DLK. The origin of the 85- and 57-kDa forms of DLK is presently unknown, although Reddy et al. (37) have proposed that they might conceivably represent proteolytic fragments derived from the full-length protein. Parallel blots processed with pre-immune serum (Fig. 1A) or DLK antiserum pre-incubated with the recombinant protein as competitor demonstrated no immunoreactivity at all (data not shown). In order to circumscribe the subcellular location of each of these immunoreactive species, we prepared membrane, nuclear, and cytosolic fractions from NIH 3T3 cells. As shown in Fig. 1B, the 150- and 85-kDa forms of DLK were detected exclusively in the membrane and nuclear fractions, respectively, whereas the 57-kDa immunoreactive band was recovered within the cytosolic and nuclear fractions. A survey of the subcellular distribution of DLK at the ultrastructural level by immunoelectron microscopy further revealed distinct labeling for DLK in both the nucleus and cytosol (Fig. 1, C and E), as well as within the Golgi complex (Fig. 1D), where DLK has been previously shown to behave like a peripheral membrane protein facing the cytosol (33). Immunogold labeling of other membranous structures such as the endoplasmic reticulum was never observed (Fig. 1E). Taken together, these data demonstrate that the 150-kDa full-length endogenous DLK protein appears to be located primarily within the membranes of the Golgi apparatus in NIH 3T3 cells, whereas the derivative lower molecular mass species appear to reside in the nucleus and/or cytosol.

View larger version (106K): [in this window] [in a new window]   Fig. 1.   Expression and localization of DLK in NIH 3T3 cells. A, 50 µg of total protein prepared from NIH 3T3 cells were fractionated on SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. Blots were then overlaid with preimmune serum (lane 1) or with DLK antiserum (lane 2) and developed by enhanced chemiluminescence. Molecular mass standards are indicated on the left. B, equal amounts of protein (40 µg) from cytosolic, membrane, and nuclear fractions prepared from NIH 3T3 cells were subjected to SDS-PAGE, followed by immunoblot analysis with the DLK antiserum. C-E, immunoelectron microscopic localization of DLK in subcellular compartments of NIH 3T3 cells. The distribution of gold particles (arrows) is shown for the cytosol (Cy; C and E), nucleus (Nu; C) and over the stacks of the Golgi complex (Gc; D). No labeling was detected over the endoplasmic reticulum (ER; E). Scale bars: C = 1µm; D and E = 0.5 µm.

Calphostin C Induces Apoptosis in NIH 3T3 Cells-- Calphostin C is a potent and selective inhibitor of PKC. The compound abrogates enzyme activity by interfering with the binding of diacylglycerol and phorbol esters within the PKC regulatory domain (27). Recent studies have also disclosed that calphostin C can trigger the activation of the stress-responsive kinase JNK (28) and induce apoptosis (i.e. programmed cell death) in a variety of cellular backgrounds, albeit by an as yet unknown mechanism (29, 30). We therefore began this section of our study by monitoring for hallmark features of apoptosis in calphostin C-treated NIH 3T3 cells. Using an antibody specific for the activated (i.e. cleaved) form of caspase-3, one of the key effector molecules in apoptotic cell death (38), we detected a gradual increase in caspase-3 activation as early as 3 h following treatment with 250 nM calphostin C; this dose is known to affect cell survival in several cell types (39, 40) (Fig. 2A, upper panel). Concurrent immunoblotting of the lysates with a antibody specifically targeted to phospho-JNK1 also revealed that calphostin C was able to stimulate the phosphorylation of JNK1 as early as 1 h after initiation of the treatment (Fig. 2A, middle panel). Under these treatment conditions, the levels of JNK1 protein remained constant (Fig. 2A, lower panel). Calphostin C exposure, therefore, appears to be able to stimulate JNK1 phosphorylation and activate caspase 3 in NIH 3T3 cells.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   Induction of apoptosis in calphostin C-treated NIH 3T3 cells. A, NIH 3T3 cells were exposed to 250 nM calphostin C (Cal.) for 0,5-4 h, lysed, and subjected to immunoblot analyses using antibodies specific for cleaved (i.e. active) caspase-3 (upper panel), phospho-JNK (middle panel), or JNK (lower panel). B-D, NIH 3T3 cells grown on coverslips were treated with Me2SO (B) or 250 nM calphostin C for 30 min (C and D), fixed in methanol/acetone, and stained with the DNA-intercalating dye acridine orange. Arrows in C and D denote cells with morphological alterations in nuclear and chromatin structure. Scale bars: B and C = 50 µm; D = 20 µm.

The induction of apoptosis was also confirmed by monitoring nuclear morphology using the DNA-intercalating dye acridine orange. As depicted in Fig. 2 (C and D), exposure to calphostin C led to chromatin condensation and to the fragmentation of nuclei into small spherical bodies typical of apoptotic cell death. Additional morphological changes characteristic of apoptosis, such as cytoplasmic shrinkage, membrane blebbing, and loss of adherence, were also observed following exposure to calphostin C (data not shown).

Exposure to Calphostin C Causes Down-regulation of the Membrane-associated Form of DLK-- Since DLK is known to act as an upstream regulator of the stress-responsive kinase JNK (16), we next wished to determine if DLK levels were subject to regulation in response to an apoptotic stimulus. For this purpose, whole cell extracts were prepared from NIH 3T3 cells exposed to calphostin C (250 nM for 30 min) and processed for immunoblot analysis with the DLK antiserum. In cells exposed to solvent (Me₂SO) alone, the antiserum recognized the expected 150-, 85-, and 57-kDa immunoreactive bands (Fig. 3A). Calphostin C treatment, however, led to the disappearance of the 150-kDa membrane-associated form of DLK (Fig. 3A) without altering the levels of the 85- and 57-kDa immunoreactive forms. To confirm that the membrane-associated form of DLK was indeed down-regulated in response to calphostin C, lysates subjected to immunoprecipitation with the DLK antiserum. The immunoprecipitates were run on SDS-PAGE and processed for immunoblotting with the same antiserum. Fig. 3B (left panel) shows that the 150-kDa membrane-associated form of DLK disappeared almost completely upon exposure to calphostin C, whereas the level of the 85-kDa form remained essentially unchanged. The 57-kDa band could not be detected under these conditions. Furthermore, calphostin C-mediated down-regulation of the 150-kDa form of DLK was also observed when the lysates were immunoprecipitated with a combination of antibodies raised against phosphoserine, phosphothreonine, and phosphotyrosine prior to immunoblotting with the DLK antiserum (Fig. 3B, right panel), suggesting that the membrane-associated form of DLK, which appears to exist as a phosphoprotein in unchallenged cells, is specifically targeted for down-regulation upon initiation of apoptosis by calphostin C. 

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Down-regulation of DLK in calphostin C-treated NIH 3T3 cells. A, cultures of NIH 3T3 cells were incubated in the absence (lane 1) or presence (lane 2) of 250 nM calphostin C (Cal.) for 30 min, lysed, and subjected to Western blotting with the DLK antiserum. B, NIH 3T3 cells were incubated in the absence or presence of 250 nM calphostin C (Cal.) for 30 min, lysed, and immunoprecipitated with preimmune serum (lanes 1 and 4), DLK antiserum (lanes 2 and 3), or with antibodies to phosphoserine, phosphothreonine, and phosphotyrosine (lanes 5 and 6). Following immunoprecipitation, immune complexes were subjected to Western blotting with the DLK antiserum. C, NIH 3T3 cells were exposed to 250 nM calphostin C (Cal.) for the indicated time periods, lysed, and subjected to immunoblot analyses using antibodies to DLK (first panel), JNK (second panel), or  actin (third panel). D, NIH 3T3 cells were treated for 30 min with increasing concentrations of calphostin C (Cal.) as indicated, lysed, and subjected to immunoblotting with antibodies to DLK (upper panel) or  actin (lower panel). E, cultures of NIH 3T3, Neuro-2a, and AtT-20 cells were exposed to 250 nM calphostin C (Cal.) for 30 min, lysed, and subjected to immunoblot analyses with antibodies to DLK (upper panel) or  actin (lower panel).

In order to better appreciate the kinetics of DLK down-regulation, we set out to monitor the levels of immunoreactive DLK protein at selected time points during the early phase of apoptotic induction. In this instance, we noted a significant down-regulation of the membrane-associated form of DLK as early as 5 min after treatment with 250 nM calphostin C (Fig. 3C, upper panel) with a gradual progression toward minimal levels being attained by 30 min. No further reduction in DLK levels were registered beyond this time point (data not shown). Immunoblots processed in parallel with antibodies targeting JNK and  actin ensured that this trend was not attributed to variations in the loading of protein samples (Fig. 3C, middle and lower panels).

In conjunction with these experiments, we also investigated the dose response of the phenomenon. Under this scheme calphostin C induced down-regulation of DLK in a dose-dependent manner. A dose of 50 nM was apparently sufficient to significantly affect the level of DLK protein within the 30-min exposure period (Fig. 3D).

Finally, we undertook to determine if calphostin C could elicit a similar down-regulation of DLK levels in murine cell lines other than fibroblasts. When this was done, a dose of 250 nM calphostin C given over 30 min resulted in the disappearance of the 150-kDa form of DLK in both neuroblastoma Neuro-2a and pituitary AtT-20 cells (Fig. 3E).

Down-regulation of DLK by Calphostin C Is Independent of PKC Activity, Proteolytic Degradation, and Subcellular Localization-- A series of experiments with inhibitors of various specificities were next performed in an attempt to identify candidate mechanisms responsible for calphostin C-induced DLK down-regulation in NIH 3T3 cells. Initially, we sought to determine whether the effects of calphostin C on DLK could be recreated by modulating of the catalytic activity of PKC. NIH 3T3 cells were therefore incubated with two potent PKC inhibitors, bisindolylmaleimide I (10 µM) or staurosporine (500 nM). These compounds, unlike calphostin C, target the ATP-binding site (41) rather than the regulatory domain. As depicted in the immunoblots presented in Fig. 4A, neither of these inhibitors, when presented at functional doses, affected the levels of DLK in NIH 3T3 cells. Having ruled out an apparent requirement for PKC signaling activity in the process, we proceeded to determine if down-regulation might by attributed to the targeted proteolysis of DLK. To do this, we pre-incubated NIH 3T3 cells with agents capable of preventing protein degradation via distinct proteolytic pathways before supplementing the culture medium with calphostin C. Data presented in Fig. 4 (B and C) reveal that pre-incubation of cells with the proteasome inhibitors lactacystin or MG-132, the calpain inhibitor calpeptin, the cysteine protease inhibitor EST, and the caspase inhibitors DEVD-CHO or Z-VAD-FMK, at doses known to antagonize target enzyme activity in a variety of cell types (28, 42) neither augmented nor decreased the magnitude of DLK down-regulation in response to calphostin C. Taken together, these results suggest that calphostin C-induces the targeted down-regulation of DLK in NIH 3T3 cells according to a mechanism(s) that is independent of PKC catalytic activity and does not appear to involve proteolytic degradation.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Calphostin C-induced down-regulation of DLK is independent of protein kinase C activity, proteolytic degradation, and subcellular localization. A, NIH 3T3 cells were incubated for 1 h in the absence or in the presence of two potent PKC inhibitors, bisindolylmaleimide I (Bis, 10 µM) and staurosporine (Stauro, 500 nM), prior to immunoblot analysis with the DLK antiserum. B, NIH 3T3 cells were pre-incubated with Me2SO (control) or the protease inhibitors lactacystin (10 µM), MG-132 (50 µM), calpeptin (200 µM), or EST (50 µM) for 1 h, followed by concurrent incubation for another 30 min with 250 nM calphostin C (Cal.). Cells were subsequently lysed and subjected to immunoblot analysis with the DLK antiserum. C, same as B except that cells were pre-treated with Me2SO (control) or the caspase inhibitors DEVD-CHO (100 µM) and Z-VAD-FMK (100 µM) for 1 h prior to the addition of calphostin C (Cal.). D, same as B except that cells were pre-treated with Me2SO (control) or the fungal metabolite BFA (5 µg/ml) for 1 h prior to the addition of calphostin C (Cal.).

Because calphostin C has recently been observed to promote the disassembly of the Golgi apparatus (43), the site where the membrane-associated form of DLK is primarily localized in NIH 3T3 cells (33), we decided to test whether disruption of the Golgi complex with brefeldin A (BFA) alone could also promote DLK down-regulation. When NIH 3T3 cells were exposed to BFA at a dose known to compromise the integrity of the Golgi apparatus in these cells (33), no effect on the levels of DLK were observed (Fig. 4D, lane 3). By contrast, addition of calphostin C to BFA-treated cells induced the dramatic decrease in DLK protein levels (Fig. 4D, lanes 4 and 5) characteristic of exposure to this compound alone (Fig. 4D, lanes 1 and 2). These findings, therefore, led us to conclude that down-regulation of the membrane-associated form of DLK in response to calphostin C was also independent of its subcellular localization to the Golgi apparatus.

Calphostin C Induces Oligomerization of DLK in NIH 3T3, as Well as in Transfected COS-1 Cells-- In order to extend these findings, we next examined whether down-regulation of DLK could be reconstituted in COS-1 cells transfected with a functional DLK expression construct. COS-1 cells were therefore transfected and exposed, 48 h later, to 250 nM calphostin C for 30 min before being processed for immunoblotting with the DLK antiserum. Although not as dramatic as in NIH 3T3 cells, the response to calphostin C treatment did bring about a noticeable decrease in ectopic DLK protein levels. In addition, exposure to calphostin led to the appearance of immunoreactive products with molecular masses exceeding 250 kDa (Fig. 5A, lanes 3 and 4). A similar accumulation of high molecular weight DLK products was also noted when COS-1 cells transfected with expression vectors encoding DLK deletion mutants lacking either a portion of the COOH-terminal region (amino acids 720-888) or the entire NH₂-terminal (amino acids 1-159) and COOH-terminal (amino acids 512-888) regions were incubated with calphostin C (Fig. 5A, lanes 5-8). Thus, the ability of calphostin C to induce the formation of these protein complexes does not appear to be significantly affected by the absence of the NH₂ and COOH-terminal domains of DLK, which contain putative docking sites for Src homology 3 domain-bearing proteins (15).

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Generation of high molecular weight forms of DLK in calphostin C-treated cells. A, COS-1 cells were transfected with expression constructs encoding wild-type (WT) and truncated forms of DLK, as indicated on the right. At 48 h after transfection, cells were exposed to 250 nM calphostin C (Cal.) for 30 min and then subjected to immunoblotting with antibodies to DLK (lanes 1-6) or the FLAG epitope (lanes 7 and 8). B, NIH 3T3 cells were exposed to 250 nM calphostin C (Cal.) for the indicated time periods prior to immunoblot analysis with the DLK antiserum. C, NIH 3T3 cells were exposed to increasing doses of UV irradiation as indicated and then incubated for 30 min at 37 °C. Cell lysates were analyzed by immunoblotting with the DLK antiserum.

In view of these observations, we next attempted to determine whether calphostin C could also induce the formation of high molecular weight immunoreactive products with the endogenous DLK expressed by NIH 3T3 cells. In a manner resembling the results of the transfection experiments in COS-1 cells, at least part of the DLK immunoreactivity from calphostin C-treated NIH 3T3 cells was found to be associated with high molecular mass complexes (greater than 250 kDa in apparent mobility) that were readily detectable, albeit after prolonged exposure to x-ray film, just below the junction of the stacking and running gels (Fig. 5B). In order to test whether the production of high molecular weight DLK complexes could occur in response to another pro-apoptotic stimulus, NIH 3T3 cells were exposed to increasing doses of UV irradiation before being subjected to immunoblot analysis with the DLK antiserum. The results presented in Fig. 5C demonstrate that irradiation with 250-1000 J/m² UV light also resulted in the progressive accumulation of the high molecular weight forms of DLK.

DLK Is a Potential Substrate for tTG in Calphostin C-treated Cells-- To characterize the molecular mechanism(s) contributing to the formation of the high molecular weight DLK products in response to calphostin C treatment, we asked whether DLK could serve as a substrate for tTGs, a family of cross-linking enzymes induced during apoptosis (31, 32). To verify this postulate, NIH 3T3 cells were pre-incubated with the tTG-specific inhibitor monodansylcadaverine (44) for 1 h before supplementing the culture medium with calphostin C, lysed, and the extracts processed for immunoblot analysis. As shown in Fig. 6A, 1 mM monodansylcadaverine almost completely antagonized the disappearance of the parent 150-kDa form of DLK as well as the formation of DLK oligomers. We nest asked if monodansylcadaverine could exert a similar effect in calphostin C-treated COS-1 cells transfected with the full-length DLK expression construct. In this instance, monodansylcadaverine almost completely prevented the calphostin C-induced accumulation of high molecular weight DLK species in transfected cells (Fig. 6B), thereby suggesting that tTGs might be responsible for the transglutamination and consequent oligomerization of DLK in response to an apoptotic stimulus.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   tTG-catalyzed cross-linking of DLK in calphostin C-treated cells. A, NIH 3T3 cells were exposed to monodansylcadaverine (MDC) at the indicated concentrations for 1 h prior to treatment with 250 nM calphostin C (Cal.) for 30 min. After incubation, cells were harvested, lysed, and subjected to immunoblotting with the DLK antiserum. B, COS-1 cells transiently transfected with an expression vector for wild-type DLK were pre-treated with Me2SO (control) or the tTG inhibitor monodansylcadaverine (MDC, 1 mM) for 1 h, followed by further incubation for 30 min with 250 nM calphostin C (Cal.). Cells were subsequently lysed and subjected to immunoblot analysis with the DLK antiserum.

DLK Is a Substrate for Purified tTG in Vitro-- In order to better gauge the involvement of tTGs in DLK oligomerization, we began by incubating NIH 3T3 cell lysates with purified guinea pig tTG and subjected the reaction products to immunoblot analysis with the DLK antiserum. The data are presented in Fig. 7A and demonstrate that addition of tTG to cell extracts contributed not only to the disappearance of the native 150-kDa membrane-associated form of DLK but appeared to favor the concomitant accumulation of oligomeric products with molecular masses higher than 250 kDa. The ability of DLK to serve as a substrate for transglutamination was further tested by incubating the purified mouse recombinant His-tagged DLK fusion protein (amino acids 30-662; carrying the intact/functional catalytic and leucine zipper domains) with guinea pig tTG. As shown in Fig. 7B, the recombinant form of DLK, which migrates in the native state as a doublet of approximately 70 kDa on Coomassie Blue-stained gels, was converted to a series of increasingly transglutaminated forms, some of which migrated within size ranges corresponding roughly to dimers and multimers, following incubation in the presence of tTG.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   DLK is a substrate for tTG in vitro. A, total proteins (100 µg) prepared from intact NIH 3T3 cells were incubated either with bovine serum albumin (4 µg, lane 1) or with purified guinea pig liver tTG (1 µg, lane 2; 4 µg, lane 3) for 1 h at 37 °C. After incubation, proteins were separated on SDS-PAGE and immunoblotted with the DLK antiserum. B, purified recombinant His-tagged DLK (2 µg) incubated in the absence (lane 1) or in the presence of tTG (1 µg, lane 2; 4 µg, lane 3) was electrophoresed through SDS-PAGE and stained with Coomassie Brillant Blue. C, purified recombinant His-tagged DLK (2 µg) immunoprecipitated either with pre-immune serum (lane 1) or with DLK antiserum (lanes 2-4) was incubated in the absence (lanes 1 and 2) or in the presence of tTG (1 µg, lane 3; 4 µg, lane 4) for 1 h at 37 °C. Following incubation, the immunoprecipitates were analyzed by Western blotting using DLK antiserum (upper panel) or assayed for DLK activity (lower panel) as described under "Experimental Procedures."

Because tTG-catalyzed protein cross-linking has been shown to alter the activity of several enzymes (45, 46), we were compelled to determine how transglutamination might affect DLK activity in vitro. To test this, recombinant DLK was immunoprecipitated and incubated either in the absence or the presence of tTG. The immunocomplexes were then subjected to immunoblotting with the DLK antiserum and to in vitro sequential protein kinase assays with GST-MKK7, His-JNK, and GST-c-Jun as substrates for the phosphorylation cascade reaction. As expected (Fig. 7C), addition of tTG to immunoprecipitated recombinant DLK promoted the appearance of immunoreactive bands with molecular sizes consistent with the formation of DLK dimers and multimers. However, the generation of these transglutaminated species was paralleled by a significant decrease in the catalytic activity of the oligomers when compared with unmodified DLK (Fig. 7C). The results of this exercise demonstrate that DLK catalytic activity can be significantly reduced following transglutamination and subsequent oligomerization.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although advances over the past few years have contributed to our general understanding of how the mixed lineage kinase DLK regulates JNK signaling, little is currently known about the mechanisms affecting its own regulation, about the proteins it is most likely to interact with within the cell, and about its physiological function(s). However, the documented involvement of JNK-dependent signaling events during cell growth and apoptosis implies equally important, or at the very least, supportive roles for involvement of DLK in the regulation of these cellular processes. In order to verify this working hypothesis, we chose to investigate candidate mechanisms for the regulation of DLK activity in cultured cells induced to undergo apoptosis following exposure to calphostin C. We provide evidence that calphostin C induces a rapid and dramatic decrease in the levels of the 150-kDa membrane/Golgi-associated form of DLK in NIH 3T3, Neuro-2a, and AtT-20 cells as well as in COS-1 cells transiently transfected with a functional DLK expression vector. We also demonstrate that the ability of calphostin C to down-regulate DLK expression is encountered during the early phases of the apoptotic response, namely during that time period preceding activation of JNK phosphorylation and caspase-3 cleavage. We also describe, for the first time, that down-regulation of DLK levels in response to calphostin C is accompanied by the appearance of SDS and mercaptoethanol-resistant high molecular weight DLK species, that this shift in DLK molecular weight is partially suppressed by the tTG inhibitor monodansylcadaverine, that DLK can serve as a substrate for tTG-catalyzed cross-linking reactions in vitro and in vivo, and that transglutamination of DLK leads to a shut-down of its catalytic activity. These findings, therefore, lead us to propose that the mixed lineage kinase DLK is subjected to tTG-dependent post-translational modification during the early phases of apoptosis.

Like all transglutaminases, tTG is a Ca²⁺-dependent cross-linking enzyme that catalyzes the post-translational modification of proteins by transamidation of specific polypeptide-bound glutamines (31, 32). This activity commonly results in the formation of -glutamyl--lysine isopeptide bonds within or between polypeptides chains that are of great physiological interest because of their exceptional stability and resistance to mechanical breakage and chemical attack. Although the precise biological functions of tTGs are not fully understood, recent reports from several laboratories have suggested a participative role for protein polymers formed via tTG-catalyzed cross-links during apoptosis (31, 32). In fact, it has been shown that expression and activity of tTG are both substantially increased at the onset of apoptosis in numerous cell types (47-50). Under these circumstances, the activation of tTG in dying cells leads to the formation of specific cross-linked protein polymers that are thought to be involved in some as yet unspecified way during the early and late stages of the apoptotic process (31, 32). Although some of the tTG substrates might presumably be involved in committing cells to apoptosis, others have been found to participate in the assembly of a detergent-insoluble cross-linked protein scaffold that prevents the leakage of macromolecules and inflammatory substances in the surrounding tissues (31, 32). Thus, the up-regulation of tTG activity observed in vitro and in vivo in apoptotic cells might conceivably constitute one of the key elements characterizing or contributing to this particular type of cell death. To our knowledge, the results presented here add, for the first time, a cell survival regulatory protein kinase to the list of known cellular proteins that serve as substrates for tTG in dying cells. Examples of cross-linked protein polymers formed by tTG in cells undergoing programmed cell death include actin, annexin, troponin, involucrin, fibronectin, histone H2B, and the retinoblastoma protein (32, 50). In contrast to most of these intracellular proteins, which have also been found to be substrates of the thiol protease caspases and calpains in dying cells (31, 32), our data indicate that tTG is the major effector element of the apoptotic response acting on the processing of DLK following exposure to calphostin C.

The finding that DLK undergoes tTG-dependent polymerization shortly after exposure to calphostin C raises the intriguing possibility that this post-translational modification may constitute a biochemical event implicated in the instructive phase of the apoptotic response. In mammalian cells, initiation of apoptosis usually involves the participation of a series of intracellular signaling molecules among which distinct families of serine/threonine protein kinases, namely MAPKs, PKC, cyclic AMP-dependent protein kinase A, and protein kinase B or Akt, are important components (51). Although the role played by each of these protein kinases during the regulation of apoptosis is largely influenced by the cell context and the nature of the death stimulus, their involvement can be tentatively categorized as either pro-apoptotic or anti-apoptotic based on their respective abilities to promote or prevent apoptosis following activation (51). With respect to the MAPKs, for example, it is generally assumed that activation of the JNK and p38 kinases promotes apoptosis, whereas induction of ERK activity has been shown to inhibit apoptosis and favor survival (52). Interestingly, many serine/threonine kinases reported to have either pro-apoptotic or anti-apoptotic properties have been found to act as caspase substrates during the apoptotic response induced by a wide range of stimuli (53, 54). The caspase-dependent cleavage of these proteins triggers the activation of pro-apoptotic kinases, such as MEKK1, PKC, or p21-activated kinase (54-56), and the inactivation of anti-apoptotic kinases, such as Akt, PKC, or Raf-1 (53, 57), an upstream activator of the ERKs. Our demonstration that DLK serves as a substrate for tTG, both in vivo and in vitro, is, therefore, consistent with the possibility that protein polymerization induced by tTG in dying cells could represent another effective way of regulating the activity of pro-apoptotic and anti-apoptotic kinases. Our data showing that the tTG-dependent oligomerization of DLK in vitro is paralleled by its functional inactivation strongly support this notion. Since there is presently no information in the literature concerning the role of DLK during programmed cell death, it is tempting to speculate that the modulation of DLK activity by cross-linking represents an important in vivo mechanism by which tTG regulates cellular growth and apoptosis. Moreover, by virtue of its localization to the Golgi apparatus in NIH 3T3 cells (33), it seems equally plausible that the tTG-dependent oligomerization of DLK in this locale might engender a form of trafficking stress stimulus in this organelle which results in apoptosis. The presence on Golgi membranes of several signaling molecules implicated in the regulation of secretory traffic, cellular growth control and/or apoptosis, including Cdc42, phosphatidylinositol 3-kinase, PKC, and Fas (58-61), supports the notion that the Golgi complex might function as a sensor and/or integrator for several varieties of stress stimuli (62, 63).

Although the molecular details surrounding DLK oligomerization and catalytic inactivation by tTG remain to be fully defined, it is not unreasonable to suspect that select glutamine and lysine residues located within or in close structural proximity to the catalytic and/or regulatory domains may serve as potential acceptor sites for transglutamination by tTG. The kinase domain of DLK actually contains 7 glutamine and 14 lysine residues, which may act as acyl donors and acceptor substrates, respectively, for tTG-catalyzed reactions.

In summary, this report provides strong in vivo and in vitro evidence that the mixed lineage kinase DLK serves as a substrate for tTG in cells undergoing programmed cell death. To our knowledge, these studies also provide the first demonstration that the tTG-dependent oligomerization of a protein kinase exerts a modulatory effect on its activity. Further studies are currently under way to investigate the relevance of these phenomena in an in vivo setting.

	ACKNOWLEDGEMENTS

We thank Drs. James Woodgett, Roger Davis, and Nathalie Rivard for pMT2 p54 SAPK-HA, GST-MKK7, and GST-c-Jun constructs, respectively.

	FOOTNOTES

^* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and by the "Fonds pour la Formation de Chercheurs et l'Aide à la Recherche" of the Province of Quebec.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dépt. de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500, boulevard de l'Université, Sherbrooke, Québec J1K 2R1, Canada. Tel.: 819-821-8000 (ext. 2062); Fax: 819-821-8049; E-mail: rblouin@courrier.usherb.ca.

Published, JBC Papers in Press, August 1, 2000, DOI 10.1074/jbc.M006528200

	ABBREVIATIONS

The abbreviations used are: JNK, c-Jun NH₂-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, mitogen-activated protein kinase kinase; MKKK, mitogen-activated protein kinase kinase kinase; MLK, mixed lineage kinase; PKC, protein kinase C; tTG, tissue transglutaminase; DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; His, hexahistidine; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; ERK, extracellular signal-regulated kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinases; BFA, brefeldin A; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline with Triton X-100; DLK, dual leucine zipper-bearing kinase.

	REFERENCES

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