Hematopoietic Progenitor Kinase 1 Is a Component of Transforming
Growth Factor
-induced c-Jun N-terminal Kinase Signaling
Cascade*
Guisheng
Zhou
,
Susan C.
Lee
,
Zhengbin
Yao§, and
Tse-Hua
Tan
¶
From the
Department of Microbiology and Immunology,
Baylor College of Medicine, Houston, Texas 77030 and
§ Amgen, Inc., Boulder, Colorado 80301
 |
ABSTRACT |
The c-Jun N-terminal kinase (JNK) signaling
pathway is involved in transforming growth factor
(TGF-
)
signaling in a variety of cell systems. We report here that
hematopoietic progenitor kinase 1 (HPK1), a novel Ste20-like protein
serine/threonine kinase, serves as an upstream mediator for the
TGF-
-activated JNK1 cascade in 293T cells. TGF-
treatment
resulted in a time-dependent activation of HPK1, which was
accompanied by similar kinetics of JNK1 activation. The activation of
JNK1 by TGF-
was abrogated by a kinase-defective HPK1 mutant but not
by a kinase-defective mutant of kinase homologous to Ste20/Sps1. This
result indicates that HPK1 is specifically required for TGF-
-induced
activation of JNK1. We also found that TGF-
-induced JNK1 activation
was blocked by a kinase-defective mutant of TGF-
-activated kinase 1 (TAK1). In addition, interaction between HPK1 and TAK1 was observed in
transient transfection assays, and this interaction was enhanced by
TGF-
treatment. Both stress-activated protein kinase/extracellular
signal-regulated kinase kinase (SEK) and mitogen-activated protein
kinase kinase 7 (MKK7) are immediate upstream activators of JNK1.
Although SEK and MKK7 acted downstream of TAK1, only a kinase-defective
SEK mutant blocked TGF-
-induced activation of JNK1, indicating that
the TGF-
signal is relayed solely through SEK, but not MKK7,
in vivo. Furthermore, TGF-
-induced activating protein 1 activation was blocked by a HPK1 mutant, as well as by TAK1 and
SEK mutants. Taken together, these studies establish a potential
cascade of TGF-
-activated interacting kinases beginning with HPK1, a
Ste20 homolog, and ending in JNK1 activation: HPK1
TAK1
SEK
JNK1.
 |
INTRODUCTION |
Transforming growth factor-
(TGF-
)1 belongs to a
family of multifunctional cytokines that regulate cell proliferation,
cellular differentiation, apoptosis, cell adhesion and motility, and
production of the extracellular matrix (reviewed in Refs. 1 and 2). TGF-
initiates its pleiotropic effects by binding a heteromeric cell
surface receptor complex composed of type I and II transmembrane serine/threonine kinase receptors. Upon ligand binding, the type II
receptor, which confers ligand binding specificity, phosphorylates the
type I receptor, which confers signal specificity, in the highly
conserved GS domain, thereby activating type I receptor kinase activity
toward its downstream effectors (reviewed in Refs. 1 and 2).
A variety of signaling pathways are involved in transducing the TGF-
signal from the membrane receptor complex to the nucleus. The highly
conserved mothers against dpp (MAD) proteins have been identified as
downstream signal transducers (reviewed in Refs. 1 and 2). The protein
kinase A pathway has been shown to mediate TGF-
-induced cAMP
response element-binding protein phosphorylation and fibronectin
expression (3), whereas the protein kinase C pathway mediates the
TGF-
-induced activation of extracellular signal-regulated kinase
(ERK) pathway (4) and the adhesion response (5). TGF-
has also been
found to activate the evolutionarily conserved mitogen-activated
protein kinase (MAPK) cascades, including ERK1 (6), ERK2 (7, 8), and
c-Jun N-terminal kinase (JNK) (7-10), in a variety of cell systems.
The core of the MAPK cascades is a three-kinase module involving a
sequential protein kinase reaction (reviewed in Refs. 11 and 12). For
example, the prototype module for the JNK pathway is MEKK1-SEK-JNK.
However, a growing list of protein kinases at every level of the
three-kinase module has been identified, such as TGF-
-activated
kinase 1 (TAK1), MAPK upstream kinase (MUK), and mixed-lineage kinase 3 (MLK3) at the MAPK kinase kinase (MAPKKK) level (11) and
mitogen-activated protein kinase kinase 7 (MKK7) (13, 14) at the MAPK
kinase (MAPKK) level. Moreover, epistasis analyses in yeast have
identified Ste20 as the further upstream kinase, MAPKKK kinase
(MAPKKKK), for the Ste11(MAPKKK)-Ste7(MAPKK)-Fus/Kss1(MAPK) module
(15). Two subgroups of mammalian Ste20-like kinases have also been
identified. One subgroup consists of p21cdc42/rac1-activated
kinase 1 (PAK1) and PAK1-related kinases, which contain a C-terminal
catalytic domain and an N-terminal regulatory domain containing a
p21cdc42/rac1-binding region (16, 17). The other subgroup
consists of germinal center kinase (GCK; Ref. 18), hematopoietic
progenitor kinase 1 (HPK1; Refs. 19 and 20), GCK-like kinase (GLK; Ref.
21), KHS/GCKR (22, 23), and HPK/GCK-like kinase (HGK; Ref. 24)/NcK interacting kinase (NIK; Ref. 25). This subgroup of Ste20-like kinases
is characterized by an N-terminal kinase domain and a C-terminal region
of unknown function and by the lack of a Rac1/Cdc42-binding domain
found in PAKs.
Although HPK1 is widely expressed in embryonic tissues, its expression
is restricted to hematopoietic tissues in adults (19, 20). In transient
transfection assays, HPK1 has been shown to interact with MEKK1 (19)
and MLK3 (20), which, in turn, can activate SEK, thereby leading to the
activation of JNK pathway. Because its physiological activator(s) has
not been identified, the biological function of HPK1 and the mechanisms
of HPK1 signaling are currently unknown. However, a potential
association between HPK1 and several protein-tyrosine kinase receptors
mediated by SH2/SH3 adaptor proteins has recently been reported
(26-28).
We have previously shown in transfected 293T cells that TAK1, a
TGF-
-activated MAPKKK level kinase, serves downstream of HPK1 and
upstream of SEK in the JNK pathway (10). Because TAK1 is a potential
mediator of TGF-
signal transduction (29), we were prompted to
investigate whether HPK1 is an in vivo component of the
TGF-
-activated JNK signaling pathway. The studies presented here
show that TGF-
treatment of 293T cells resulted in a marked and
persistent increase in HPK1 activity and that HPK1 was required for the
activation of JNK and activating protein 1 (AP-1) by TGF-
. These
studies establish that HPK1 acts as an upstream activator for the
TAK1-SEK-JNK1 module in relaying the TGF-
signal into the nuclei in
293T cells.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
[
-32P]ATP was purchased from ICN
Biomedicals (Irvine, CA). An enhanced chemiluminescence system was
purchased from Amersham Pharmacia Biotech. TGF-
was purchased from
R & D Systems (Minneapolis, MN). Anti-HA antibody (12CA5) and
anti-FLAG antibody (M2) were purchased from Roche Molecular
Biochemicals and Eastman Kodak Co., respectively. Monoclonal anti-GST
and polyclonal anti-MEKK1 antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Polyclonal anti-JNK1 antibody (Ab101;
Ref. 33), polyclonal anti-HPK1 antibody (Ab2025; Ref. 19), and
polyclonal anti-HGK antibody (Ab2228; Ref. 24) were described
previously. All other reagents were purchased from Sigma unless
otherwise indicated.
Plasmids--
The GST-Jun (1-79) is a gift from M. Karin (UCSD,
San Diego, CA). D. Templeton (Case Western Reserved University,
Cleveland, OH) kindly provided the pUna3-MEKK1(KR) plasmid. The
pEF-TAK1 and pEF-TAK1-K63W plasmids are gifts from K. Matsumoto (Nagoya University, Japan). pEBG-KHS-KR is a gift from J. Blenis (Harvard Medical School, Boston, MA). p5xTRE-CAT was kindly provided by Dr. J. Bruder (Gen Vec, Rockville, MD). Dr. L. I. Zon (Children's Hospital, Boston, MA) kindly provided pEBG-SEK1-AL encoding a dominant-negative SEK1 mutant. pHA-JNK1 is a gift from Dr. J. Woodgett
(Ontario Cancer Institute, Toronto, Canada). pCI-FLAG-HPK1 and
pCI-FLAG-HPK1-M46 (19), pCR-FLAG-MKK7-K76E (14), and pCR-HGK-KR (24)
were described previously. pT7tag-MUK-KN encoding a dominant-negative MUK mutant was provided by S. Ohno (Yokohama City University, Yokohama, Japan).
Cells and Transfection--
293 and 293T cells were provided by
Dr. M. C.-T. Hu (Amgen, Thousand Oaks, CA) and grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and 1× streptomycin/penicillin at 37 °C in a humidified
atmosphere of 5% CO2. 293T cells were plated at a density
of 1.5 × 105 cells/35-mm plate well and transfected
the next day using the modified calcium phosphate precipitation
protocol (Specialty Media, Inc., Lavallette, NJ). Cells were
transfected with plasmids encoding
-galactosidase (0.15 µg) in
combination with an empty vector or various amounts of plasmids
encoding kinases or kinase mutants as indicated in the figure legends.
Cell Fractionation and Western Blot Analysis--
293 cells were
fractionated into cytosolic and nuclear fractions according to the
protocol as described previously (30). Western blot analysis was
performed using an enhanced chemiluminescence detection kit according
to the manufacturer's protocol (Amersham Pharmacia Biotech).
CAT Assays and Immunocomplex Kinase Assays--
CAT assays (31)
and immunocomplex kinase assays (32-35) were performed as described previously.
 |
RESULTS |
Concomitant Activation of HPK1 with JNK1 Following TGF-
Treatment--
The JNK signaling pathway is activated by TGF-
in a
variety of cellular systems, including 293T cells (7-10). We have
previously demonstrated that TAK1, a TGF-
-activated kinase, is a
potent activator for JNK and serves downstream of HPK1, a MAPKKKK level kinase, in the JNK pathway (10). We were thus prompted to address whether HPK1 is involved in TGF-
signaling in vivo. Using
an HPK1-specific antibody Ab2025 (19), we detected a high level of HPK1
expression in human embryonic kidney 293 and 293T cells (Fig.
1A). Cell fractionation
analysis indicates that HPK1 is localized in the cytosol but not in the
nucleus of 293 cells (Fig. 1A). To study the role of HPK1 in
TGF-
signaling, 293T cells were transfected with FLAG-tagged HPK1,
and following TGF-
treatment, the FLAG-HPK1 was immunoprecipitated
with anti-FLAG antibody (M2). HPK1 activity was determined by an
immunocomplex kinase assay using myelin basic protein as substrate.
TGF-
treatment resulted in a time-dependent activation
of HPK1 (Fig. 1B). The increased kinase activity was not due
to various levels of HPK1 because an equivalent expression of FLAG-HPK1
was detected by immunoblotting analysis with anti-FLAG antibody (M2)
(Fig. 1B, bottom panel). Endogenous HPK1
immunoprecipitated with an anti-HPK1 antibody (Ab2025) was also
activated by TFG-
with similar kinetics (data not shown). To address
a possible link between HPK1 and JNK in response to TFG-
, endogenous
JNK1 was immunoprecipitated with an anti-JNK1 antibody (Ab101) from the
FLAG-HPK1-transfected cell lysates, and its activity was determined by
an immunocomplex kinase assay using GST-c-Jun (1-79) as substrate.
Fig. 1C shows that endogenous JNK1 was activated by TFG-
with kinetics similar to that of HPK1. Thus, HPK1 was activated
concomitant with JNK1 in response to TGF-
in 293T cells.

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Fig. 1.
Concomitant activation of HPK1 with JNK
occurs following TGF- treatment.
A, HPK1 is expressed in 293 and 293T cells. 50 µg of whole
cell lysate from 293T cells and whole cell, cytosolic, and nuclear
lysates from 293 cells were resolved by 8% SDS-polyacrylamide gel
electrophoresis and immunoblotted with purified anti-HPK1 antibody,
Ab2025. B, HPK1 was activated by TGF- in a
time-dependent manner. 293T cells (1.5 × 105 cells in 35-mm wells) were transfected with 50 ng of
FLAG-tagged HPK1 and treated with TGF- (10 ng/ml) for various times
as indicated. The cells were collected 48 h after transfection.
FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2), and
immunocomplex kinase assays were performed using myelin basic protein
(MBP) as a substrate. Equivalent levels of FLAG-HPK1
expression were verified by immunoblotting with an anti-FLAG antibody
(M2). C, TGF- resulted in JNK activation with kinetics
similar to HPK1 activation. Endogenous JNK1 was immunoprecipitated with
an anti-JNK1 antibody (Ab101) from the cells transfected with FLAG-HPK1
after TGF- treatment, and immunocomplex kinase assays were performed
using GST-c-Jun (1-79) as a substrate.
|
|
TGF-
-induced JNK1 Activation Is Specifically Blocked by the HPK1
Mutant but Not by the KHS Mutant--
To investigate the involvement
of HPK1 in TGF-
-induced JNK1 activation, we attempted to block the
signaling cascade with HPK1-M46, an HPK1 kinase-defective mutant in
which methionine is substituted for lysine 46 (19). 293T cells were
transfected with HA-JNK1 alone or with HA-JNK1 plus various amounts of
HPK1-M46. Following TGF-
treatment, HA-JNK1 was immunoprecipitated
with an anti-HA antibody (12CA5), and the JNK1 activity was measured. Fig. 2A shows that
TGF-
-induced JNK1 activation was blocked by cotransfected HPK1
mutant (HPK1-M46) in a dose-dependent manner. In contrast,
cotransfection of HA-JNK1 with KHS-KR, a kinase-defective mutant of KHS
(22), another MAPKKKK level kinase, had no inhibitory effect on JNK1
activation by TGF-
. These data indicate that HPK1 is specifically
required for the transduction of TGF-
signal to JNK1 activation. To
further confirm the specific involvement of HPK1 in TGF-
-induced
JNK1 activation, we tested the effect of the HPK1 mutant on JNK1
activation by UV-C, a potent stimulus for the JNK pathway (33, 34).
UV-C-induced JNK1 activation was blocked by HGK-KR, a kinase-defective
mutant of HGK, a MAPKKKK level kinase (24). However, cotransfected HPK1
mutant HPK1-M46 had no effect on UV-C-induced JNK1 activation (Fig.
2B). Thus, HPK1 acts as a specific mediator for
TGF-
-induced JNK1 signaling pathway.

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Fig. 2.
JNK activation by TGF-
is specifically blocked by a kinase-defective HPK1 mutant
(HPK1-M46). A, 293T cells (1.5 × 105
cells in 35-mm wells) were transfected with either HA-JNK (0.1 µg)
alone or HA-JNK plus various amounts of HPK1-M46, as indicated by the
italic numbers. As a control, cotransfections of HA-JNK with
various amounts of a kinase-defective mutant of KHS (KHS-KR) were
included. Empty vector was used to normalize the amount of transfected
DNA. 36 h post-transfection, the cells were treated with TGF-
(10 ng/ml) for 12 h. Cell lysates were prepared, HA-JNK1 was
immunoprecipitated with an anti-HA antibody (12CA5), and immunocomplex
kinase assays were performed using GST-c-Jun (1-79) as a substrate.
Equivalent levels of HA-JNK, HPK1-M46, and KHS-KR expression were
verified by immunoblotting using anti-HA (12CA5), anti-FLAG (M2), and
anti-GST antibodies, respectively (three bottom panels).
B, UV-C-induced JNK activation is not sensitive to HPK1-M46.
293T cells (1.5 × 105 cells in 35-mm wells) were
transfected with HA-JNK alone (0.1 µg), HA-JNK plus 2 µg of
HPK1-M46, or HA-JNK plus 2 µg of HGK-KR. Empty vector was used to
normalize the amount of transfected DNA. 44 h post-transfection,
the cells were treated with UV-C (300 J/m2) for 30 min.
Cell lysates were prepared, HA-JNK1 was immunoprecipitated with an
anti-HA antibody (12CA5), and immunocomplex kinase assays were
performed using GST-c-Jun (1-79) as a substrate. Equivalent levels of
HA-JNK1, HPK-M46, and HGK-KR expression were verified by immunoblotting
using anti-HA (12CA5), anti-FLAG (M2), and anti-HGK (Ab2228)
antibodies, respectively (three bottom panels).
|
|
TAK1 Mutant Blocks TGF-
-induced JNK1 Activation--
TAK1, a
member of the MAPKKK family, has been shown as a potential mediator of
TGF-
signal transduction (29). Our previous studies have shown that
TAK1 is a potent JNK activator and a mediator of HPK1-induced JNK
activation (10). Given that HPK1 is required for the activation of JNK1
by TGF-
(shown above), we wondered whether TAK1 is an in
vivo component of the JNK1 signaling cascade triggered by TGF-
in 293T cells. To address this question, we investigated whether JNK1
activation by TGF-
is blocked by a kinase-defective mutant of TAK1,
TAK1-K63W. Cotransfection of 293T cells with HA-JNK1 and TAK1-K63W
abolished TGF-
-induced JNK1 activation (Fig.
3, lanes 3 and 4).
We did not observe interference with TGF-
-induced activation of JNK1
by a dominant-negative mutant of MEKK1 (lanes 5 and 6).
Further evidence for the specific involvement of TAK1 in
TGF-
-induced JNK1 activation is provided by the inability of the
kinase-defective mutant of MUK, another MAPKKK level kinase (36), to
block the JNK1 activation by TGF-
(data not shown). Therefore, TAK1
is likely an in vivo signaling component of
TGF-
-activated JNK1 pathway in 293T cells.

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Fig. 3.
TGF- -induced JNK
activation is blocked by the dominant-negative TAK1 mutant,
TAK-K63W. 293T cells (1.5 × 105 cells in 35-mm
wells) were transfected with either HA-JNK1 (0.1 µg) alone
(lanes 1 and 2) or HA-JNK1 plus various amounts
of TAK-K63W (lanes 3 and 4) and MEKK1-KR
(lanes 5 and 6), as indicated by the italic
numbers. Empty vector was used to normalize the amount of
transfected DNA. 36 h post-transfection, the cells were treated
with TGF- (10 ng/ml) for 12 h. Cell lysates were prepared,
HA-JNK1 was immunoprecipitated with an anti-HA antibody (12CA5), and
immunocomplex kinase assays were performed using GST-c-Jun (1-79) as a
substrate. To ensure equivalent levels of HA-JNK, HA-TAK-K63W, and
MEKK1-KR expression, an equal amount of each cell lysate was resolved
by 10% SDS-polyacrylamide gel electrophoresis, and immunoblotting was
performed with anti-HA (12CA5) and polyclonal anti-MEKK1antibodies
(three bottom panels).
|
|
The HPK1-TAK1 Interaction Is Enhanced by TGF-
--
Complex
formation exists widely in signaling processes and is an important
mechanism to facilitate signal transduction and maintain signaling
specificity. This led us to investigate whether HPK1 interacts with
TAK1 in vivo by a coimmunoprecipitation assay. HA-tagged
TAK1 and FLAG-tagged HPK1 were overexpressed by transient transfection
in 293T cells. When an anti-HA antibody (12CA5) was used to precipitate
HA-TAK1, FLAG-HPK1 was co-precipitated (Fig. 4A, left panel).
Conversely, HA-TAK1 was co-precipitated with FLAG-HPK1 when an
anti-FLAG antibody (M2) was used to precipitate FLAG-HPK1 (Fig.
4A, right panel). These data indicate that a
complex was formed between HPK1 and TAK1. Moreover, TGF-
treatment
significantly enhanced the complex formation between HPK1 and TAK1
(Fig. 4B). Thus, HPK1 interacted with TAK1 in 293T cells,
and this interaction was enhanced in response to TGF-
.

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Fig. 4.
TGF- enhances the
association of HPK1 with TAK1. A, 293T cells (1 × 106 cells in 100-mm dish) were transfected with FLAG-HPK1
(10 µg) alone or FLAG-HPK1 (10 µg) plus HA-TAK1 (10 µg). Empty
vector was used to normalize the amount of transfected DNA. 36 h
post-transfection, the cell lysates were prepared. FLAG-HPK1 and
HA-TAK1 were precipitated with an anti-FLAG antibody (M2) and an
anti-HA antibody (12CA5), respectively. The precipitants were then
immunoblotted with 12CA5 and M2, respectively. B, 293T cells
(1 × 106 cells in 100-mm dish) were transfected with
vector alone (lane 1), FLAG-HPK1 plus HA-TAK1 (lanes
2 and 3), FLAG-HPK1 alone (lane 4), and
HA-TAK1 alone (lane 5). 10 µg of each plasmid was used,
and empty vector was used to normalize the amount of transfected DNA.
36 h post-transfection, the cells were treated either with
(lane 3) or without (lanes 1, 2,
4, and 5) TGF- (10 ng/ml) for 12 h. Cell
lysates were prepared, and FLAG-HPK1 was precipitated with an anti-FLAG
antibody (M2). The precipitants were then immunoblotted with an anti-HA
antibody (12CA5). IB, immunoblotted; IP,
immunoprecipitated.
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|
TGF-
-induced Activation of JNK1 Is Only Blocked by a SEK Mutant
but Not by an MKK7 Mutant--
Both SEK (37) and MKK7 (13, 14) are
kinases that lie immediately upstream of JNK. We were interested in
determining which MAPKK (either SEK, MKK7, or both) relays the TGF-
signal from TAK1 to JNK1 activation in 293T cells. We examined the
ability of kinase-defective SEK and MKK7 mutants to block JNK1
activation by TAK1 and TGF-
. TAK-induced JNK1 activation was
abrogated by both SEK and MKK7 mutants in transient transfection assays
(Fig. 5A). However, only the
SEK mutant blocked TGF-
-induced JNK1 activation, whereas the MKK7
mutant exerted no effect (Fig. 5B). These data indicate that
it is SEK but not MKK7 that acts downstream of TAK1 and serves as an
endogenous regulator that relays the TGF-
signal to activate JNK1 in
293T cells.

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Fig. 5.
JNK activation by TGF-
is blocked by the SEK mutant but not by the MKK7 mutant.
A, both SEK and MKK7 mutants blocked TAK1-induced JNK
activation. 293T cells (1.5 × 105 cells in 35-mm
wells) were transfected with HA-JNK (0.1 µg) alone (lane
1), HA-JNK plus TAK1 (2 µg, lane 2), or HA-JNK plus
TAK1 and various amounts of either SEK (lanes 3 and
4) or MKK7 (lanes 5 and 6) mutants, as
indicated by the italic numbers. Empty vector was used to
normalize the amount of transfected DNA. Cell lysates were prepared at
48 h post-transfection, HA-JNK1 was precipitated with an anti-HA
antibody (12CA5), and immunocomplex kinase assays were done using
GST-c-Jun (1-79) as a substrate. Equivalent levels of HA-JNK, HA-TAK1,
MKK7-K76E, and SEK-AL expression were verified by immunoblotting using
anti-HA (12CA5), anti-HA (12CA5), anti-FLAG (M2), and monoclonal
anti-GST antibodies, respectively (four bottom panels).
B, only the SEK mutant blocked JNK1 activation by TGF- .
293T cells (1.5 × 105 cells in 35-mm wells) were
transfected with HA-JNK1 (0.1 µg) alone (lanes 1 and
2), HA-JNK1 plus various amounts of either SEK (lanes
3 and 4) or MKK7 (lanes 5 and 6)
mutants, as indicated by the italic numbers. Empty vector
was used to normalize the amount of transfected DNA. 36 h
post-transfection, the cells were treated with TGF- (10 ng/ml) for
12 h. Cell lysates were prepared, HA-JNK1 was precipitated with an
anti-HA antibody (12CA5), and immunocomplex kinase assays were
performed using GST-c-Jun (1-79) as a substrate. Equivalent levels of
HA-JNK, MKK7-K76E, and SEK-AL expression were verified by
immunoblotting using anti-HA (12CA5), anti-FLAG (M2), and monoclonal
anti-GST antibodies, respectively (three bottom
panels).
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|
HPK1 Is Required for TGF-
-induced, AP-1-dependent
Gene Expression--
The JNK signaling pathway has been implicated in
AP-1-dependent gene expression because c-Jun, one component
of the AP-1 complex, is a substrate for JNK in vivo (38).
TGF-
also exerts many of its effects through AP-1 gene expression
(39, 40). To further confirm the functional involvement of HPK1 in
TGF-
-induced JNK1 activation, we investigated the effect of the HPK1
mutant on TGF-
-induced AP-1 activity. AP-1 activity was increased by
TGF-
in a time-dependent manner, peaking at 24 h
(data not shown). We cotransfected a chloramphenicol acetyltransferase
(CAT) reporter construct containing multiple c-Jun-binding sites
(5xTRE-CAT) with or without HPK1-M46 into 293T cells.
TGF-
-stimulated AP-1 activity was blocked by HPK1-M46 (Fig.
6). As expected, cotransfection of the
TAK1 mutant or the SEK1 mutant also abrogated the TGF-
-induced AP-1
activity (Fig. 6). The CAT activity of the various transfectants
correlated closely with the activation of JNK1 (data not shown),
indicating that JNK1 activation contributed to the TGF-
-induced,
AP-1-dependent CAT activity. Taken together, these data
indicate that HPK1 is required for TGF-
-induced, JNK1-mediated AP-1
activation.

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Fig. 6.
TGF- -induced AP-1
activation is abrogated by the HPK1, TAK1, and SEK mutants. 293T
cells (1.5 × 105 cells in 35-mm wells) were
transfected with 5xTRE-CAT (0.1 µg) alone, 5xTRE-CAT plus 2 µg of
HPK1-M46, TAK1-K63W, and SEK-AL. Empty vector was used to normalize the
amount of transfected DNA. 36 h post-transfection, the cells were
treated with TGF- (10 ng/ml) for 12 h. Cells were harvested
48 h after transfection, and CAT activity was measured and
compared with that induced by transfection of empty vector. The
pCMV- -gal reporter plasmid (0.15 µg) was used in all transfection
assays to normalize variations in transfection efficiency. The results
are expressed as fold induction.
|
|
 |
DISCUSSION |
There is a growing interest in the identification and
characterization of Ste20-like kinases, because they might represent the first step in a number of linear cascades of sequentially acting
serine/threonine kinases leading to the stimulation of members of the
MAPK superfamily. Here we present the evidence that HPK1, a novel
Ste20/PAK-like protein, serine/threonine kinase, and a potent activator
of JNK, acts as an upstream regulator for the JNK1 signaling cascade
triggered by TGF-
. The evidence includes: (i) HPK1 activation by
TGF-
was accompanied by similar kinetics of JNK1 activation; (ii)
TGF-
-induced activation of JNK1 was specifically blocked by an HPK1
kinase-defective mutant; (iii) TGF-
enhanced the interaction between
HPK1 and TAK1, a TGF-
-activated MAPKKK level kinase, which is also
required for TGF-
-induced JNK1 activation; and (iv) TGF-
-induced, JNK1-mediated AP-1 activation was abrogated by an HPK1
kinase-defective mutant. The functional significance of the activation
of HPK1 by TGF-
has not been completely understood. It has been
recently shown that HPK1 specifically interacts with adaptor proteins,
such as Crk, CrkL, and Grb2, and through these adaptor proteins
connects to the membrane tyrosine kinase receptors (26-28). It is thus
conceivable that the JNK cascade is activated through a specific
interaction between HPK1 and an adaptor protein that connects it to the
TGF-
receptor complex. This hypothesis is supported by the recent
finding that GCK, another member of the HPK1 subfamily of mammalian
Ste20 homologs, interacts in vivo with the tumor necrosis
factor-
receptor 1-associated factor-2 and with MEKK1, thereby
acting as a molecular bridge and coupling tumor necrosis factor-
receptor 1-associated factor-2 to the JNK signaling pathway (41).
TAK1 was originally identified as a TGF-
-responsive MAPKKK level
kinase (29). It has been shown that TAK1 can activate SEK, an immediate
upstream activator kinase of JNK, in vitro (29), suggesting
that TAK1 might be involved in mediating TGF-
-induced JNK
activation. However, no direct evidence for this has been given so far,
although TAK1 has been shown to mediate JNK activation by other
stimuli, such as ceramide (42). Here we presented data showing that
TGF-
-stimulated JNK1 and AP-1 activation in 293T cells was
completely abrogated by a kinase-defective TAK1 mutant. Moreover, by
using an immunoprecipitation approach, we found that HPK1 interacted
with TAK1 and that this interaction was greatly enhanced by TGF-
.
Thus, these results indicate that TAK1 mediates TGF-
signaling to
JNK1 activation and subsequent AP-1 activation, although we have not
yet examined the activation of TAK1 by TGF-
in our system. This
conclusion is also in line with our previous observation that TAK1 acts
downstream of HPK1 and mediates HPK1-induced JNK activation (10).
The MAPK cascades can be activated by multiple extracellular stimuli
with little apparent commonality in the cellular effects, such as
cytokines, hormones, growth factors, and stresses (11, 12). The
mechanism that determines MAPK signaling specificity in response to
diverse extracellular stimuli has not been fully characterized. We
report here that TGF-
-induced JNK1 activation was only blocked by
the SEK1 mutant but not the MKK7 mutant at the MAPKK level, and by the
TAK1 mutant but not the MEKK1 and MUK mutants at the MAPKKK level.
These results imply that a mechanism might exist to ensure the correct
recruitment of TGF-
-specific JNK signaling components in
vivo. This idea is supported by the recent discovery that the
mammalian scaffold protein JIP1 preferentially binds MKK7 but not SEK,
thus assembling the selective MAPK module MLK3-MKK7-JNK (43). The
differential responses of SEK and MKK7 to extracellular stimuli have
also been reported. For example, UV and anisomycin strongly activate
SEK; however, MKK7 is only weakly activated (44). In addition, the
differential activation of SEK and MKK7 was observed in
MST/MLK2-dependent activation of JNK in which MST/MLK2
activates recombinant MKK7 more efficiently than recombinant SEK,
whereas MEKK1 activates both to a similar extent (45). Therefore, the
signaling leading to JNK activation may diverge upstream of JNK at the
various levels of the kinase module.
An emerging property of signal transduction pathways that might account
for signaling specificity is the formation of signaling complexes.
These complexes may result from the preferred physical interaction
between kinases within a module (12, 46). The inducible interaction
between HPK1 and TAK1 by TGF-
, combined with the requirement of HPK1
for TGF-
-induced JNK activation and the specific blockage of
TGF-
-induced JNK activation by the TAK1 mutant, strongly indicates
that HPK1 might have a recruiting function, coupling a TGF-
signal
to TAK1.
 |
ACKNOWLEDGEMENTS |
We thank Drs. J. Blenis, J. Bruder, M. C.-T. Hu, M. Karin, 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 and critical reading of the
manuscript; Roshi Afshar for technical assistance; and Mary Lowe for
secretarial assistance.
 |
FOOTNOTES |
*
This work was supported by the National Institutes of Health
Grants R01-AI38649 and R01-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.
¶
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: 713-798-3033; E-mail:
ttan{at}bcm.tmc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor
;
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase;
MAPKKK, MAPKK kinase;
MAPKKKK, MAPKKK kinase;
ERK, extracellular signal-regulated kinase;
JNK, c-Jun N-terminal kinase;
MEKK 1, MAPK kinase kinase 1;
HPK1, hematopoietic progenitor kinase 1;
KHS, kinase homologous to Ste20;
GCK, germinal center kinase;
HGK, HPK1/GCK-like kinase;
SEK, stress-activated protein kinase/ERK kinase;
MKK7, MAPK kinase 7;
TAK1, TGF-
-activated kinase 1;
MLK-3, mixed
lineage kinase 3;
HA, hemagglutinin;
GST, glutathione
S-transferase;
AP-1, activating protein 1;
MUK, MAPK
upstream kinase;
PAK1, p21cdc42/rac1-activated kinase 1;
CAT, chloramphenicol acetyltransferase.
 |
REFERENCES |
-
Massague, J.,
Hata, A.,
and Liu, F.
(1997)
Trends Cell Biol.
7,
187-197[CrossRef]
-
Derynck, R.,
and Feng, X.-H.
(1997)
Biochim. Biophys. Acta
1333,
F105-F150[CrossRef][Medline]
[Order article via Infotrieve]
-
Wang, L.,
Zhu, Y.,
and Sharma, K.
(1998)
J. Biol. Chem.
273,
8522-8527[Abstract/Free Full Text]
-
Axmann, A.,
Seidel, D.,
Reimann, T.,
Hempel, U.,
and Wenzel, K. W.
(1998)
Biochem. Biophys. Res. Commun.
249,
456-460[CrossRef][Medline]
[Order article via Infotrieve]
-
Chakrabarty, S.,
Rajagopal, S.,
and Moskal, T. L.
(1998)
Lab. Invest.
78,
413-421[Medline]
[Order article via Infotrieve]
-
Mulder, K. M.,
and Morris, S. L.
(1992)
J. Biol. Chem.
267,
5029-5031[Abstract/Free Full Text]
-
Frey, R. S.,
and Mulder, K. M.
(1997)
Cancer Lett.
117,
41-50[CrossRef][Medline]
[Order article via Infotrieve]
-
Frey, R. S.,
and Mulder, K. M.
(1997)
Cancer Res.
57,
628-633[Abstract]
-
Atfi, A.,
Djelloul, S.,
Chastre, E.,
Davis, R.,
and Gespach, C.
(1997)
J. Biol. Chem.
272,
1429-1432[Abstract/Free Full Text]
-
Wang, W.,
Zhou, G.,
Hu, M. C.-T.,
Yao, Z.,
and Tan, T.-H.
(1997)
J. Biol. Chem.
272,
22771-22775[Abstract/Free Full Text]
-
Fanger, G. R.,
Gerwins, P.,
Widmann, C.,
Jarpe, M. B.,
and Johnson, G. L.
(1997)
Curr. Opin. Genet. Dev.
7,
67-74[CrossRef][Medline]
[Order article via Infotrieve]
-
Ip, Y. T.,
and Davis, R. J.
(1998)
Curr. Opin. Cell Biol.
10,
205-219[CrossRef][Medline]
[Order article via Infotrieve]
-
Tournier, C.,
Whitmarsh, A. J.,
Cavanagh, J.,
Barrett, T.,
and Davis, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7337-7342[Abstract/Free Full Text]
-
Yao, Z.,
Diener, K.,
Wang, X. S.,
Zukowski, M.,
Matsumoto, G.,
Zhou, G.,
Mo, R.,
Sasaki, T.,
Nishina, H.,
Hui, C. C.,
Tan, T.-H.,
Woodgett, J. P.,
and Penninger, J. M.
(1997)
J. Biol. Chem.
272,
32378-32383[Abstract/Free Full Text]
-
Leberer, E.,
Dignard, D.,
Harcus, D.,
Thomas, D. Y.,
and Whiteway, M.
(1992)
EMBO J.
11,
4815-4824[Abstract]
-
Manser, E.,
Leung, T.,
Salihuddin, H.,
Zhao, Z. S.,
and Lim, L.
(1994)
Nature
367,
40-46[CrossRef][Medline]
[Order article via Infotrieve]
-
Martin, G. A.,
Bollag, G.,
McCormick, F.,
and Abo, A.
(1995)
EMBO J.
14,
1970-1978[Abstract]
-
Katz, P.,
Whalen, G.,
and Kehrl, J. H.
(1994)
J. Biol. Chem.
269,
16802-16809[Abstract/Free Full Text]
-
Hu, M. C.-T.,
Qiu, W. R.,
Wang, X.,
Meyer, C. F.,
and Tan, T.-H.
(1996)
Genes Dev.
10,
2251-2264[Abstract]
-
Kiefer, F.,
Tibbles, L. A.,
Anafi, M.,
Janssen, A.,
Zanke, B. W.,
Lassam, N.,
Pawson, T.,
Woodgett, J. R.,
and Iscove, N. N.
(1996)
EMBO J.
15,
7013-7025[Abstract]
-
Diener, K.,
Wang, X. S.,
Chen, C.,
Meyer, C. F.,
Keesler, G.,
Zukowski, M.,
Tan, T.- H.,
and Yao, Z.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9687-9692[Abstract/Free Full Text]
-
Tung, R. M.,
and Blenis, J.
(1997)
Oncogene
14,
653-659[CrossRef][Medline]
[Order article via Infotrieve]
-
Shi, C.-S.,
and Kehrl, J. H.
(1997)
J. Biol. Chem.
272,
32102-32107[Abstract/Free Full Text]
-
Yao, Z.,
Zhou, G.,
Wang, X. S.,
Brown, A.,
Diener, K.,
Gan, H.,
and Tan, T.-H.
(1999)
J. Biol. Chem.
274,
2118-2125[Abstract/Free Full Text]
-
Su, Y.-C.,
Han, J.,
Xu, S.,
Cobb, M.,
and Skolnik, E. Y.
(1997)
EMBO J.
16,
1279-1290[Abstract/Free Full Text]
-
Anafi, M.,
Kiefer, F.,
Gish, G. D.,
Mbamalu, G.,
Iscove, N. N.,
and Pawson, T.
(1997)
J. Biol. Chem.
272,
27804-27811[Abstract/Free Full Text]
-
Oehrl, W.,
Kardinal, C.,
Ruf, S.,
Adermann, K.,
Groffen, J.,
Feng, G. S.,
Blenis, J.,
Tan, T.-H.,
and Feller, S. M.
(1998)
Oncogene
17,
1893-1901[CrossRef][Medline]
[Order article via Infotrieve]
-
Ling, P.,
Yao, Z.,
Meyer, C. F.,
Wang, X. S.,
Oehrl, W.,
Feller, S. M.,
and Tan, T.-H.
(1999)
Mol. Cell. Biol.
19,
1359-1368[Abstract/Free Full Text]
-
Yamaguchi, K.,
Shirakabe, K.,
Shibuya, H.,
Irie, K.,
Oishi, I.,
Ueno, N.,
Taniguchi, T.,
Nishida, E.,
and Matsumoto, K.
(1995)
Science
270,
2008-2011[Abstract]
-
Zhou, G.,
Seibenhener, M. L.,
and Wooten, M. W.
(1997)
J. Biol. Chem.
272,
31130-31137[Abstract/Free Full Text]
-
Lai, J.-H.,
Horvath, G.,
Subleski, J.,
Bruder, J.,
Ghosh, P.,
and Tan, T.-H.
(1995)
Mol. Cell. Biol.
15,
4260-4271[Abstract]
-
Meyer, C. F.,
Wang, X.,
Chang, C.,
Templeton, D.,
and Tan, T.-H.
(1996)
J. Biol. Chem.
271,
8971-8976[Abstract/Free Full Text]
-
Chen, Y.-R.,
Meyer, C. F.,
and Tan, T.-H.
(1996)
J. Biol. Chem.
271,
631-634[Abstract/Free Full Text]
-
Chen, Y.-R.,
Wang, X.,
Templeton, D.,
Davis, R. J.,
and Tan, T.-H.
(1996)
J. Biol. Chem.
271,
31929-31936[Abstract/Free Full Text]
-
Chen, Y.-R.,
Wang, W.,
Kong, A.-N. T.,
and Tan, T.-H.
(1998)
J. Biol. Chem.
273,
1769-1775[Abstract/Free Full Text]
-
Hirai, S.,
Izawa, M.,
Osada, S.,
Spyrou, G.,
and Ohno, S.
(1996)
Oncogene
12,
641-650[Medline]
[Order article via Infotrieve]
-
Derijard, B.,
Raingeaud, J.,
Barrett, T.,
Wu, I. H.,
Han, J.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
Science
267,
682-685[Medline]
[Order article via Infotrieve]
-
Whitmarsh, A. J.,
and Davis, R. J.
(1996)
J. Mol. Med.
74,
589-607[CrossRef][Medline]
[Order article via Infotrieve]
-
Zhang, Y.,
Feng, X.-H.,
and Derynck, R.
(1998)
Nature
394,
909-913[CrossRef][Medline]
[Order article via Infotrieve]
-
Martin, M.,
Vozenin, M. C.,
Gault, N.,
Crechet, F.,
Pfarr, C. M.,
and Lefaix, J. L.
(1997)
Oncogene
15,
981-989[CrossRef][Medline]
[Order article via Infotrieve]
-
Yuasa, T.,
Ohno, S.,
Kehrl, J. H.,
and Kyriakis, J. M.
(1998)
J. Biol. Chem.
273,
22681-22692[Abstract/Free Full Text]
-
Shirakabe, K.,
Yamaguchi, K.,
Shibuya, H.,
Irie, K.,
Matsuda, S.,
Moriguchi, T.,
Gotoh, Y.,
Matsumoto, K.,
and Nishida, E.
(1997)
J. Biol. Chem.
272,
8141-8144[Abstract/Free Full Text]
-
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674[Abstract/Free Full Text]
-
Lu, X.,
Nemoto, S.,
and Lin, A.
(1997)
J. Biol. Chem.
272,
24751-24754[Abstract/Free Full Text]
-
Hirai, S.,
Noda, K.,
Moriguchi, T.,
Nishida, E.,
Yamashita, A.,
Deyama, T.,
Fukuyama, K.,
and Ohno, S.
(1998)
J. Biol. Chem.
273,
7406-7412[Abstract/Free Full Text]
-
Madhani, H. D.,
and Fink, G. R.
(1998)
Trends Genet.
14,
151-155[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.