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INTRODUCTION |
A large body of work has focused on signal transduction via
protein kinases generically termed mitogen-activated protein kinases (MAPK)1 that link a variety
of extracellular signals to cellular responses as diverse as
proliferation, differentiation, and apoptosis (reviewed in Refs. 1-3).
Biochemical and genetic evidence has demonstrated that activation of a
prototypical MAPK occurs through sequential activation of a series of
upstream kinases: a serine/threonine MAPK kinase kinase (MAPKKK)
phosphorylates a dual specificity protein kinase (MAPKK or MKK or
MEK) that in turn phosphorylates and activates a MAPK. Three groups of
mammalian MAPKs and the upstream kinases and stimuli that activate them
have been studied most extensively. These include the
p42/p44MAPKs (extracellular signal-regulated kinases, ERK1
and ERK2), that are generally activated by mitogens and differentiation
inducing stimuli, the p46/p54SAPKs (stress-activated
protein kinases, SAPKs), and the p38MAPKs. Stress-activated
protein kinases were discovered as the principal c-Jun
NH2-terminal kinases and therefore have also been termed JNKs. Distinct from ERK1 and ERK2, the SAPKs are predominantly activated by cell stress-inducing signals such as heat shock, ultraviolet irradiation, proinflammatory cytokines, hyperosmolarity, ischemia/reperfusion, and axonal injury.
Like previously identified MAPK pathways in mammalian cells and yeast,
the SAPK pathways were initially thought to lead in a linear fashion
from activation of a Rho-like small GTPase through a series of
intermediate protein kinases to SAPK activation. However, with the
identification of two MAPKKs (MKK4/SEK1, MKK7/JNKK2) many MAPKKKs (four
MEKKs, five MLKs, ASK1, Tpl-2, and TAK1) and multiple additional MAPKKK
kinases that appear to lie in pathways proximal to the SAPKs, it is
clear that the organization, regulation, and function of these protein
kinase pathways remain poorly understood.
The five identified members of the mixed lineage kinase (MLK) family
share two common structural features (4, 5). Each has a kinase
catalytic domain whose primary structure is hybrid between those found
in serine/threonine and tyrosine protein but most closely resemble MAPK
kinase kinases. Second, closely juxtaposed COOH-terminal to the
catalytic domain, each MLK protein has a domain that is predicted to
form two leucine/isoleucine zippers separated by a short spacer region.
It has been proposed that all members of the MLK family behave
functionally as MAPK kinase kinases in pathways leading to activation
of SAPKs (6). In support of this hypothesis, MLK2, MLK3, DLK, and LZK
have all been shown to activate p46SAPK when overexpressed
in cultured cells (6-12). Furthermore, MLK2 and MLK3 have been shown
to associate with, phosphorylate, and activate MKK4 (6, 12, 13).
Despite the common features of the MLK family, the various members of
the family are likely to have diverse biological behavior. This is
predicted by comparison of structure that shows that MLK1, MLK2, and
MLK3 form one closely related MLK subfamily while DLK and LZK form a
second distinct subfamily (5, 11). MLK2 and MLK3 have highly conserved
kinase catalytic and leucine zipper domains, sharing more than 70%
sequence identity. Both possess an SH3 domain in their
NH2-terminal region, and both have a functional CRIB domain
that mediates GTP-dependent association with Rac1 and
Cdc42Hs (9, 14). DLK and LZK share highly conserved kinase catalytic
and leucine zipper domains that are more than 90% identical, whereas
these domains have only 36% identity to those of MLK2 and MLK3. Given
the noted structural differences, DLK and LZK substrates may be
distinct from those utilized by MLK2 and MLK3. Indeed, DLK and LZK
substrates have not been identified. Moreover, DLK and LZK lack both
CRIB motifs and SH3 domains and possess COOH-terminal regions that are
structurally distinct from those of MLK2 and 3. These observations
predict that protein-protein interactions unique to each protein kinase
lead to distinct regulation and function by determining different
subcellular compartmentalization and by providing access to different substrates.
The studies reported here provide evidence in support of this
hypothesis. In vitro and complementary in vivo
biochemical studies demonstrated that both DLK and MLK3 directly
associate with, phosphorylate, and activate MKK7. Unlike MLK3, however,
DLK did not phosphorylate or activate recombinant MKK4 in
vitro. To investigate the physiological relevance of these
observations, the cellular and subcellular localization of
endogenous DLK, MLK3, MKK7, and MKK4 was investigated both
in neuron cell culture and in the nervous system. Whereas MLK3 was
identified predominantly in the periventricular ependyma and meninges,
DLK and MKK7 were predominantly localized in neurons. DLK and MKK7 were
identified in similar nuclear and extranuclear compartments. However,
MKK4 occupied compartments distinct from that of DLK and MKK7. The
dissimilar cellular and subcellular compartmentalization and
differential substrate utilization of DLK and MLK3 provide evidence
that specific mixed lineage kinases participate in unique signal
transduction events.
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MATERIALS AND METHODS |
Antibodies--
A rabbit polyclonal antiserum directed against
the carboxyl-terminal 223 amino acids of DLK (C1) was described
previously (5). Affinity purified rabbit anti-MKK7 polyclonal serum was a gift of Professor Eisuke Nishida (15). Monoclonal antibodies against
the FLAG epitope (M2, Kodak/IBI), the Myc epitope (9E10, Oncogene
Science), and the hemagglutinin epitope (12CA5, BMB) were obtained
commercially as were rabbit polyclonal antibodies against SAPK
/JNK3
(Upstate Biotechnology, Inc.), MLK3 (Santa Cruz), phospho-MKK4 (New
England Biolabs), and MKK4 (Upstate Biotechnology, Inc.). Mouse
monoclonal antibodies for SAPK
/JNK1 (Pharmagen) were obtained
commercially. In immunoblotting experiments, GST-MKK proteins were
detected using the C1 polyclonal serum that was raised against a
GST-DLK fusion protein.
Plasmids and Plasmid Constructions--
Previously described
plasmids used herein included DLK(K185A) and DLK (16), MEKK4 and
MKK4-KM (Ref. 17, gifts of Dr. G. Johnson), FLAG-MLK3, FLAG-MLK3(K144E)
(Ref. 12, gifts of Dr. J. Woodgett), GST-MKK1 and GST-MKK3 (Ref. 18,
gifts of Dr. K.-L. Guan), GST-MKK5 (Ref. 19, gift of Dr. J. Dixon),
GST-MKK7 (Ref. 20, gift of Dr. R. Davis), Myc-p46SAPK (7),
and GST-c-Jun (1-79) (7). GST-MKK7(K76A) was created by introducing
point mutations into the GST-MKK7 template using sequential polymerase
chain reaction steps as described (16). Synthetic oligonucleotides used
included 1) a 5' MKK7 oligonucleotide 5'-CCCGGATCCATGCTGGGGCTCCCATCA-3', 2) a 3' MKK7 antisense
oligonucleotide 5'-GCCGAATTCCTACCTGAAGAAGGGCAG-3', and 3)
5'-CATTGCTGTTGCGCAAATGCG-3' and 4) 5'-CGCATTTGCGCAACAGCAATG-3'
as antisense and sense K-A mutagenesis oligonucleotides, respectively.
The BamHI-EcoRI fragment of the resultant
amplification product was subcloned into the BamHI-EcoRI prepared PGEX-KT plasmid (21).
FLAG-MKK7 and FLAG-MKK7-KA expression constructs were prepared using
polymerase chain reaction employing Expand High-Fidelity DNA polymerase
(Boehringer Mannheim), GST-MKK7 or GST-MKK7(KA) plasmid as templates,
and synthetic oligonucleotides, including a 5' oligonucleotide encoding
a Kozak's consensus and a FLAG-epitope
(5'-ATAAAGCTTCCAGAGGCCATGGACTACAAGGACGACGATGACAAGCTGGGGCTCCCATCAACCTTGTTCA-3'). Amplified fragments were subcloned into pCR3.1 (Invitrogen) following the manufacturer's recommendations. All new constructs were sequenced to assure Taq polymerase fidelity.
Cell Culture and Transfections--
COS 7 cells (ATCC) were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, penicillin, and streptomycin. Experiments involving
transient transfections were carried out as described previously (7)
with a total of 2 µg of the appropriate combinations of eukaryotic
expression plasmid using Lipofectamine (Life Technologies, Inc.)
according to the manufacturer's protocol.
Primary embryonic cortical neuron culture was established from
dissected cortices obtained from Sprague-Dawley rat fetuses at
embryonic day 17. Cortical cells were dissociated in Neurobasal Medium
containing B-27, GutaMax-I and AlbuMax II (Life Technologies, Inc.) and
2 × 105 cells/well of a 24-well plate were seeded on
glass coverslips precoated with 100 µg/ml poly-D-lysine.
Cultures were incubated for 2 weeks at 37 °C in humidified 95% air,
5% CO2. Half of the medium was replaced twice
weekly. Cultures were characterized by immunocytochemistry using neuron
specific antibodies against neurofilament protein (SMI31,
Steinberger Meyer Immunochemicals) and astrocyte-specific antibody,
glial fibrillary acidic protein (Sigma). Cultures typically were
composed of 95% neurons and 5% astrocytes as described previously
(22).
Immunoprecipitations and
Immunoblotting--
Immunoprecipitations were performed as described
elsewhere (7) using the indicated antibodies. For immunoblotting,
immunoprecipitates or 30 µl of cell lysate were separated under
reducing conditions by SDS-polyacrylamide gel electrophoresis and
immunoblotted as described previously (7, 16).
Expression and Purification of GST Fusion Proteins--
With the
exception of GST-MKK7 and GST-MKK7(KA), GST fusion proteins were
expressed in Escherichia coli and were prepared, purified,
analyzed, and quantified as described previously (5). Purified
recombinant GST-MKK4, GST-MKK4(KA), and GST-p46SAPK were
obtained commercially (Upstate Biotechnology, Inc.). Recombinant GST-MKK7 and GST-MKK7(KA) were prepared as described by Frangioni et al. (23). Briefly, following expression in culture,
bacterial pellets were washed in 50 ml of ice-cold STE (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM
EDTA) and resuspended in STE containing 100 µg of lysozyme. After a
15-min incubation on ice, dithiothreitol (5 mM final
concentration) and Sarkosyl (1.5% final concentration) were added, and
the resultant suspension was pulled several times through a 18-gauge
needle. Supernatant obtained following centrifugation of the suspension
at 10,000 × g for 5 min was adjusted to contain 4%
Triton X-100 and purified on glutathione-Sepharose beads. Fusion proteins were eluted from beads with 10 mM reduced
glutathione in a buffer containing 75 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM dithiothreitol, 0.1% Triton
X-100 and were concentrated by ultrafiltration using Centricon-10
(Amicon, Inc.)
In Vitro Kinase Assays--
Cells were transfected as indicated
in figure legends. After 24-48 h, cell lysates were prepared as
described previously (7) in 1 ml of a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM sodium vanadate, 50 mM sodium fluoride, 20 mM
-glycerophosphate, 10% glycerol, 1% Triton X-100,
and protease inhibitors. Protease inhibitors included 2 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml pepstatin, 0.5 µg/ml
leupeptin, and 1 µg/ml aprotinin. p46SAPK immune-complex
kinase assays were performed as described previously (7). Complexes
were incubated for 30 min at 30 °C in 30 µl of kinase buffer (25 mM HEPES, pH 7.2, 10% glycerol, 100 mM NaCl, 20 mM MgCl2, 0.1 mM sodium
vanadate, and protease inhibitors) containing 25 µM ATP,
5 µCi of [
-32P]ATP (3000 Ci/mmol, Amersham) and 2 µg of GST-c-Jun(1-79). Reactions were terminated by addition of
Laemmli buffer, boiled, resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and autoradiographed. Where indicated,
incorporated counts were counted with a Bio-Rad phosphorimager.
Equivalent expression of transfected p46SAPK was assessed
by immunoblotting. DLK and MLK3 immune-complex kinase assays were
performed as above except that 2 µg of GST-MKK4, GST-MKK1, GST-MKK3,
GST-MKK5, GST-MKK7, or their kinase negative mutants were substituted
as substrate were indicated in figure legends. MKK7 immune-complex
kinase assays were carried out as above except that 2 µg of GST-SAPK
was substituted as substrate.
In Vitro SAPK Activation Assays--
In vitro
activation of SAPK was determined essentially as described (7). FLAG
epitope-tagged DLK, DLK(K185A), MLK3, or MLK3(K144E) were
immunoprecipitated from 500 µg of transiently transfected COS 7 cell
lysate. In vitro reconstituted coupled kinase assays were
performed by combining indicated MLK immune complexes with either
recombinant GST-MKK4 or GST-MKK7 or their kinase negative mutants as
indicated in kinase buffer containing 50 µM ATP and 5 µCi of [
-32P]ATP (3000 Ci/mmol, Amersham Pharmacia
Biotech) and incubated at 30 °C for 60 min. Recombinant GST-SAPK and
GST-c-Jun (1-79)-Sepharose beads were added, and reaction mixes were
incubated for an additional 15 min at 30 °C. Reactions were
terminated by addition of Laemmli buffer, boiled, resolved by SDS-PAGE,
transferred to polyvinylidene difluoride membranes, and autoradiographed.
Affinity Precipitation--
FLAG-DLK, FLAG-MLK3, or vector only
were expressed and labeled with [35S]methionine in a
single reaction reticulocyte lysate-based in vitro
transcription/translation system according to the directions of the
manufacturer (Promega). One-tenth of each reaction mix was incubated
for 30 min at room temperature with or without 10 µg of GST-MKK7 and
25 µl of glutathione-agarose in a PBST buffer containing
phosphate-buffered 150 mM NaCl (pH 7.4), 1% Triton X-100,
and protease inhibitors. Beads were washed five times with PBST then
eluted in 5 mM reduced glutathione. Eluates were separated on SDS-PAGE under reducing conditions. Gels were fixed, treated with
ENHANCE (DuPont), dried, and autoradiographed.
Immunocytochemistry--
Adult Sprague-Dawley rats were perfused
intracardially with 4% paraformaldehyde in phosphate-buffered saline.
Nervous tissues were removed, washed in cold PBS, and cryoprotected in
sucrose prior to freezing in liquid nitrogen. Spinal cords were cut in 6-µm sections on a cryostat; 40-µm sections were obtained with a
sliding microtome of brain and cerebellum. Free-floating sections of
brain and cerebellum were used for immunocytochemistry as described (24). Primary antibody was detected with species-appropriate horseradish peroxidase-conjugated anti-IgG secondary antibodies and
developed with nickel enhanced 3,3'-diaminobenzidene tetrahydrochloride using the Vector Elite detection kit (Vector Laboratories). Indirect immunoflourescence studies utilized complementary secondary
antibodies conjugated to Cy2 or Cy3 (The Jackson Laboratory) as
indicated in the legends to Figs. 6-9. In studies of primary cortical
neurons in culture, cells were fixed with 4% paraformaldehyde and
permeabilized with 0.2% Triton X-100 in PBS. After exposure to the
primary antibody at 4 °C overnight, the bound antibody was detected
using a secondary antibody conjugated to Cy3 or Cy2.
Subcellular Fractionation Studies--
The preparation of intact
nuclei from adult rat cortex and from primary cortical neurons was
carried out by isopycnic banding at the 30-35% Iodoxanol interface,
in order to cause the least possible disruption of the nucleoprotein
complexes within them, following the manufacturers protocol (OptiPrep).
Rat Model of Sciatic Nerve Injury--
Adult male Sprague-Dawley
rats weighing 175-225 g were anesthetized with chloral hydrate. The
sciatic nerve was exposed in the gluteal region and cut. Control rats
were anesthetized and the sciatic nerve was exposed but not cut. At 5 min, 30 min, and 1 day after axotomy, animals were perfused
intracardially with 4% paraformaldehyde in phosphate-buffered saline.
The spinal cord in the region of the lumbar bulge was removed, washed
in cold PBS, cryoprotected in sucrose, and frozen in liquid nitrogen. Three animals at each time point were employed. A minimum of three sections per spinal cord were analyzed by immunoflourescence.
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RESULTS |
Distinct from MLK3, DLK Neither Phosphorylates Nor Activates MKK4
in Vitro--
Preliminary experiments demonstrated that catalytically
inactive MKK4 attenuated the ability of DLK to activate
p46SAPK when overexpressed in co-transfected COS 7 cells
(data not shown and Ref. 6). To more closely examine the potential
epistatic relationship between DLK and MKK4, the ability of DLK to
phosphorylate MKK4 in vitro was investigated.
Immunoprecipitated overexpressed DLK, MLK3, or their inactive mutants
were incubated with catalytically inactive recombinant GST-MKK4-KR in a
buffer containing radiolabeled ATP and magnesium. As reported by others
(10, 12), MLK3, but not its inactive mutant, phosphorylated GST-MKK4-KR
in vitro (Fig. 1A).
Contrary to expectation, DLK did not phosphorylate GST-MKK4-KR. In
similar experiments, the capacity of immunoprecipitated DLK to
phosphorylate in vitro catalytically inactive mutants of GST fusion proteins of MKK1, MKK3, or MKK5 was also tested (Fig.
1B). DLK did not phosphorylate recombinant MKK1, MKK3, or
MKK5 in vitro.

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Fig. 1.
MLK3 but not DLK phosphorylates recombinant
MKK4 in vitro. A, FLAG-tagged DLK
(F-DLK), MLK3 (F-MLK3), or catalytically inactive
DLK(K185A) (F-K185A) or MLK3(K144E) (F-K144E)
were immunoprecipitated from lysates prepared from transiently
transfected COS 7 cells. Indicated immunoprecipitates were assayed
in vitro for their capacity to phosphorylate purified
recombinant GST-MKK4(KR). Reaction mixes were separated on 12.5%
SDS-PAGE, transferred to nitrocellulose, and autoradiographed.
Immunoblots from corresponding experiments were used to evaluate
relative abundance of GST-MKK4(KR) in each reaction. B,
FLAG-tagged DLK (F-DLK) or DLK(K185A) (F-K185A)
were immunoprecipitated from lysates prepared from transiently
transfected COS 7 cells. Indicated immunoprecipitates were assayed
in vitro for their capacity to phosphorylate bacterially
expressed and purified recombinant kinase-negative GST-MKK1, GST-MKK3,
or GST-MKK5 as indicated. Immunoblots from corresponding experiments
were used to evaluate relative abundance of indicated GST-MKK in each
reaction. Experiments were repeated three times with similar
results.
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These unexpected results were confirmed by studying the ability of
immunoprecipitated DLK to activate MKK4 in an in vitro reconstitution experiment. Immunoprecipitated overexpressed DLK, MLK3,
or their catalytically inactive mutants were sequentially combined in
the presence of ATP and magnesium with recombinant MKK4 (or its kinase
negative mutant) and SAPK. SAPK activation was assayed by capture of
SAPK onto GST-c-Jun-agarose beads followed by kinase assay in the
presence of [
-32P]ATP. As reported previously, MLK3
but not its kinase negative mutant activated MKK4 and ultimately SAPK
(Fig. 2) (10, 12). However, in three
repeated experiments, DLK did not activate MKK4 in
vitro.

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Fig. 2.
MLK3 but not DLK activates MKK4 in
vitro. FLAG-tagged DLK (DLK wt), MLK3
(MLK3 wt), or catalytically inactive DLK(K185A)
(K185A) or MLK3(K144E) (K144E) were
immunoprecipitated from lysates prepared from transiently transfected
COS 7 cells. Immunoprecipitates were combined as indicated with
purified bacterially expressed GST-MKK4 (MKK4 wt) and
GST-p46SAPK (SAPK wt), or catalytically inactive
MKK4 (MKK4-KR), and incubated at 30 °C for 20 min in the presence of
ATP. Activation of SAPK was assayed in an in vitro kinase
assay following incubation of reaction mixes with GST-c-Jun-Sepharose
beads as described under "Materials and Methods." Experiments were
repeated three times with similar results.
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DLK Phosphorylates and Activates MKK7 in Vitro--
By analysis of
primary structure, MLK2 and MLK3 form a closely related group
structurally distant from DLK. Therefore, it was hypothesized that DLK
utilizes substrate or substrates distinct from MKK4. The recently
identified MAP kinase kinase MKK7/JNKK2 shown to specifically activate
SAPK was considered a potential candidate substrate (15, 25, 26). To
test this possibility, in vitro kinase assays were repeated
as above. Indeed, immunoprecipitated DLK phosphorylated a recombinant
catalytically inactive GST-MKK7 fusion protein in vitro;
immunoprecipitated MLK3 behaved in a similar fashion (Fig.
3A). In control experiments,
neither immunoprecipitated catalytically inactive DLK (K185A) nor
catalytically inactive MLK3 (K144E) phosphorylated GST-MKK7-KA.

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Fig. 3.
DLK and MLK3 phosphorylate and activate MKK7
in vitro. A, FLAG-tagged DLK
(F-DLK), MLK3 (F-MLK3), or catalytically inactive
DLK(K185A) (F-K185A) or MLK3(K144E) (F-K144E)
were immunoprecipitated from lysates prepared from transiently
transfected COS 7 cells. Indicated immunoprecipitates were assayed
in vitro for their capacity to phosphorylate bacterially
expressed and purified recombinant GST-MKK7-KA or GST-MKK4-KA. Reaction
mixes were separated on 12.5% SDS-PAGE, transferred to nitrocellulose,
and autoradiographed. Immunoblots from corresponding experiments were
used to evaluate relative abundance of GST-MKK7(KA) or GST-MKK4(KA) in
each reaction. B, FLAG-tagged DLK (F-DLK wt),
MLK3 (F-MLK3 wt), DLK(K185A) (F-K185A), or
MLK3(K144E) (F-K144E) were immunoprecipitated from lysates
prepared from transiently transfected COS 7 cells. Immunoprecipitates
were combined as indicated with purified GST-MKK7 (MKK7 wt)
or catalytically inactive GST-MKK7 (MKK7-KA) and incubated
at 30 °C for 60 min in the presence of ATP. Samples were incubated
with recombinant GST-p46SAPK (SAPK wt) and
GST-c-Jun and activation of SAPK was assayed in an in vitro
kinase assay. Experiments were repeated three times with similar
results.
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In vitro reconstitution coupled kinase assays were used to
confirm and extend these observations. As reported previously by others
(15, 26), bacterially expressed and purified wild type GST-MKK7
activated rSAPK in vitro without known prior activation by a
MAPKKK. To confirm that MKK7 catalytic activity was required for SAPK
activation in these experiments, a mutant GST-MKK7 was created in which
the invariant lysine in subdomain II (Lys76, known to
stabilize ATP in the ATP binding site in other kinases) was replaced
with alanine. GST-MKK7(KA) mutant failed to activate rSAPK in
vitro. Given these results, we examined whether overexpressed and
immunoprecipitated DLK or MLK3 would potentiate the activation of
GST-MKK7 in vitro. Immunoprecipitated DLK and MLK3, but not their catalytically inactive mutants, further activated recombinant GST-MKK7 and ultimately SAPK (Fig. 3B). Substitution of the
mutant GST-MKK7-KA in reconstitution experiments blocked the ability of
immunoprecipitated DLK and MLK3 to activate SAPK.
DLK Activates MKK7 in Vivo--
Experiments were performed to
confirm that DLK activates MKK7 in vivo. COS 7 cells were
transiently co-transfected with plasmid encoding FLAG-tagged MKK7 and
either Myc-tagged DLK or catalytically inactive Myc-DLK (K185A).
Activation of immunoprecipitated MKK7 was evaluated in an in
vitro coupled kinase assay using recombinant SAPK and GST-c-Jun
(1-79) as substrate (Fig. 4). Expression
of DLK but not catalytically inactive K185A resulted in the activation of MKK7. In control experiments, when DLK was co-expressed with catalytically inactive MKK7-KA, immunoprecipitated MKK7-KA failed to
phosphorylate rSAPK in vitro. These results demonstrate that overexpressed DLK can activate overexpressed MKK7 in
vivo.

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Fig. 4.
Overexpressed DLK activates MKK7 in
vivo. COS 7 cells were co-transfected as indicated with
plasmids encoding FLAG-tagged MKK7 or catalytically inactive
FLAG-tagged MKK7-KA and with plasmids encoding vector only, Myc-tagged
DLK, MEKK1 , or catalytically inactive myc-DLK(K185A) or MEKK1 -KM.
Cells were lysed after 24 h and immunoprecipitated FLAG-MKK7 was
combined with recombinant GST-SAPK and GST-c-Jun and assayed for
catalytic activity in vitro in a coupled kinase assay.
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DLK Associates with MKK7 in Vivo and in Vitro--
To examine
whether overexpressed DLK interacts in vivo with
overexpressed MKK7, myc-tagged DLK was co-expressed with FLAG-tagged MKK7 in COS 7 cells. As shown, FLAG-tagged MKK7 co-immunoprecipitated with myc-tagged DLK (Fig. 5A).
To confirm a direct interaction between DLK and MKK7, metabolically
labeled DLK expressed in a reticulocyte lysate system was incubated
with bacterially expressed and purified GST-MKK7 or control. As shown,
DLK co-purified only in the presence of GST-MKK7 when isolated by
glutathione-agarose affinity chromatography (Fig. 5B). MLK3
behaved in a similar fashion. Taken together with the results of the
experiments described above, these results provide evidence that MKK7
may serve as a necessary intermediate in a pathway between DLK or MLK3
and SAPK activation in vivo.

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Fig. 5.
DLK associates with MKK7. A,
COS 7 cells were cotransfected with indicated combinations of plasmid
encoding Myc-tagged DLK (0.5 µg), FLAG-tagged MKK7 (0.5 µg),
FLAG-MKK7-KA (0.5 µg), or empty vector (to 2 µg total). Cell
lysates were immunoprecipitated as indicated with anti-Myc (9E10) or
anti-FLAG (M2) antibody. Immunoprecipitates were separated by SDS-PAGE,
transferred to nitrocellulose, then immunoblotted as indicated with
anti-DLK polyclonal serum (C1) or anti-FLAG (M2) antibody. Twenty µg
of whole cell lysate obtained from Myc-DLK and FLAG-MKK7 transfected
cells were run in the left lanes and after immunoblotting
with the indicated antibodies served as mobility markers. B,
DLK and MLK3 or vector-only control were in vitro
transcribed and translated in the presence of
[35S]methionine. Samples were either mixed or not mixed
with GST-MKK7 as indicated. Following incubation with
glutathione-agarose, samples were washed and eluted with reduced
glutathione. Eluates were separated by SDS-PAGE and visualized by
autoradiography.
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Distribution of DLK, MLK3, and MKK7 Proteins in Normal Adult Rat
Nervous System--
To obtain evidence supporting the epistatic
relationship between endogenous DLK and MLK3 and their
potential substrates in brain, the subcellular localization of DLK,
MLK3, and MKK7 proteins was examined by immunohistochemistry (Fig.
6). Using the previously characterized C1
polyclonal serum generated against a COOH-terminal DLK-GST fusion
protein (5), DLK immunoreactivity was detected in neurons of the
central nervous system (Fig. 6, A-D). This finding was
consistent with previous observations using in situ RNA
hybridization in which DLK transcript was widely detected in neurons of
the central and peripheral nervous system (16). In pyramidal cells of
the cerebral cortex and in Purkinje cells of the cerebellum, strong DLK
immunoreactivity was also detected in apical dendrites. MLK3
immunoreactivity could not be distinguished above background in
neurons. Instead, MLK3 protein was identified primarily in cells of the
periventricular ependyma, choroid plexus, and meninges. (Fig. 6,
E and F and not shown). Using a previously
characterized MKK7 rabbit antiserum (24), MKK7, like DLK, was
identified predominantly in neurons of the central nervous system (Fig.
6, G-J). Whereas DLK and MKK7 immunoreactivity was
identified in the same cells of the cerebral cortex and cerebellum,
regional differences were detected in the intensity of MKK7 and DLK
staining (compare granule cells of the dentate gyrus of hippocampus,
Fig. 6, D, I, and J).

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Fig. 6.
Indirect immunohistochemistry demonstrating
localization of DLK, MLK3, and MKK7 in normal adult rat nervous
system. DLK (A-D) and MKK7 protein (G-J)
co-localized within neurons of the central and peripheral nervous
system including those of the cerebral cortex (compare A and
G) and cerebellum (B, H). MLK3 protein
was identified in the ependyma (E, F) but could
not be identified in neurons even by immunoflourescence (F).
Note DLK and MKK7 immunoreactivity within dendritic processes and
accentuated staining in a perinuclear and nuclear distribution
(C, H). Within the neurons of the hippocampus,
MKK7 immunoreactivity was most prominent within the neurons of the
lower leaflet of the dentate gyrus (I, J),
whereas DLK staining was more uniformly distributed (compare
D and I, J). Purkinje cells of the
cerebellum and their apical dendrites stained intensely for DLK and
MKK7 (B, H). Note differences in staining
intensity between DLK and MKK7 within the granular cell layer
(B, H).
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Identification of DLK, MKK7, and MKK4 in Cellular
Subfractions--
Several experiments were undertaken to study the
subcellular localization of DLK, MKK7, and MKK4. Normal rat cerebral
cortex was disrupted and cells were subfractionated by isopycnic
banding. Nuclear, cytosolic, and microsomal fractions were identified
by immunoblotting using subfraction specific markers (Fig.
7A). The purity of isolated
nuclear preparations were demonstrated as shown in Fig. 7, B
and C. Following separation of subfraction lysates on
SDS-PAGE and immunoblotting, DLK was identified most abundantly in the
microsomal fraction but was also prominent in the cytosol. Like MKK4,
MKK7 was particularly enriched in the cytosolic fraction. A small but
detectable quantity of DLK and MKK7 were identified within the nuclear
fraction (Fig. 7A). Neither antibodies directed against
phosphorylated-MKK4 (New England Biolabs) nor
phosphorylation-independent antibodies directed against MKK4 (Sigma and
Santa Cruz) recognized MKK4 in the nuclear fraction.

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Fig. 7.
Localization of DLK, MKK7, and MKK4 in
subfractionated cerebral cortex. A, cerebral cortex was
homogenized and cells were subfractionated as described under
"Materials and Methods." Twenty-µg aliquots of nuclear
(N), microsomal (M), and cytosolic (C)
fractions were separated on SDS-PAGE, transferred to nitrocellulose,
then immunoblotted as indicated with antibodies directed against DLK,
MKK7, MKK4, and phosphorylated MKK4. Fractions were also immunoblotted
with antibodies specific for 3 subunit of
sodium-potassium ATPase (alpha3), synaptophysin
(synapt), and histone 1 to demonstrate suitability of
fractionation. In order to demonstrate the purity of the nuclear
preparation, an aliquot was stained with DAPI indicating the presence
of DNA. Shown is an identical field visualized both by phase
(B) and immunoflourescence microscopy (C).
Immunoflourescence and confocal microscopy were used to analyze
additional aliquots for the presence of DLK (D) and MKK7
(E). Insets represent reconstructions of confocal
images in the z plane. Control experiments lacking primary
antibody or using primary antibody preadsorbed with a molar excess of
appropriate fusion protein failed to stain isolated nuclei (data not
shown).
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To assure that the nuclear localization of DLK and MKK7 suggested by
the immunoblotting experiments above was not due to contamination of
nuclear fractions with associated microsomal membranes, isolated nuclei
from rat cerebral cortex were fixed and analyzed by indirect immunoflourescence. Confocal microscopy demonstrated homogeneous distribution of DLK and MKK7 protein throughout the nucleus (Fig. 7,
D, E, and insets).
DLK Translocates to the Nucleus in an in Vivo Model of Neuronal
Injury--
Several components of the various MAP kinase pathways have
been demonstrated to translocate within cells following appropriate stimulation. For this reason, the subcellular localization of DLK was
also examined by immunoflourescence microscopy in an in vivo
model of neuronal injury. Axotomy of the rat sciatic nerve led to
translocation of DLK from a predominantly non-nuclear cell compartment
to a distinctly nuclear localization within motor neurons of the lumbar
spinal cord beginning within 5 min of axonal injury (Fig.
8). Strong nuclear DLK immunoreactivity
was maintained at 30 min and at 24 h following injury. Three
animals at each time point were examined and a minimum of three
sections per spinal cord was evaluated by indirect immunoflourescence
for each animal studied. Sham-operated animals were used as controls at
each time point studied. In these control animals, the intensity of DLK immunoreactivity in the nucleus was never observed to be above that
observed in the cytoplasm. However, in sections from injured animals,
from 2 to 14 cells per section exhibited dramatically increased nuclear
localization of DLK. Phosphorylated MKK4 was not identified in the cell
nucleus either in control animals or following axotomy at any time
point studied (data not shown). However, MKK7, like DLK, accumulated
within the nucleus following axotomy.

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Fig. 8.
DLK translocates to the nucleus following
truncation of the sciatic nerve in an in vivo model of
cellular injury. At indicated times following sciatic nerve
axotomy, normal adult rats were perfused fixed with 4%
paraformaldehyde by left ventricular puncture. Spinal cord in the
region of the lumbar bulge was removed, prepared, and studied by
immunoflourescence microscopy. Shown are representative sections of
immunoflourescence staining of motor neurons for DLK (A,
C, E, G, I, J)
and MKK7 (B, D, F, H) in
control animals (A, B, I) and at 5 min
(C, D), 30 min (E, F), and
24 h (G, H) following axotomy.
Arrows emphasize location of nuclei. Note DLK
immunoreactivity along plasma membranes best visualized in
A, E, and J. I and
J show a larger field of lumbar spinal cord sections 20 min
after sham operation or sciatic nerve axotomy, respectively.
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DLK Immunoreactivity Co-localizes with That of MKK7 in Neuronal
Culture--
To investigate the subcellular localization of DLK, MKK7,
and MKK4 proteins in an additional system, embryonic rat cortical neurons in primary culture were examined by immunocytochemistry. DLK
immunoreactivity was detected in a prominent perinuclear and intranuclear distribution in neurons (Fig.
9, A, G,
I, and K). DLK was also detected within all
neuronal processes and was prominently associated with plasma membrane
(Fig. 9, A, G, I, K, and
Fig. 9A, inset). This was consistent with our published
observations that DLK was associated with plasma membrane fractions
isolated from synaptosomal preparations (16). Like DLK, MKK7
immunoreactivity was identified in perinuclear and intranuclear
compartments as well as in cell processes (Fig. 9, B,
H, J, L). Using three distinct commercially obtained antibodies, MKK4 immunoreactivity was detected in
neuron cell processes and in a non-nuclear distribution within the
neuron cell body that was distinct from that of DLK and MKK7 (Fig. 9,
C and D).

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Fig. 9.
DLK immunoreactivity co-localizes with that
of MKK7 in embryonic cortical neurons in primary culture. Indirect
immunoflourescence microscopy was used to investigate the subcellular
localization of DLK (A, G), MKK7 (B,
H), MKK4 (C), phosphorylated-MKK4 (D),
SAPK /JNK1 (E), and SAPK /JNK3 (F) in rat
cortical neurons in primary culture. Enlargement of neuronal process in
A (inset) demonstrates localization of DLK protein along
plasma membrane. Cells were doubled-labeled for neurofilament protein
(Cy3, red) (I, J) and for either DLK
(G) or MKK7 (H) (both Cy2, green).
Double photographic exposures to demonstrate superimposition of
staining patterns for DLK and neurofilament protein (K) or
MKK7 and neurofilament protein (L) are shown.
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The observations that DLK and MKK7 occupied compartments distinct
from MKK4 suggested the hypothesis that spatially and functionally distinct SAPK pathways composed of unique components exist within individual cells. To examine this hypothesis further, the subcellular distribution of SAPK
/JNK3 and SAPK
/JNK1 were investigated by immunohistochemistry in primary neuronal culture. SAPK
/JNK3
immunoreactivity was identified exclusively in the neuronal nuclei
(Fig. 9F). In contrast, SAPK
/JNK1 immunoreactivity was
extranuclear and localized predominantly to the neuronal
processes (Fig. 9E). Therefore, different SAPK species and
different MKK species occupy distinct compartments within neurons.
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DISCUSSION |
This paper establishes DLK as a MAPK kinase kinase capable of
associating with, phosphorylating, and activating MKK7. That DLK
functions in this role in a physiologic setting is supported by
observations that endogenous DLK and MKK7 are present in the same
compartments within neurons. Moreover, both biochemical evidence and
analysis of subcellular localization indicate that DLK does not use
MKK4 as an intermediate in SAPK activation.
MAPKKK substrate specificity appears to be defined by the combination
of several factors that include (but may not be limited to) the
biochemical affinity of the kinase for its substrate, cell
type-specific expression, and differential targeting of unique MAPKKKs
to specific subcellular compartments. The results presented herein
provide an example of each of these determinants. In a fashion
reminiscent of the specificity of MKK3 and MKK6 for
p38mapks (27-29) and of MKK7 for the
p46/p54SAPKs (15, 25, 26, 30), DLK appears to possess
enzymatic specificity for MKK7. Conversely, our results demonstrate
that MKK7 may serve as a substrate for several MAPKKKs, including MLK3
and MEKK1. Precedence from the SAPK pathway literature suggests that
this is not an unexpected paradox since MKK4 appears to behave as a substrate for multiple MAPKKKs at least in biochemical assays (reviewed
in Ref. 2). However, by imposing a number of additional determinants of
interaction specificity, nature appears to have restricted interactions
between specific kinases and substrates in the SAPK-like pathways.
That DLK and MLK3 are expressed in different cell types in the nervous
system provides one example by which interactions between members of
the MLK family and specific MAPK kinases may be restricted. Specification of subcellular compartmentation within individual cells
may provide a second mechanism determining these interactions. Recognition that various MAPK kinases and MAPK kinase kinases are
targeted to distinct subcellular compartments supports this hypothesis.
The subcellular localization of endogenous DLK within neurons is
complex. In the nerve terminal, endogenous DLK is present both in
cytosol and in subcellular fractions enriched in plasma membrane (16).
In the cell body, DLK occupies compartments that include the plasma
membrane, cytosol, and nucleus. Recently, the distinct subcellular
compartmentation of other MAPKKKs has also been described. Endogenous
MEKK family members occupy distinct subcellular compartments in COS
cells including Golgi, post-Golgi, and nuclear compartments (31), and
the mixed lineage kinase MLK2 co-localizes with phosphorylated JNK1/2
along microtubules in injected Swiss 3T3 cells (9). Our results show
that like the MAPKKKs, endogenous MKK4 and MKK7 also occupy distinct
neuronal subcellular compartments.
Our intent in examining the subcellular localization of DLK and its
potential MAPK kinase substrates was to provide evidence supporting the
biochemical data suggesting that endogenous DLK uses MKK7 and not MKK4
as substrate. Consistent with the biochemical data, the subcellular
compartmentation of DLK and MKK7 are similar in neurons; the
subcellular compartmentation of MKK4 is distinct from that of DLK and
MKK7. Because DLK and MKK7 have overlapping but not identical
subcellular compartmentation in neurons, genetic and other experimental
approaches will be necessary to further validate the hypothesis that
endogenous DLK specifically uses MKK7 as substrate.
SAPK isoforms also appear to be differentially compartmentalized within
neurons. Ten splice isoforms derived from three SAPK/JNK genes have
been isolated from brain (32). Although in most cases the specific
splice isoform has not been studied, SAPK species have been identified
associated with several cellular compartments, including cytosol,
microtubules (9), and nuclei (13, 33-36). Our observation that
endogenous SAPK
/JNK1 and SAPK
/JNK3 occupy distinct subcellular
compartments in the same cell extends these observations. Beyond
differences in subcellular compartmentation, limited evidence presently
exists to support the hypothesis that discretely localized SAPK species
within the same cell have dissociated function. The most direct
evidence presently available in this regard derives from the
observation that genetic deletion of JNK3, but not deletion of JNK1 or
2, protects mice from hippocampal neuron injury and apoptotic cell
death associated with chronic administration of a glutamate
receptor-agonist (37). That SAPK
/JNK3 and SAPK
/JNK1 are
differentially compartmentalized within individual neurons is
consistent with the results of these JNK null-mutant experiments.
Together, these observations suggest the existence of spatially and
functionally discrete SAPK species within the same cell.
Translocation between subcellular compartments may provide an
additional mechanism by which MLK protein kinases gain access to their
specific substrates. This report documents that DLK and MKK7 can
translocate to the cell nucleus. The mechanism and functional significance of translocation of DLK and MKK7 to the neuronal nucleus
following axonal injury will require additional detailed study. Each of
these protein kinases is present in neuronal nuclei in vivo
under basal conditions, yet each accumulates following axotomy. That
nuclear accumulation of DLK occurs rapidly suggests that this kinase
translocates from extranuclear pools of pre-existing protein. The
nuclear translocation of MAP kinase kinase kinases following a cellular
stimulus has not been observed previously, although MEKK1 has been
identified in the nucleus of COS cells under basal conditions (7). It
is not known whether stimulus-induced nuclear translocation is a
response common to other MAPKKK proteins.
Activation of SAPK (particularly SAPK
/JNK3) has been implicated in
the neuronal response to various types of cellular injury (19, 21, 35).
SAPK-activating and -interfering mutant overexpression studies
performed in PC12 cells first implicated SAPK in the neuronal apoptotic
response to nerve growth factor withdrawal (34). Recent studies
demonstrated that SAPK
/JNK3 translocates to the nucleus of
hippocampal neurons following hypoxic injury in humans (37). Indeed,
deletion of SAPK
/JNK3 protects mice from hippocampal neuron injury
and programmed cell death associated with chronic administration of a
glutamate receptor agonist (35). Finally, it was recently reported that
sciatic nerve axotomy induces sustained SAPK, c-Jun, and AP-1
activation in lumbar dorsal root ganglion axons (19). Rapid and
sustained translocation of DLK corresponds temporally with this
activation. Combined with these observations, the results reported
herein provide provocative preliminary evidence suggesting that DLK
participates in a cellular response to axonal injury mediated by MKK7
and SAPK
/JNK3.