From Amgen, Inc., Boulder, Colorado 80301 and the
¶ Department of Microbiology and Immunology, Baylor College of
Medicine, Houston, Texas 77030
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ABSTRACT |
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The yeast serine/threonine kinase STE20 activates
a signaling cascade that includes STE11 (mitogen-activated protein
kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals
from both Cdc42 and the heterotrimeric G proteins associated with
transmembrane pheromone receptors. Using degenerate polymerase chain
reaction, we have isolated a human cDNA encoding a protein kinase
homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun
N-terminal kinase (JNK) signaling pathway when transfected into 293T
cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced
JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK
upstream kinase (MUK), strongly inhibited HGK-induced JNK activation.
TNF- The c-June N-terminal kinase
(JNK)1 family members belong
to the mitogen-activated protein kinase (MAPK) superfamily, which also
includes extracellular signal-regulated kinases (ERKs) and the p38
family (reviewed in Ref. 1). MAPK superfamily members are
serine/threonine-directed kinases that target other kinases, transcription factors, and membrane receptor tails, causing diverse effects such as cell proliferation, transformation, differentiation, and apoptosis. Activation of MAPK members occurs through an assembly of
kinase cascades in response to stimuli. The original MAPK cascade (c-Raf The JNK family members were characterized biochemically as two kinases
and cloned from human fetal brain and rat brain libraries (2, 3).
Presently, the JNK family consists of three genes, JNK1, JNK2, and
JNK3, which can be subdivided further into 10 isoforms (4). JNK1 and
JNK2 have four isoforms, while JNK3 has two isoforms; these isoforms
arise from alternative splicing. Their murine homologues are called
stress-activated protein kinases (3). Active JNKs target Ser-Pro and
Thr-Pro motifs on their substrates. Substrates for JNK family members
include the transcription factors c-Jun, JunD, ATF-2, Elk-1, Sap-1a,
and p53; phosphorylation of c-Jun, ATF-2, Elk-1, and Sap-1a increases
their transcriptional activity (reviewed in Ref. 1).
JNK plays a crucial role in stress responses, cell proliferation,
apoptosis (5-9), and oncogenesis (10). JNK kinase activity can be
activated by various agents, including the proinflammatory cytokines
(TNF- The large number and differential nature of the JNK-activating agents
suggest multiple pathways leading to JNK activation. Recent work has
added complexity to this simple cascade paradigm. There are multiple
upstream kinases (MAPK kinase kinases or MAPKKKs), including MEKK1
(13), transforming growth factor A family of further upstream protein kinases, including germinal center
kinase (GCK; Refs. 23 and 24), hematopoietic progenitor kinase (HPK1;
Refs. 25 and 26), GCK-like kinase (GLK; Ref. 27), and kinase homologous
to STE20 (KHS; Refs. 28 and 29), which have homology to
Saccharomyces cerevisiae STE20, have been identified and
shown to activate the JNK signaling pathway (reviewed in Ref. 30). The
serine/threonine kinase STE20 sequentially activates STE11 and STE7 in
the signaling cascade that includes STE11 (MAPKKK), STE7 (MAPKK), and
FUS3/KSS1 (MAPK) in the yeast S. cerevisiae pheromone
response. STE20 activates this pathway in response to signals from both
Cdc42 and the heterotrimeric G proteins associated with transmembrane
pheromone receptors (reviewed in Ref. 31). Thus far, p21
(Rac1/Cdc42)-activated kinases (PAKs) and HPK/GCK-related kinases
(HPK1, GCK, GLK, and KHS) have been identified as potential upstream
kinases for MEKK1. This kinase cascade has been suggested by many
laboratories as follows: HPK1, GCK, PAKs cDNA Library Screening and Cloning of HGK--
Degenerate
oligonucleotide primers were designed to the conserved regions (kinase
subdomains VI and VIII) of the serine/threonine protein kinase and used
in a polymerase chain reaction (PCR) as described previously (32).
Replicate filters from a human Northern Blot Analysis of HGK--
Filters containing
poly(A)+ RNA (2 µg/lane) from various tissues were
purchased from CLONTECH. Filters were probed with
an HGK probe corresponding to the non-catalytic region of HGK.
Hybridization was performed at 68 °C in Express Hybridization Buffer
(CLONTECH), followed by three washes in 0.1% SSC,
0.1% SDS at 55 °C. Blots were exposed for 24 h at
Cells, Transfections, Antibodies, and Reagents--
Human
embryonic kidney 293T cells were kindly provided by Dr. M. C.-T.
Hu (Amgen, Inc., Thousand Oaks, CA). 293T cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and 1× penicillin/streptomycin. 293T cells were plated at a
density of 1 × 106 cells/100-mm dish (or 1.5 × 105 cells/35-mm plate as indicated) and transfected the
next day using the calcium phosphate precipitation protocol (Specialty Media, Inc.) The amounts of DNA used for each transfection were as
indicated, and empty vector was used to achieve equal amounts of DNA in
each transfection. A monoclonal antibody (12CA5) against the
hemagglutinin (HA) epitope was purchased from Boehringer Mannheim. The
monoclonal antibody (M2) against the Flag epitope was purchased from
Kodak, Inc. To generate polyclonal rabbit anti-HGK antibody, a
synthetic peptide (CNPTNTRPQSDTPEIRKYKKRFN) corresponding to the
peptide sequence 823-844 of HGK was synthesized, then coupled to
keyhole limpet hemocyanin via the cysteine residue at the N terminus,
and used to immunize rabbits. Antibody affinity purification was done
as described previously (27).
Plasmid Construction--
Full-length HGK was cloned into pCR3.1
(Invitrogen) and pCI (Promega, Madison, WI) vectors by PCR using two
oligonucleotide primers. An N-terminal Flag epitope sequence was added.
A catalytically inactive HGK mutant (pCR-HGK-KR) was created by
substituting lysine 54 with an arginine (K54R mutant) by site-directed
mutagenesis using the overlapping PCR method as described previously
(34). A second catalytically inactive HGK mutant (pCI-HGK-KE) was
created by substituting lysine 54 with a glutamic acid (K54E mutant). MKK7-K76E encoding a dominant-negative MKK7 mutant was described previously (17). pUna3-FL-MEKK-KR encoding a dominant-negative MEKK1
mutant was kindly provided by Dr. D. Templeton (Case Western Reserve
University, Cleveland, OH). pEBG-SEK1-AL encoding a dominant-negative SEK1 mutant was kindly provided by Dr. L.I. Zon (Children's Hospital, Boston, MA). pHA-p38 and pHA-JNK1 were kindly provided by Dr. J. Woodgett (Ontario Cancer Institute, Toronto, Canada). pEF-TAK1-K63W encoding a dominant-negative TAK1 mutant was kindly given by Dr. K. Matsumoto (Nagoya University, Nagoya, Japan). pT7tag-MUK/KN encoding a
dominant-negative MUK mutant was provided by Dr. S. Ohno (Yokohama City
University School of Medicine, Yokohama, Japan). GST-ATF2 and
pCEV-HA-ERK2 were kindly provided by Dr. J. S. Gutkind (National
Institutes of Health, Bethesda, MD). cRaf-BXB and p5xTRE-CAT were
kindly provided by Dr. J. Bruder (GenVec, Rockville, MD).
Immunocomplex Kinase Assay and Western Blot
Analysis--
Immunocomplex kinase assays were carried out as
described previously (33). MAPKs were precipitated by incubation with
specific antibodies and protein A-agarose beads (Bio-Rad) in an
incubation buffer (20 mM HEPES, pH 7.4, 2 mM
EGTA, 50 mM glycerophosphate, 1% Triton X-100, 10%
glycerol, 1 mM dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4). The precipitates were
washed twice with incubation buffer, twice with LiCl buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100), and twice with kinase buffer (20 mM MOPS, pH 7.6, 2 mM EGTA, 10 mM MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100, and 1 mM
Na3VO4), then mixed with 5 µg of the
indicated substrates, 15 µM ATP, and 10 µCi of
[ Phosphoamino Acid Analysis--
After the immunocomplex kinase
assay, proteins were separated using a 12% SDS-PAGE, transferred to a
polyvinylidene difluoride (PVDF) membrane at 40 V overnight in 10 mM Tris, 1 mM glycine, and 20% methanol. The
radioactive myelin basic protein (MBP) bands were identified and
excised by alignment of the autoradiogram to PVDF membrane. The PVDF
membrane was soaked first in methanol and then in deionized water. The
proteins on the PVDF membrane were then hydrolyzed in 250 µl of 6 N HCl for 1 h at 110 °C. The acid containing
partially hydrolyzed phosphoamino acids were pooled and lyophilized.
The lyophilized samples were washed twice with 50 µl of deionized
water. The final dried pellets were resuspended in 5 µl of 5.9%
glacial acetic acid, 0.8% formic acid, 0.3% pyridine, and 0.3 mM EDTA, pH 2.5, spiked with phosphorylated serine,
threonine, and tyrosine standards, and spotted on a 20 × 20-cm
TLC plate. The phosphoamino acids were separated in the pH 2.5 buffer
at 20 mA for 2 h. The phosphoamino acid standards were visualized by spraying the plate with 0.3% ninhydrin in acetone. The
32P-labeled residues were detected by autoradiography.
Molecular Cloning of HGK--
In order to identify novel members
of the mammalian STE20 family of protein kinases, we employed a
degenerate PCR strategy to amplify cDNA from a human macrophage
cDNA library. A PCR fragment containing novel sequences with
homology to the STE20 family of protein kinases was identified. This
cDNA fragment was subsequently used to screen a human macrophage
cDNA library. Four cDNA clones of varying sizes were isolated,
and the one containing the longest insert (3950 base pairs) was
sequenced on both strands. The complete nucleotide sequence of the
cDNA predicted an open reading frame of 1165 amino acids with a
predicted molecular mass of 133.40 kDa. The deduced amino acid sequence
encoded a kinase catalytic domain in the N terminus that contained 11 kinase subdomains (Fig. 1, A
and B). Comparison of the amino acid sequence of the
putative kinase domain of HGK with other sequences found that the HGK
kinase domain was most similar to HPK1, GLK, GCK, KHS, and STE20.
Within the kinase domain, HGK displays 47% amino acid identity to the catalytic domain of the HPK1, 52% amino acid identity to the catalytic domain of the human GLK, 48% amino acid identity to the catalytic domain of the GCK, 48% amino acid identity to the catalytic domain of
the KHS, and 39% amino acid identity to S. cerevisiae Ste20. The amino acid sequence alignment of the
catalytic domain of HGK with that of related kinases is shown in Fig.
1B. Unlike HPK1 and GCK, HGK does not contain proline-rich
regions: the putative Src homology 3 domain binding regions. However,
one of the HGK cDNA clones contains an additional 77 amino acids
with two proline-rich regions (Fig. 1C). To determine
whether other forms of HGK exist that contain the proline-rich regions,
reverse transcriptase PCR using two primers to flank the region was
employed to amplify cDNA from human brain, liver, skeletal muscle,
and placenta. Sequencing analysis of the PCR products found that,
although the longer form appears to be more abundant in the brain, the
short form is the predominant form in human liver, skeletal muscle, and
placenta (data not shown).
Expression Pattern and Catalytic Activity of HGK--
The
expression of HGK was examined in a variety of human tissues by
Northern blot analysis using a probe from the non-conserved C-terminal
coding region of HGK. The HGK probe hybridized to three transcripts of
approximately 4.6, 6.5, and 8.5 kilobase pairs in all tissues examined,
suggesting a ubiquitous tissue distribution (Fig.
2A). To determine the
expression of endogenous HGK protein, a polyclonal antibody to HGK was
generated and purified by affinity chromatography. This antibody
specifically recognized a protein of approximately 130 kDa in MCF-7,
293, HeLa, PC3, LNCaP, and Jurkat cells, as shown by Western blot
analysis (Fig. 2B). The specificity of the anti-HGK antibody
was further confirmed in 293T cells transfected with a Flag-tagged HGK
cDNA expression plasmid. Both the anti-HGK antibody and the
anti-Flag antibody recognized specifically the Flag-tagged HGK proteins
in the HGK-transfected 293T cells (Fig. 2C). HGK
phosphorylated the substrate MBP (Fig. 3A). As controls, the HGK
catalytically inactive mutants (HGK-KR and HGK-KE, which contain lysine
54 to arginine substitution and lysine 54 to glutamic acid
substitution, respectively) did not phosphorylate the substrate MBP
(Fig. 3A), indicating that the HGK kinase activity observed
was not attributable to coimmunoprecipitated kinases. Furthermore,
phosphoamino acid analysis of in vitro phosphorylated MBP
detected phosphorylated serines and threonines only (Fig. 3B), establishing HGK as a serine/threonine protein
kinase.
HGK Specifically Activates JNK, but Not ERK or p38 Kinase, in
Transfected 293T Cells--
To determine whether HGK can activate JNK,
293T cells were co-transfected with mammalian expression vectors
encoding HGK 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-(1-79) as a substrate. Transfection with HGK resulted
in strong JNK1 activation (Fig.
4A, lane
3), while cells transfected with vector showed little JNK1
activation (lanes 1 and 2).
Transfection of a kinase-inactive form of HGK, in which lysine 54 in
the ATP binding domain was mutated to a glutamic acid, did not result
in JNK activation (Fig. 4A, lane 4).
Thus, the kinase activity of HGK is required for JNK activation.
To determine whether HGK can also activate the p38 kinase or ERK, 293T
cells were transiently transfected with HGK along with HA-tagged p38 or
HA-tagged ERK2. p38 kinase and ERK2 were then immunoprecipitated and
their activities assayed using ATF-2 or MBP peptides as substrates.
Even though MKK6 activated p38 (Fig. 4B, lane
4) and the activated c-Raf1, Raf-BXB, strongly activated ERK2 (Fig. 4C, lane 4), neither p38 or
ERK kinase activities were increased when HGK was overexpressed in 293T
cells (Fig. 4, B and C, lane
3). These data suggest that the activation of JNK by HGK is specific.
HGK Activates c-Jun Transcriptional Activity--
Because c-Jun is
a substrate of JNK and the expression of HGK can activate the JNK
signaling pathway, we tested whether the expression of HGK can result
in the activation of c-Jun transcriptional activity in vivo.
Cells were transfected with an HGK expression plasmid and a
chloramphenicol acetyltransferase (CAT) reporter construct (5xTRE-CAT)
containing five copies of the c-Jun-binding sites (AP1-binding sites).
CAT assays were performed 44 h after transfection. Co-transfection
of HGK plus JNK1 stimulated the transcriptional activity mediated by
AP-1 (about 7-fold) as compared with cells transfected with vector
control (Fig. 4D). JNK1 or HGK alone showed minimal AP1-CAT
induction (about 1.5-fold). Due to the low levels of JNK expression in
293T cells, inclusion of the JNK plasmid in the transfection assay was
necessary in order to detect optimal AP-1 induction by HGK via the HGK
HGK Functions Upstream of MKK4 and MKK7--
MKK4 is an upstream
activator of JNK which phosphorylates and activates JNK. Recently,
another MAPKK/MKK, called MKK7, has been cloned and found to
specifically activate JNK, but not p38 and ERK. To determine whether
HGK activates JNK through MKK4 and MKK7, we co-transfected cells with
an expression plasmid expressing HGK and a dominant-negative mutant of
MKK4/SEK1 (SEK-AL) or a dominant-negative mutant of MKK7 (MKK7-K76E) to
determine whether they can inhibit HGK-induced JNK activation. The
expression of a dominant-negative form of MKK4 or MKK7 inhibited JNK
activity induced by HGK (Fig.
5A) and GLK (Fig.
5B), indicating that HGK functions upstream of MKK4 and/or
MKK7.
HGK-Induced JNK Activation Is Blocked by a Dominant-negative Mutant
of TAK1, but Not MEKK1 or MUK--
The MAPK kinase kinase MEKK1 is a
direct activator of MKK4, which in turn phosphorylates and activates
JNK. In addition, MEKK1 was shown to function downstream of HPK1 and
GLK (25, 27), two kinases that are highly homologous to HGK. To
determine whether HGK also activates JNK through MEKK1, we analyzed the
effect of the kinase-inactive mutant MEKK1 (MEKK-KR) on HGK-induced JNK activation. In repeated experiments, we found that, although the MEKK1-KR mutant strongly inhibited HPK-induced JNK activation (Fig.
6B), it did not efficiently
block HGK-stimulated JNK activity (Fig. 6A). These results
suggest that HGK utilizes different downstream kinases for signaling.
TAK1 was shown to function downstream of HPK1 (20); hence, we tested
whether HGK activates JNK through TAK1. We co-transfected cells with
HGK plus the dominant-negative mutant of either TAK1 (TAK-K63W) or MUK
(MUK-KN), and determined their effects on HGK-induced JNK activation.
We found that the kinase-negative TAK1 mutant (TAK-K63W), but not the
MUK mutant, significantly inhibited HGK-induced JNK activation (Fig.
7). This result strongly suggests that
TAK1 is a downstream target for HGK activity in the JNK signaling
cascade.
Activation of HGK by TNF- TNF- A human kinase homologous to yeast STE20, called HGK, was cloned.
HGK shares both sequence and structural homology with several known
members of the mammalian STE20 family of kinases, including HPK1, GLK,
GCK, and KHS. HGK has a catalytic domain in its N terminus and a long
putative regulatory region. The C terminus of HGK does not appear to
contain the Cdc42/Rac1 binding motifs found in the PAK family of
protein kinases (35). Like HPK1, GLK, GCK, and KHS, overexpression of
HGK in 293T cells strongly activates the JNK pathway, but has no effect
on either the ERK or p38 signaling pathways, suggesting that
HGK-induced JNK activation is specific. Although the C-terminal domain
of HGK does not contain the Cdc42/Rac1 binding motifs, it appears to
act as a dominant-negative mutant. Therefore, the C-terminal domain may
interact with other signaling molecules that are important for HGK
kinase activation. Interestingly, HGK-mediated JNK activation was not
affected by the dominant-negative mutant of MEKK1, which is a potent
JNK activator. In contrast, JNK activation stimulated by the highly
homologous kinase, HPK1, can be efficiently blocked by this mutant.
Similarly, our previous studies found that GLK-induced JNK activation
is also inhibited by a dominant-negative mutant of MEKK1 (27). These
data suggest that HGK may function independently of MEKK1 and that
different STE20-like protein kinases may signal through different
downstream MAPKKKs. At the MAPKKK level, there are several kinases that
can activate the JNK pathway. Among several MAPKKKs tested, we found that only the dominant-negative TAK1 mutant strongly inhibited HGK-induced JNK activation. TAK1 was originally identified as a kinase
that can activate the p38 kinase signaling pathway. More recent data
found that TAK1 can also potently activate the JNK pathway (19, 20).
Thus, HGK likely utilizes TAK1 as its downstream kinase.
Two isoforms of human HGK were detected, one of which has a deletion in
the region that contains two proline-rich domains. The proline-rich
region, contained in the long isoform of HGK, may mediate its
interaction with other Src homology 3 domain-containing adaptors. The
fact that human HGK that is missing this region can activate the JNK
pathway indicates that this region is not essential for JNK activation
in transfected 293T cells. It appears that the isoform that contained
the proline-rich region was predominantly expressed in the brain, while
the shorter form was more abundantly expressed in other tissues such as
human liver, skeletal muscle, and placenta (data not shown). The
differential expression of different isoforms of HGK suggests that
these different isoforms might have tissue- and/or cell-specific
functions and may be regulated by different mechanisms.
The specificity of JNK activation is not well understood at the present
time. Given that the JNK pathway is activated by a variety of
extracellular stimuli including UV irradiation, ionizing radiation,
heat shock, osmotic stress, protein synthesis inhibitors, apoptosis-inducing agents, and proinflammatory cytokines, it should not
be surprising that multiple mammalian STE20-like protein kinases can
mediate JNK activation. One plausible mechanism of controlling the
specificity of JNK activation is to have different upstream kinases,
each responding to distinct stimuli. In addition, different mammalian
STE20-like protein kinases might have cell-type specific functions; for
example, HPK1 may have a specific function in hematopoietic progenitor
cells, as its transcripts are preferentially expressed in hematopoietic
organs and cell lines (25, 26). This study indicates that HGK is
involved in the TNF- activated HGK in 293T cells, as well as the dominant-negative
HGK mutants, inhibited TNF-
-induced JNK activation. These results
indicate that HGK, a novel activator of the JNK pathway, may function
through TAK1, and that the HGK
TAK1
MKK4, MKK7
JNK kinase
cascade may mediate the TNF-
signaling pathway.
INTRODUCTION
Top
Abstract
Introduction
References
MEK1/2
ERK1/2) includes activation of the
serine/threonine kinase c-Raf, which activates a dual-specificity MAPK
kinase (MEK1/MEK2). MAPK/ERK is then activated via tyrosine and
threonine phosphorylation of the T-X-Y motif on the
phosphorylation loop of the enzyme. This phosphorylation event causes
local and global conformational changes in the protein, resulting in an
active enzyme structure.
and interleukin-1), G protein-coupled receptors, lymphocyte
co-stimulatory receptors (CD28 and CD40), osmotic shock, heat shock,
protein synthesis inhibitors, ceramides, DNA-damaging chemicals, UV
irradiation, and ionizing radiation (reviewed in Ref. 1). Activation of
JNK involves the upstream kinases MKK4 (also called SEK1) (11, 12) and
MEKK1 (13), and together these proteins constitute a kinase cascade
(MEKK1
MKK4/SEK
JNK) in which the serine/threonine kinase MEKK1
phosphorylates and activates the dual specificity kinase MKK4/SEK (14,
15). MKK4/SEK then activates JNK via phosphorylation of the T-P-Y motif on its phosphorylation loop (11, 12). Recently, another
MKK4/SEK-related kinase, called MKK7, has been cloned and found to
specifically activate JNK, but not p38 and ERKs (16, 17).
-activated kinase (TAK1; Ref.
18-20) and MAPK upstream kinase (MUK; Ref. 21), that activate the JNK
pathway via MKK4/SEK (reviewed in Ref. 22).
MEKKs
MKK4, MKK7
JNKs. There is also evidence suggesting that MAPKKKs (e.g.
MEKKs and MLK-3) are parallel to, but not downstream of, PAKs and
HPK/GCK family kinases (reviewed in Ref. 22). We report here the
isolation and characterization of a novel human protein kinase, HGK
(HPK/GCK-like kinase), which is homologous to STE20. Expression of HGK
specifically activates the JNK signaling pathway.
EXPERIMENTAL PROCEDURES
phage macrophage library were
prehybridized for 2 h at 42 °C in 5× standard saline citrate
(SSC), 1× Denhardt's solution containing 100 µg/ml salmon sperm
DNA, 50% formamide, 0.1% SDS, and then hybridized to the
[
-32P]dCTP-labeled, 180-base pair probe (overnight in
the same solution). After hybridization, the filters were washed twice
for 30 min in 0.1% SSC, 0.1% SDS at 55 °C. Positive clones were
isolated and sequenced on both strands.
70 °C.
-32P]ATP in 30 µl of kinase buffer. The kinase
reaction was performed at 30 °C for 30 min and terminated with an
equal volume of SDS sampling buffer. The reaction mixtures were
resolved by SDS-PAGE analysis. The Western blot analysis, antibody
incubation, and ECL detection were described previously (25).
RESULTS
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Fig. 1.
Amino acid sequence of human HGK and sequence
comparison of HGK with other related kinases. A, the
deduced amino acid sequence of HGK is shown. The catalytic domain is
underlined. B, the putative catalytic domain of
HGK was aligned and compared with human GLK, GCK, HPK1, and STE20 by
the PILEUP and PRETTY programs of the University of Wisconsin Genetics
Computation Group (GCG). The kinase subdomains are indicated with
Roman numerals. The conserved residues are in
capital letters, while the non-conserved amino
acid residues are shown in lowercase letters.
C, sequence comparison between the long and short forms of
human HGK. The numbers in parentheses
are the amino acid position; dashes are the
gaps.
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Fig. 2.
Expression of HGK. A,
Northern blot analysis of HGK mRNA. Poly(A)+ RNA from
the indicated tissues was hybridized with radioactive HGK probes from
the 3' non-conserved region. RNA size markers are shown on the
left. B, Western blot analysis of HGK protein. 30 µg of cell lysate from several cell lines was resolved by 7%
SDS-PAGE and immunoblotted with purified anti-HGK antibody, Ab2228.
C, specificity of the anti-HGK antibody (Ab2228). 293T cells
(1.5 × 105/35-mm well) were transfected with 2 µg
of an empty vector (pCI) or pCI-Flag-HGK. The cells were collected
40 h after transfection. 40 µg of total cellular proteins were
resolved by SDS-PAGE electrophoresis and immunoblotted with an anti-HGK
antibody (Ab2228; left panel) and an anti-Flag
antibody (M2; right panel).
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Fig. 3.
Catalytic activity of HGK. A,
293T cells (1.5 × 105/35-mm well) were transfected
with 2 µg of an empty vector (pCI), HGK, HGK-KR, and HGK-KE. The
cells were collected 40 h after transfection. Immunocomplex kinase
assays were performed with an anti-Flag antibody (M2) using MBP as the
substrate. Equivalent levels of wild-type and mutant HGK expression
were verified by immunoblotting using an anti-Flag antibody (M2;
bottom panel). B, HGK is a
serine/threonine protein kinase. 293T cells (1.5 × 105) were transfected with 2 µg of a Flag-HGK plasmid.
The cells were harvested at 44 h after transfection. Immunocomplex
kinase assays were performed with an anti-Flag antibody (M2) using MBP
as a substrate. After electrophoresis in 12% SDS-PAGE, the protein was
transferred to PVDF. Phosphorylated MBP was excised from the blot and
used to perform phosphoamino acid analysis. The positions of
phosphorylated serine (p-Ser), phosphorylated threonine
(p-Thr), and phosphorylated tyrosine (p-Tyr) are
indicated by arrows.
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Fig. 4.
Activation of JNK, but not p38 or ERK, by HGK
in transfected 293T cells. A, 293T cells (1 × 106 cells/100-mm dish) were transfected with 15 µg of the
empty vector pCI alone (lane 1), 5 µg of HA
epitope-tagged JNK1 plasmid (pHA-JNK1) plus 10 µg of the empty vector
pCI (lane 2), 10 µg of pCI-HGK (lane
3), or 10 µg of kinase-dead mutant pCI-HGK-KE
(lane 4). The cells were collected 48 h
later, and immunocomplex kinase assays were performed with anti-HA
antibody (12CA5) using GST-c-Jun-(1-79) as the substrate.
B, 293T cells were transfected with 15 µg of the empty
vector pCI alone (lane 1), 5 µg of HA
epitope-tagged p38 (pHA-p38) plus 10 µg of the empty vector pCI
(lane 2), 10 µg of pCI-HGK (lane
3), or 10 µg of pCI-MKK6 (lane 4).
Immunocomplex kinase assays were performed with anti-HA antibody
(12CA5) using GST-ATF2-(1-96) as the substrate. C, 293T
cells were transfected with 20 µg of the empty vector pCI
(lane 1), 10 µg of HA epitope-tagged ERK2
(pHA-ERK2) plus either 10 µg of the empty vector pCI (lane
2), 10 µg of pCI-HGK (lane 3), or 10 µg of pcRaf-BXB (lane 4). Immunocomplex kinase
assays were performed with anti-HA antibody, using MBP as the
substrate. Equivalent expression levels of HA-JNK1, HA-p38, and HA-ERK2
were verified by immunoblotting using an anti-HA antibody (12CA5;
bottom panels). D, activation of AP-1
activity by HGK. 293T cells were transfected with 1 µg of 5xTRE-CAT
reporter construct plus empty vectors (lane 1), 2 µg of pHA-JNK1 (lane 2), 4 µg of pCI-HGK
(lane 3), 4 µg of pCI-HGK-KE (lane
4), 2 µg of pHA-JNK1 and 4 µg of pCI-HGK
(lane 5), or 2 µg of pHA-JNK1 and 4 µg of
pCI-HGK-KE (lane 6). Empty vector was used to
normalize the amount of transfected DNA. The cells were harvested
44 h after transfection, and the CAT activity was measured and
compared with that of the empty vector. The results are expressed as
-fold induction.
TAK1
MKK4,7
JNK kinase cascade. Co-transfection of cells
with JNK1 and a kinase-negative mutant of HGK did not increase the
transcriptional activity of AP-1. This result indicates that HGK can
activate JNK, which in turn stimulates the transcriptional activity of c-Jun in vivo.
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Fig. 5.
HGK-induced JNK activation is blocked by
either SEK-AL or MKK7-K76E. A, 293T cells (1.5 × 105/35-mm cell well) were transfected with 1 µg of
pHA-JNK1 plus 4 µg of the empty vector pCI (lane
1), 2 µg of pCI-HGK (lane 2), 2 µg
of pCI-HGK and 2 µg of dominant-negative pEBG-SEK-AL (lane
3), or 2 µg of pCI-HGK and 2 µg of dominant-negative
MKK7-K764 (lane 4). B, for comparison,
293T cells were transfected with 1 µg of pHA-JNK1 plus 4 µg of the
empty vector pCI (lane 1), 2 µg of pCI-GLK
(lane 2), 2 µg of pCI-GLK and 2 µg of
pEBG-SEK-AL (lane 3), or 2 µg of pCI-GLK and 2 µg of MKK7-K764 (lane 4). The empty vector pCI
was used to normalize the amount of transfected DNA. Immunocomplex
kinase assays were performed 44 h after transfection with anti-HA
(12CA5) antibody using GST-c-Jun-(1-79) as the substrate. Equivalent
expression levels of HA-JNK1, Flag-HGK, and Flag-GLK were verified by
immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies,
respectively (bottom panels).
View larger version (12K):
[in a new window]
Fig. 6.
HGK-induced JNK activation is not
blocked by dominant-negative mutant of MEKK1. A, 293T
cells (1 × 106 cells/100-mm dish) were transfected
with the empty vector pCI alone (lane 1), 1 µg
of pHA-JNK1 plus empty vector (pCI) (lane 2), 10 µg of pCI-HGK (lane 3), or 10 µg of pCI-HGK
and dominant-negative pUna3-FL-MEKK1-KR (lane 4).
The empty vector pCI was used to normalize the amount of transfected
DNA. The cells were collected 48 h later, and immunocomplex kinase
assays were performed with an anti-HA antibody using GST-c-Jun-(1-79)
as the substrate. Equivalent expression levels of HA-JNK1 and Flag-HGK
were verified by immunoblotting using anti-HA (12CA5) and anti-Flag
(M2) antibodies, respectively (bottom panels).
B, for comparison, the same procedures were performed on
293T cells transfected with empty vector (pCI) alone (lane
1), 1 µg of pHA-JNK1 plus empty vector (lane
2), 10 µg of pCI-HPK1 (lane 3), or
10 µg of pCI-HPK1 and dominant-negative pUna3-FL-MEKK-KR
(lane 4). Equivalent expression levels of HA-JNK1
and Flag-HPK were verified by immunoblotting using anti-HA (12CA5) and
anti-Flag (M2) antibodies, respectively (bottom
panels).
View larger version (29K):
[in a new window]
Fig. 7.
HGK-induced JNK activation is specifically
blocked by the dominant-negative TAK1 mutant, TAK-K63W. 293T cells
(1.5 × 105/35-mm cell wells) were transfected with
0.1 µg of pHA-JNK1 alone (lane 1), 0.1 µg of
pHA-JNK1 and 2 µg of pCI-HGK plus empty vector (lane
2), 2 µg of dominant-negative pEF-TAK1-K63W
(lane 3), and 2 µg of dominant-negative
pT7tag-MUK-KN (lane 4). Empty vector (pCI) was
used to normalize the amount of transfected DNA. Immunocomplex kinase
assays were done 44 h after transfection with an anti-HA antibody
(12CA5) using GST-c-Jun as the substrate (top
panel). Equivalent expression levels of HA-JNK1 and Flag-HGK
were verified by immunoblotting using anti-HA (12CA5) and anti-Flag
(M2) antibodies, respectively (bottom
panels).
--
Since TNF-
stimulates TAK1
and JNK kinase activity (19) and HGK was found to be an upstream kinase
of TAK1 and JNK (this study), we tested whether TNF-
can also
regulate HGK kinase activity. To study the activation of HGK, an
expression construct encoding the Flag epitope-tagged HGK was
transiently transfected into 293T cells. The HGK-transfected 293T cells
were treated with TNF-
, and the cell lysates were prepared and
subjected to in vitro immune complex kinase assays. After
TNF-
stimulation, HGK kinase activity increased within 1 min and
peaked at 5 min (Fig. 8A).
Equivalent levels of overexpressed HGK were verified by immunoblotting
with anti-Flag (M2) antibody (bottom panel). This
result indicates that TNF-
stimulated HGK kinase activity.
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[in a new window]
Fig. 8.
HGK is activated by TNF-
and the TNF-
-induced JNK activation is
blocked by dominant-negative HGK mutants. A,
stimulation of the HGK kinase activity by TNF-
. 293T cells (1.5 × 105) were transfected with 50 ng of Flag-tagged HGK.
After 44 h, the cells were treated with TNF-
(100 ng/ml) for
various periods of time as indicated. The cell lysates were prepared
and immunocomplex kinase assays were performed with an anti-Flag
antibody (M2) using MBP as the substrate (top
panel). Equivalent levels of HGK expression were verified by
immunoblotting using an anti-Flag antibody (M2; bottom
panel). B, TNF-
-induced JNK activation is
blocked by HGK mutants (HGK-KR and HGK-KE). 293T cells (1.5 × 105) were transfected with HA-JNK (0.1 µg) alone, HA-JNK1
plus HGK-KR (2 µg), or HA-JNK1 plus HGK-KE (2 µg), as indicated.
The empty vector pCI was used to normalize the total amount of
transfected DNA. The cells were treated with TNF-
(100 ng/ml) for 10 min at 44 h after transfection. The cell lysates were prepared,
and immunocomplex kinase assays were performed with an anti-HA antibody
(12CA5) using GST-c-Jun as the substrate (top
panel). Equivalent levels of HA-JNK1 expression were
verified by immunoblotting using an anti-HA antibody (12CA5;
bottom panel).
-induced JNK Activation Is Blocked by the Dominant-negative
HGK Mutants--
Like many other STE20-like and MEKK-like kinases, HGK
kinase regulation using in vitro kinase assays was extremely
difficult to detect (see above). Hence, we used the dominant-negative
HGK mutants to further address the role of HGK in the TNF-
-mediated JNK activation (Fig. 8B, top panel).
293T cells were transfected with HA-JNK plus empty vector, HA-JNK plus
the dominant-negative HGK mutant HGK-KR, or HA-JNK plus the the
dominant-negative HGK mutant HGK-KE. Transfected cells were then
treated with TNF-
, followed by immunocomplex JNK kinase assays. We
found that the dominant-negative HGK mutants (HGK-KR or HGK-KE)
inhibited TNF-
-stimulated JNK activation (Fig. 8B,
top panel, lanes 3 and
4). Equivalent levels of HA-JNK1 expression were verified by
immunoblotting using anti-HA (12CA5) antibody (bottom
panel). Taken together, our results indicate that HGK is
involved in the TNF-
-induced signaling pathway.
DISCUSSION
-mediated signaling pathway. Our preliminary
result also suggests that HGK is involved in the UV-C-mediated
signaling pathway. Current studies are focused on elucidating the
underlying mechanisms of the HGK
TAK1
MKK4, MKK7
JNK kinase
cascade in UV-C and TNF-
signaling pathways.
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ACKNOWLEDGEMENTS |
---|
We thank Dean Jannuzi for the DNA sequencing; Marynette Rihanek for generating the HGK antibodies; Drs. J. Bruder, R. J. Davis, J. S. Gutkind, S.-i. Hirai, M.C.-T. Hu, K. Matsumoto, S. Ohno, D. Templeton, J. Woodgett, and L. I. Zon for providing valuable reagents; members of the Tan laboratory for the helpful discussions; Susan Lee and Roshi Afshar for technical assistance; and M. Lowe for secretarial assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants RO1-AI38649 and RO1-AI42532 (to T.-H. T.).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) AF096300.
§ The first two authors contributed equally to this work.
A Scholar of the Leukemia Society of America. To whom
correspondence should be addressed: Dept. of Microbiology and
Immunology, Baylor College of Medicine, M929, One Baylor Plaza,
Houston, TX 77030. Tel: 713-798-4665; Fax: 713-798-3700. E-mail:
ttan{at}bcm.tmc.edu.
The abbreviations used are:
JNK, c-Jun
N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MKK, MAPKK, or MEK, MAPK kinase; SEK, stress-activated protein kinase/ERK kinase; GCK, germinal center
kinase; GLK, GCK-like kinase; KHS, kinase homologous to STE20; HPK1, hematopoietic progenitor kinase-1; MAPKKK or MEKK, MAPK kinase kinase; TAK1, transforming growth factor -activated kinase; MUK, MAPK-upstream kinase; TNF-
, tumor necrosis factor
; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; PAK, p21-activated kinase; GST, glutathione
S-transferase; CAT, chloramphenicol acetyltransferase; PVDF, polyvinylidene difluoride; MOPS, 4-morpholinepropanesulfonic acid; MBP, myelin basic protein; HGK, HPK/GCK-like kinase.
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REFERENCES |
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