Originally published In Press as doi:10.1074/jbc.M001009200 on May 4, 2000
J. Biol. Chem., Vol. 275, Issue 27, 20533-20539, July 7, 2000
NESK, a Member of the Germinal Center Kinase Family That
Activates the c-Jun N-terminal Kinase Pathway and Is Expressed
during the Late Stages of Embryogenesis*
Kuniko
Nakano,
Junji
Yamauchi,
Kazuhiro
Nakagawa
,
Hiroshi
Itoh, and
Naomi
Kitamura§
From the Department of Life Science, Faculty of Bioscience and
Biotechnology, Tokyo Institute of Technology, Nagatsuta,
Midori-ku, Yokohama 226-8501, Japan
Received for publication, February 8, 2000, and in revised form, April 7, 2000
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ABSTRACT |
The c-Jun N-terminal kinase (JNK) signaling
pathway plays a crucial role in cellular responses stimulated by
stress-inducing agents and proinflammatory cytokines. The group I
germinal center kinase family members selectively activate the JNK
pathway. In this study, we have isolated a mouse cDNA encoding a
protein kinase homologous to Nck-interacting kinase (NIK), a member of
the group I germinal center kinase family. This protein kinase is
expressed during the late stages of embryogenesis, but not in adult
tissues, and thus named NESK (NIK-like
embryo-specific kinase). NESK
selectively activated the JNK pathway when overexpressed in HEK 293 cells but did not stimulate the p38 kinase or extracellular
signal-regulated kinase (ERK) pathways. NESK-induced JNK activation was
inhibited by the dominant negative mutants of MEKK1 and MKK4. Tumor
necrosis factor (TNF)-
or TNF receptor-associated factor 2 (TRAF2)
stimulated the NESK activity. Furthermore, the dominant negative NESK
mutant inhibited the JNK activation induced by TNF- or TRAF2. These results suggest that NESK, a novel activator of the JNK pathway, functions in coupling TRAF2 to the MEKK1 
MKK4 JNK kinase
cascade during the late stages of mammalian embryogenesis.
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INTRODUCTION |
The germinal center kinase
(GCK)1 family is a subfamily
of the Ste20 family of protein kinases (1). GCK family members have an
N-terminal kinase domain and a C-terminal regulatory domain. The GCK
family is divided into two structurally and functionally distinct
groups (1). Group I GCKs include GCK itself, hematopoietic progenitor
kinase-1, GCK-like kinase, GCK-related kinase, Nck-interacting kinase
(NIK), kinase homologous to SPS1/STE20, hematopoietic progenitor kinase/GCK-like kinase, and TNF receptor-associated factor 2 (TRAF2)- and Nck-interacting kinase. These kinases selectively activate the
c-Jun N-terminal kinase (JNK) signaling pathway when overexpressed in
cultured cells but do not stimulate the p38 kinase or extracellular signal-regulated kinase (ERK) signaling pathways (2-13). Group II GCKs
share catalytic domain homology with group I GCKs, but their C-terminal
regulatory domains differ significantly from those of group I GCKs
(14-17). Group II GCKs do not activate any of the known
mitogen-activated protein kinase pathways.
The JNK pathway plays a crucial role in cellular responses stimulated
by a variety of stress-inducing agents, including osmotic and heat
shock, UV irradiation, and proinflammatory cytokines such as tumor
necrosis factor (TNF)- and interleukin 1 (18). JNKs are activated
through threonine and tyrosine phosphorylations by mitogen-activated
protein kinase kinases such as MKK4 and MKK7, which are in turn
phosphorylated and activated by mitogen-activated protein kinase kinase
kinase, including MEKK1, mixed lineage kinase 2, and mixed lineage
kinase 3 (18). Many members of the group I GCK family, including GCK,
GCK-related kinase, GCK-like kinase, hematopoietic progenitor
kinase/GCK-like kinase, and TRAF2- and Nck-interacting kinase, have
been shown to mediate the TNF--induced JNK activation (5, 7, 10, 11,
13). Among them, GCK, GCK-related kinase, and TRAF2- and
Nck-interacting kinase have been implicated in mediating the
TNF--induced JNK activation through TRAF2 (7, 10, 13). The JNK
activation by group I GCKs such as GCK and GCK-like kinase is mediated
through the MEKK1 and MKK4 kinase cascade (5, 10), whereas TAK1, but not MEKK1, functions as a mitogen-activated protein kinase kinase kinase in the hematopoietic progenitor kinase/GCK-like kinase-induced JNK activation (11). NIK and TRAF2- and Nck-interacting kinase interact
with the Src homology 2-Src homology 3 domain-containing adapter
protein Nck and have been proposed to link protein tyrosine kinase
signals to the JNK activation (8, 13).
The Drosophila Misshapen and C. elegans Mig-15
proteins are highly homologous to mammalian NIK, both within and
outside of the kinase domain (19). The Misshapen protein functions
upstream of basket, the Drosophila homologue of
JNK, and hemipterous, a homologue of MKK7, to stimulate
dorsal closure in the Drosophila embryo (19). Dorsal closure
occurs during the later stages in Drosophila embryogenesis
and involves cell migrations and shape changes that position and fuse
the lateral epidermal primordia over the aminoserosa. The
Drosophila TRAF binds Misshapen in vitro, and
coexpression of Misshapen and Drosophila TRAF leads to the synergistic activation of JNK (20). The C. elegans Mig-15 is necessary for several developmental processes in C. elegans.
mig-15 mutants have a variety of developmental defects including
defects in Q-neuroblast migration and muscle arm targeting (19). Thus, GCKs may be responsible for some of the developmental processes in mammals.
We report here a novel mammalian group I GCK family kinase isolated
from mouse embryo by a PCR-based screen for cDNA clones of protein
kinases. It is expressed during the late stages of mouse embryogenesis,
but not in various adult tissues, and has the highest homology to NIK.
Thus, this newly identified kinase was designated NESK
(NIK-like embryo-specific
kinase). We demonstrate in this report that NESK, like
other group I GCK family members, can selectively activate the JNK
pathway when overexpressed in HEK 293 cells. Furthermore, dominant
negative forms of MEKK1 and MKK4 inhibited the NESK-induced JNK
activation, and a dominant negative NESK mutant inhibited TRAF2-induced
JNK activation, suggesting that NESK functions downstream of TRAF2 and
upstream of MEKK1 and MKK4 in the JNK signaling pathway.
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EXPERIMENTAL PROCEDURES |
PCR Cloning of Partial cDNAs for Protein
Kinases--
Degenerate oligonucleotide primers corresponding to
subdomains VIb and IX (amino acid sequences VHRDL and
D(V/M)W(S/A)(F/Y), respectively) of the catalytic domain of protein
kinases were chemically synthesized. The sequences of the primers were
as follows: forward primer,
5'-GT(G/T)CA(C/T)(A/C)G(A/G/C/T)GA(C/T)(C/T)T-3'; reverse primer,
5'-G(A/T)A(G/T)(G/C)(A/T)CCA(A/G/C/T)A(T/C)(G/A)TC-3'. Double-stranded
cDNA from mouse 11-day embryo (QUICK-CloneTM;
CLONTECH) was amplified by PCR with the primers,
using a Gene AmpTM DNA amplification reagent kit (Takara Shuzo Co.).
Thirty cycles were performed with a step cycle profile of 30 s at
94 °C, 30 s at 35 °C, and 2 min 30 s at 72 °C. The
PCR products were purified by agarose gel electrophoresis and subcloned
into pT7Blue vector (Novagen) for DNA sequencing.
Library Screening and Sequence Analysis--
The cloned PCR
product was used as a probe to screen a 
gt11 mouse whole embryo (15 days) cDNA library (CLONTECH) to obtain a
full-length cDNA clone. Hybridization to nylon replica membranes (Hybond-N+; Amersham Pharmacia Biotech) was performed at
42 °C for 16 h with 32P-labeled probe in a solution
containing 50% formamide, 5× Denhardt's solution (0.1% bovine serum
albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficoll), 5× SSPE (0.75 M NaCl, 50 mM sodium phosphate, pH 7.4, 6 mM EDTA), 0.5% SDS, and 0.2 mg/ml salmon sperm DNA. The
probe was labeled using the Rediprime DNA labeling system (Amersham
Pharmacia Biotech). The membranes were washed twice with 1× SSPE
containing 0.1% SDS at 65 °C for 15 min. Hybridization-positive phage clones were isolated by repeated plaque purification. The DNA
sequences were determined from both strands by the chain termination method.
Northern Blot Analysis--
Mouse adult and embryo
multiple-tissue Northern blot membranes (CLONTECH)
were hybridized in the same hybridization solution used for library
screening at 42 °C for 16 h with the 32P-labeled
probe. The membranes were washed twice with 1× SSPE containing 0.1%
SDS at 65 °C for 15 min. The hybridization probe was the PCR clone
of NESK.
Expression Plasmids--
Full-length NESK and the kinase domain
of NESK (
NESK) were cloned into mammalian FLAG tag expression
vectors (pME18S-FLAG and pCMV-FLAG) by PCR using two oligonucleotide
primers. A catalytically inactive NESK mutant (pME18S-FLAG-NESK(K54E)
and pCMV-FLAG-NESK(K54E)) was created with the QuickChangeTM
mutagenesis kit (Stratagene). Complementary DNAs for human TRAF2 and a
region of human MEKK1 corresponding to mouse MEKK1 (21) were
amplified from human fetal brain cDNA
(CLONTECH). Dominant negative human MEKK1 was constructed as described previously (21). The DNAs were cloned into
mammalian VSVG or FLAG tag expression vector (pCMV-VSVG, pCMV-FLAG).
pCMV-FLAG-MKK4(K95R), pCMV-HA-p38, pCMV-Ras(G12V), and
pGEX2T-ATF2-(1-76) were described previously (22, 23). SR-HA-JNK,
SR-HA-ERK, and pGEX2T-c-Jun-(1-223) were kindly provided by Dr. M. Karin (University of California, San Diego).
Cell Culture and Transfection--
COS7 and HEK 293 cells were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum. The cells were cultured at 37 °C in a humidified
atmosphere containing 5% CO2. Plasmid DNAs were
transfected into COS7 or HEK 293 cells by FuGENETM6 transfection
Reagent (Roche Molecular Biochemicals). The final amount of transfected
DNA was adjusted with empty vector, pME18S or pCMV.
In Vitro Kinase Assays--
At 48 h after transfection, the
cells were lysed with 400 µl of lysis buffer A (20 mM
HEPES-NaOH, pH 7.5, 3 mM MgCl2, 100 mM NaCl2, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM EGTA, 1 mM Na3 VO4,
10 mM NaF, 20 mM 
-glycerophosphtate, and
0.5% Triton X-100). The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was incubated with 0.2 µg of an
anti-FLAG antibody (M2 monoclonal antibody; Eastman Kodak Co.) and 20 µl of a 50% slurry of protein A-Sepharose (Amersham Pharmacia
Biotech) for 1.5 h at 4 °C. The immune complexes were precipitated and washed twice with lysis buffer A and twice with reaction buffer A (20 mM HEPES-NaOH, pH 7.5, 1 mM dithiothreitol, 10 µM
Na3VO4, 2 mM -glycerophosphtate,
0.1 mM phenylmethanesulfonyl fluoride, 0.1 µg/ml
leupeptin, and 0.1 mM EGTA). The precipitates were
incubated in 30 µl of reaction buffer A containing 5 µg of myelin
basic protein (MBP) (Sigma), 20 µM ATP, and 5 µCi of
[
-32P]ATP (NEN Life Science Products) at 30 °C for
20 min. The reaction was stopped by adding 10 µl of 4× Laemmli
sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 30 mM dithiothreitol, and 10% glycerol). The mixture was
heated at 95 °C for 5 min. The proteins were separated by
SDS-polyacrylamide gel electrophoresis. The radioactivity incorporated into MBP was detected by autoradiography. Assays were performed at
least three times, and representative results are shown in the figures.
Mitogen-activated Protein Kinase Assays--
The cells were
starved with serum-free medium 24 h post-transfection. At 48 h after transfection, the cells were lysed with 400 µl of lysis
buffer A. The lysates were centrifuged at 14,000 rpm for 10 min at
4 °C. The supernatant was incubated with 0.2 µg of an anti-HA
antibody (Roche Molecular Biochemicals) and 20 µl of a 50% slurry of
protein A-Sepharose for 1.5 h at 4 °C. The immune complexes
were precipitated and washed twice with lysis buffer A and twice with
reaction buffer A. The precipitates were incubated in 30 µl of
reaction buffer A containing 5 µg of GST-c-Jun-(1-223) for JNK
assay, 5 µg of GST-ATF2 for p38 assay, or 5 µg of MBP for ERK
assay, 20 µM ATP, and 5 µCi of
[-32P]ATP at 30 °C for 20 min. The reaction was
stopped by adding 10 µl of 4× Laemmli sample buffer. The mixture was
heated at 95 °C for 5 min. The proteins were separated by
SDS-polyacrylamide gel electrophoresis. The radioactivity incorporated
into GST-c-Jun-(1-223), GST-ATF2, or MBP was detected by
autoradiography. Assays were performed at least three times, and
representative results are shown in the figures.
Immunoblotting--
Aliquots of cell lysates were boiled in
Laemmli sample buffer. The boiled samples were electorophoresed on
polyacrylamide gels, and the proteins were electrophoretically
transferred to nitrocellulose membranes. After the membranes were
blocked, the separated proteins were visualized by the enhanced
chemiluminescence detection system (Amersham Pharmacia Biotech), using
anti-rabbit or anti-mouse Ig antibody conjugated with horseradish
peroxidase (Amersham Pharmacia Biotech) as a secondary antibody.
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RESULTS |
Isolation of a cDNA Clone Encoding a Novel Protein Kinase,
NESK--
To isolate cDNA clones encoding novel protein kinases,
we used the strategy of PCR amplification with degenerative
oligonucleotide primers corresponding to conserved amino acid sequences
in the catalytic domain of protein kinases. The amplified PCR products of approximately 200 base pairs from mouse 11-day embryo cDNA were
cloned in the plasmid vector and identified by DNA sequencing. Sequences of 20 different protein kinases were cloned. Two of them were
distantly related with other known protein kinases. We previously
reported one of them (24). We designated the other clone NESK and
characterized it further.
Northern blot analysis using the PCR clone as a probe revealed that the
mouse embryo produced significant amounts of NESK mRNA. Thus, to
obtain a full-length cDNA clone for mouse NESK, a cDNA library
from the mouse embryo was screened using the PCR clone as a probe.
Eight hybridization-positive clones were obtained from about 8 × 105 phage. The clone that contained the largest cDNA
was sequenced to determine the primary structure of mouse NESK.
Amino Acid Sequence of NESK--
The nucleotide sequence of the
cDNA predicted an open reading frame of 1455 amino acids with a
calculated molecular mass of 167,325 (Fig.
1A). The deduced amino acid
sequence contained a kinase catalytic domain in the N terminus that
included 12 kinase subdomains (Fig. 1B). Comparison of the
amino acid sequence of the kinase domain of NESK with other sequences
showed it to be most similar to that of NIK (8), a member of the group
I GCK family, with 57% identity (Fig. 1B). In addition,
NESK had a C-terminal germinal center kinase homology region. It shared
38% amino acid identity with NIK (Fig. 1C). However, NESK
was only 20% identical to NIK in the intermediate region. While NIK
has two potential Src homology 3 domain binding sites (8), NESK
contained three possible Src homology 3 binding sites in the
intermediate region. NESK shared 46 and 56% amino acid identity in the
kinase domain and 33 and 32% identity in the germinal center kinase
homology region with C. elegans Mig-15 and
Drosophila Misshapen proteins, respectively (Fig. 1,
B and C).
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Fig. 1.
Deduced amino acid sequence of NESK and
alignment with related proteins. A, NESK contains an
open reading frame of 1455 amino acids. The kinase domain is marked by
a single underline. The C-terminal domain is
marked by a broken underline. Three proline-rich
motifs are shown in black. B, amino acid
alignment of the kinase domain of NESK with related proteins. Identical
amino acids are shown in black. The kinase subdomains are
indicated with Roman numerals. C,
amino acid alignment of the C-terminal domain of NESK with related
proteins. Identical amino acids are shown in black.
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Expression Pattern of NESK mRNA--
The expression of NESK
mRNA was examined in a variety of mouse adult tissues and at
various stages of mouse embryogenesis by Northern blot analysis using
the first PCR product of NESK as a probe. The probe hybridized to a
transcript of approximately 9.0 kilobases in the late stages of
embryogenesis. However, no hybridized transcript was detected in any
adult tissues examined (Fig. 2). These
results suggest that NESK functions in the late stages of
embryogenesis.
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Fig. 2.
Northern blot analysis of NESK mRNA.
An embryo (A) and an adult tissue (B) mouse
Northern blot were hybridized with a radioactive NESK probe.
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Kinase Activity of NESK--
To determine whether NESK has kinase
activity, COS7 cells were transfected with the FLAG-tagged full-length
NESK or the FLAG-tagged truncated NESK containing only the kinase
domain, and an immune complex kinase assay was performed on NESK
immunoprecipitates using MBP as a substrate. Although the
immunoprecipitates from cells transfected with vector alone
phosphorylated MBP, the kinase activity was markedly increased in
immunoprecipitates from cells transfected with the full-length NESK
(Fig. 3, lanes 1 and 2). The kinase activity was much higher in
immunoprecipitates from cells transfected with the kinase domain of
NESK (Fig. 3, lane 4). To rule out the
possibility that an associated kinase coprecipitating with NESK may
account for the kinase activity, a FLAG-tagged kinase-defective NESK
mutant in which Lys54 in the ATP binding domain was
replaced with a glutamic acid (K54E) was expressed in COS7 cells. The
level of phosphorylation of MBP by the K54E mutant was much lower than
that of the kinase-active NESK (Fig. 3, lane 3).
These results indicate that NESK is a functional protein kinase.
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Fig. 3.
Immune complex kinase assay of NESK.
COS7 cells were transfected with 2 µg of an empty vector (pME18S)
(lane 1), pME18S-FLAG-NESK (lane
2), pME18S-FLAG-NESK(K54E) (lane 3),
and pME18S-FLAG-NESK (lane 4). At 48 h
after transfection, the cells were collected, and immune complex kinase
assays were performed with an anti-FLAG antibody (M2) using MBP as a
substrate (upper panel). Expression levels of
proteins were verified equivalent by immunoblotting using an anti-FLAG
antibody (bottom panel).
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NESK Activates JNK, but Not p38 Kinase or ERK, in Transfected HEK
293 Cells--
To examine whether NESK can activate the mammalian JNK
cascade, we co-transfected HEK 293 cells with mammalian expression vectors encoding the full-length NESK, the kinase-inactive NESK (K54E),
or the kinase domain of NESK (NESK) and an HA epitope-tagged JNK1.
Recombinant JNK was then immunoprecipitated from cell lysates and used
in a protein kinase assay with GST-c-Jun as a substrate. Transfection
of cells with the full-length NESK resulted in JNK1 activation (Fig.
4A, lane
2), while cells transfected with vector alone showed little
activation (Fig. 4A, lane 1).
Transfection of cells with the kinase-inactive form of NESK (K54E) did
not result in JNK1 activation (Fig. 4A, lane 3).
Transfection of cells with the kinase domain of NESK (NESK) resulted
in strong JNK1 activation (Fig. 4A, lane
4). Thus, the kinase activity of NESK is required for JNK
activation.
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Fig. 4.
Activation of JNK, but not p38 or ERK, by
NESK in transfected HEK 293 cells. A, HEK 293 cells
(5 × 105 cells/60-mm dish) were transfected with 1 µg of SR-HA-JNK1 (lanes 1-5) plus 2 µg of
the empty vector pCMV alone (lane 1), 2 µg of
pCMV-FLAG-NESK (lane 2), 2 µg of
pCMV-FLAG-NESK(K54E) (lane 3), 2 µg of
pCMV-FLAG-NESK (lane 4), and 2 µg of
pCMV-FLAG-MEKK1 (lane 5). The cells were
collected 48 h after transfection, and immune complex kinase
assays were performed with an anti-HA antibody using GST-c-Jun-(1-223)
as a substrate (upper panel). B, HEK
293 cells were transfected with 1 µg of pCMV-HA-p38 (lanes
1-5), plus 2 µg of the same expression vectors
(lanes 1-4) as described in A.
Otherwise, the cells were treated with anisomycin (20 µg/ml) for 20 min (lane 5). The cells were collected 48 h
after transfection or after anisomycin treatment, and immune complex
kinase assays were performed with an anti-HA antibody using GST-ATF2 as
a substrate (upper panel). C, HEK 293 cells were transfected with 1 µg of SR-HA-ERK (lanes
1-5), plus 2 µg of the same expression vectors
(lanes 1-4) as described in A and 2 µg of pCMV-Ras(G12V) (lane 5). The cells were
collected 48 h after transfection, and immune complex kinase
assays were performed with an anti-HA antibody using MBP as a substrate
(upper panel). Expression levels of proteins were
verified equivalent by immunoblotting using an anti-HA antibody
(middle panel) and an anti-FLAG antibody
(bottom panel).
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To examine whether NESK can also activate p38 kinase or ERK, HEK 293 cells were transiently co-transfected with the full-length NESK,
NESK(K54E), or NESK along with HA epitope-tagged p38 kinase or ERK2.
p38 kinase or ERK2 was then immunoprecipitated, and its activity was
assayed by phosphorylation of either GST-ATF2 or MBP. Treatment of
cells with anisomycin strongly activated p38 kinase (Fig.
4B, lane 5). However, no increase in
p38 kinase activity was observed when NESK and its mutants were
overexpressed in HEK 293 cells (Fig. 4B, lanes
1-4). Similarly, although expression of Ras(G12V) in HEK
293 cells strongly activated ERK2 (Fig. 4C, lane
5), ERK2 activity was not increased when NESK and its
mutants were overexpressed in HEK 293 cells (Fig. 4C,
lanes 1-4). These results suggest that NESK does
not play a role in the p38 kinase and ERK pathways and that the
activation of the JNK pathway by NESK is specific.
Inhibition of NESK-induced JNK Activation by the Dominant Negative
Mutants of MKK4 and MEKK1--
MKK4 is an upstream activator of JNK,
which phosphorylates and activates JNK. To determine whether NESK
activates JNK through MKK4, HEK 293 cells were co-transfected with
expression vectors encoding NESK and a dominant negative mutant of MKK4
to determine whether the dominant negative mutant of MKK4 could inhibit
the NESK-induced JNK activation. The expression of the dominant
negative form of MKK4 inhibited the JNK activity induced by NESK (Fig. 5, lanes 2 and
4), suggesting that NESK functions upstream of MKK4.
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Fig. 5.
Inhibition of NESK-induced JNK activation by
dominant negative kinase mutants of MEKK1 and MKK4 in HEK 293 cells. HEK 293 cells (5 × 105 cells/60-mm dish)
were transfected with 1 µg of SR-HA-JNK1 (lanes
1-4) plus 2 µg of pCMV-FLAG-NESK (lanes
2-4), 2 µg of pCMV-FLAG-MEKK1(KR) (lane
3), and 2 µg of pCMV-FLAG-MKK4(KR) (lane
4). The final amount of DNA was adjusted to 5 µg with
empty vector. The cells were collected after 48 h, and immune
complex kinase assays were performed with an anti-HA antibody using
GST-c-Jun-(1-223) as a substrate (top panel).
Expression levels of proteins were verified equivalent by
immunoblotting using an anti-HA antibody (middle
panel) and an anti-FLAG antibody (bottom
panel).
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MEKK1 is a physiological activator of MKK4, which phosphorylates and
activates MKK4. To determine whether NESK activates JNK through MEKK1,
HEK 293 cells were co-transfected with expression vectors encoding NESK
and a dominant negative mutant of MEKK1. The expression of the dominant
negative form of MEKK1 inhibited the JNK activity induced by NESK (Fig.
5, lane 3), suggesting that MEKK1 is a downstream
target for the NESK activity in the JNK signaling cascade.
Inhibition of TNF-- or TRAF2-induced JNK Activation by the
Dominant Negative Mutant of NESK--
To determine whether TNF- can
regulate NESK kinase activity, HEK 293 cells were transfected with an
expression vector encoding NESK and exposed to TNF- for various
periods of time. Then an immune complex kinase assay was performed
using MBP as a substrate. NESK immune complexes from TNF--treated
cells exhibited an elevation in in vitro protein kinase
activity (Fig. 6A). Next, to
determine whether NESK is involved in the TNF--induced JNK
activation, HEK 293 cells were transfected with an expression vector
encoding a dominant negative mutant of NESK. Reverse transcriptase-PCR analysis showed that the NESK mRNA was expressed in HEK 293 cells (data not shown), suggesting that the endogenous NESK protein is
present in HEK 293 cells. We expected that the dominant negative mutant
of NESK inhibited the activity of the endogenous NESK. The expression
of the dominant negative mutant of NESK inhibited the JNK activity
induced by TNF- (Fig. 6B), suggesting that NESK is
involved in the TNF--induced signaling pathway.
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Fig. 6.
Inhibition of
TNF--induced JNK activation by dominant
negative mutants of NESK in HEK 293 cells. A, HEK 293 cells (5 × 105 cells/60-mm dish) were transfected
with 2 µg of pCMV-FLAG-NESK. The cells were treated with TNF- (50 ng/ml) for 0, 5, 15, 30, and 60 min (lanes 1-5)
at 48 h after transfection, and immune complex kinase assays were
performed with an anti-FLAG antibody using MBP as a substrate
(upper panel). Expression levels of NESK were
verified equivalent by immunoblotting using an anti-FLAG antibody
(bottom panel). B, HEK 293 cells
(5 × 105 cells/60-mm dish) were transfected with 1 µg of SR-HA-JNK1 (lanes 1-3), plus 2 µg
of pCMV-FLAG-NESK(K54E) (lane 3). The final
amount of DNA was adjusted to 3 µg with empty vector. The cells were
treated with (lanes 2 and 3) and
without (lane 1) TNF- (50 ng/ml) for 10 min at
48 h after transfection, and immune complex kinase assays were
performed with an anti-HA antibody using GST-c-Jun-(1-223) as a
substrate (upper panel). Expression levels of
proteins were verified equivalent by immunoblotting using an anti-HA
antibody (middle panel) and an anti-FLAG antibody
(bottom panel).
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Because TNF--induced JNK activation requires TRAF2 (25), the effect
of TRAF2 on NESK activity was examined by co-transfecting expression
vectors encoding NESK and TRAF2. The presence of TRAF2 markedly
increased NESK kinase activity (Fig.
7A). To determine whether NESK
is involved in the TRAF2-induced JNK activation, HEK 293 cells were
co-transfected with expression vectors encoding TRAF2 and a dominant
negative mutant of NESK. The expression of the dominant negative NESK
inhibited the JNK activity induced by TRAF2 (Fig. 7B). These
results suggest that NESK is a downstream target of TRAF2 in TNF-
signaling.
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Fig. 7.
Inhibition of TRAF2-induced JNK activation by
dominant negative mutants of NESK. A, HEK 293 cells
(5 × 105 cells/60-mm dish) were transfected with 2 µg of the empty vector pCMV alone (lane 1),
pCMV-FLAG-NESK (lanes 2 and 3), and
pCMV-VSVG-TRAF2 (lane 3). The final amount of DNA
was adjusted to 4 µg with empty vector. After 48 h, the cells
were collected, and immune complex kinase assays were performed with an
anti-FLAG antibody using MBP as a substrate (upper
panel). Expression levels of proteins were verified
equivalent by immunoblotting using an anti-FLAG antibody
(middle panel) and an anti-TRAF2 antibody (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA) (bottom
panel). B, HEK 293 cells (5 × 105 cells/60-mm dish) were transfected with 1 µg of
SR-HA-JNK1 (lanes 1-3), 2 µg of
pCMV-VSVG-TRAF2 (lanes 2 and 3), 2 µg of pCMV-FLAG-NESK(K54E) (lane 3). The final
amount of DNA was adjusted to 5 µg with empty vector. The cells were
collected after 48 h, and immune complex kinase assays were
performed with an anti-HA antibody using GST-c-Jun-(1-223) as a
substrate (upper panel). Expression levels of
proteins were verified equivalent by immunoblotting using an anti-HA
antibody (middle panels) and an anti-FLAG
antibody (bottom panel).
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DISCUSSION |
In this study, we identified a novel protein kinase, NESK, from
mouse embryo. NESK shares high sequence homology with members of the
group I GCK family in both the N-terminal kinase domain and the
C-terminal putative regulatory domain. All members of the group I GCK
family selectively activate the JNK pathway when overexpressed in
cultured cells but do not stimulate the p38 kinase or ERK signaling
pathways (1). NESK also activates the JNK pathway when overexpressed in
HEK 293 cells but does not activate the p38 kinase or ERK pathways.
Based on these structural and functional properties, it can be
concluded that NESK is a novel member of the group I GCK family.
NESK mRNA is expressed exclusively during the late stages of mouse
embryogenesis. Further, NESK is highly homologous to the Drosophila Misshapen and C. elegans Mig-15
proteins, both in the kinase domain and in the C-terminal regulatory
domain. The Misshapen protein functions upstream of the JNK pathway to
stimulate dorsal closure in the Drosophila embryo (19).
Dorsal closure occurs during the late stages of Drosophila
embryogenesis. The Mig-15 protein is necessary for several
developmental processes in C. elegans (19). Thus, NESK is
suggested to function as an intracellular signaling molecule in a
developmental process during the late stages of mammalian
embryogenesis. More recently, a novel member of the group I GCK family,
named NRK, was cloned from mouse embryo (26). NESK is 99% identical to
NRK. Moreover, the expression pattern of NESK mRNA is similar to
that of NRK mRNA. Thus, although the role of NRK in the JNK pathway
has not been determined, NESK is probably the same protein as NRK.
In situ hybridization revealed that NRK was predominantly
expressed in skeletal muscle during the late stages in mouse
embryogenesis (26). Dorsal closure in Drosophila
embryogenesis involves cell migrations. mig-15 mutants have
developmental defects in Q-neuroblast migration and muscle arm
targeting. Thus, NESK may participate in intracellular signaling involved in muscle cell migration.
It has been shown that the dominant negative mutant of MEKK1 inhibited
GCK-, NIK-, GCK-like kinase-, or GCK-related kinase-induced JNK
activation (5, 7, 8, 10). Hematopoietic progenitor kinase/GCK-like
kinase-induced JNK activation has been shown to be inhibited by the
dominant negative mutant of TAK1 but not by that of MEKK1(11).
Moreover, the dominant negative mutant of mixed lineage kinase 3 inhibited hematopoietic progenitor kinase-1-induced JNK activation (4).
Thus, MEKK1, TAK1, and mixed lineage kinase 3 are downstream kinases of
group I GCKs. In this study, we demonstrated that the dominant negative
form of MEKK1 inhibited NESK-induced JNK activation, suggesting that
MEKK1 is a downstream target kinase of NESK. It is known that MEKK1
stimulates MKK4, which in turn activates JNK. JNK activation induced by
NESK was inhibited by the dominant negative mutant of MKK4, suggesting
that MKK4 is a downstream kinase of MEKK1 whose activity is induced by
NESK. Group I GCKs have an N-terminal kinase domain and a C-terminal domain. The C-terminal domain of GCK and NIK interacts with MEKK1 and
overexpression of this domain alone can activate JNK (8, 10). The
C-terminal domain of GCK-like kinase seems to be essential for maximal
activation of JNK because the C-terminal truncation mutant of GCK-like
kinase has a greatly reduced ability to activate JNK (5). Because the
C-terminal domain of NESK shares high sequence homology with that of
NIK, it is possible that it functions as a regulatory domain for a
downstream substrate. However, the kinase domain of NESK alone was able
to activate the JNK pathway. Further characterization is required to
determine a role of the C-terminal domain of NESK in regulating the JNK pathway.
Because the JNK pathway is stimulated by a variety of stress-inducing
agents and proinflammatory cytokines, it is possible that NESK
activates the pathway in response to upstream signals. A crucial role
for TRAF2 in coupling the TNF receptor to JNK activation has been
substantiated by a study of cells from TRAF2 knockout mice;
TRAF2-deficient cells do not stimulate JNK activity in response to
TNF- (27). It has been shown that two group I GCK family members,
GCK and TRAF2- and Nck-interacting kinase, interact with TRAF2. In the
present study, we have demonstrated that TNF- and TRAF2 stimulated
the NESK activity, and the dominant negative mutant of NESK blocked
TNF-- or TRAF2-induced JNK activation. Thus, NESK may interact with
TRAF2 and play a role in coupling TRAF2, as well as TNF-, to JNK activation.
In this study, we have found candidates of components functioning
upstream and downstream of NESK in mammalian cells. However, NESK seems
to be expressed in specific types of cells at the late stages of mouse
embryogenesis. Thus, the identification of upstream and downstream
signaling molecules of NESK in these cells will provide valuable
insights into the signaling pathway regulated by NESK.
| |
ACKNOWLEDGEMENT |
We thank Dr. M. Karin for providing plasmids.
| |
FOOTNOTES |
*
This work was supported in part by research grants from the
Ministry of Education, Science, Sports and Culture of Japan, Uehara Memorial Foundation, and Core Research for Evolutional Science and
Technology.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB035267.
Present address: Dept. of Molecular Biology, Institute of
Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
§
To whom correspondence should be addressed: Dept. of Life Science,
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel.:
81-45-924-5701; Fax: 81-45-924-5771; E-mail:
nkitamur@bio.titech.ac.jp.
Published, JBC Papers in Press, May 4, 2000, DOI 10.1074/jbc.M001009200
| |
ABBREVIATIONS |
The abbreviations used are:
GCK, germinal center
kinase;
NIK, Nck-interacting kinase;
JNK, c-Jun N-terminal kinase;
ERK, extracellular signal-regulated kinase;
TNF, tumor necrosis factor;
TRAF2, TNF receptor-associated factor 2;
MBP, myelin basic protein;
PCR, polymerase chain reaction;
GST, glutathione
S-transferase;
HA, hemagglutinin.
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