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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3347-3354
Activation of Hematopoietic Progenitor Kinase-1 by Erythropoietin
By
Yuka Nagata,
Friedemann Kiefer,
Takeshi Watanabe, and
Kazuo Todokoro
From the Tsukuba Life Science Center, The Institute of Physical and
Chemical Research (RIKEN), Ibaraki, Japan; the Max-Planck-Institute for
Physiological and Clinical Research, W.G. Kerckhoff-Institute, Bad
Nauheim, Germany; and the Medical Institute of Bioregulation, Kyushu
University, Fukuoka, Japan.
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ABSTRACT |
Hematopoietic progenitor kinase-1 (HPK1), which is expressed
predominantly in hematopoietic cells, was identified as a mammalian Ste20 homologue that, upon transfection, leads to activation of JNK/SAPK in nonhematopoietic cells. The JNK/SAPK pathway is activated by various environmental stresses and proinflammatory and hematopoietic cytokines. Upstream activators of HPK1 currently remain elusive, and
its precise role in hematopoiesis has yet to be defined. We therefore
examined the possible involvement of HPK1 in erythropoietin (Epo) and
environmental stress-induced JNK/SAPK activation in the Epo-dependent
FD-EPO cells and Epo-responsive SKT6 cells. We found that Epo, but not
environmental stresses, induced rapid and transient activation of HPK1,
whereas both induced activation of JNK/SAPK. A screen for HPK1 binding
proteins identified the hematopoietic cell-specific protein 1 (HS1) as
a potential HPK1 interaction partner. We found HPK1 constitutively
associated with HS1 and that HS1 was tyrosine-phosphorylated in
response to cellular stresses as well as Epo stimulation. Furthermore,
antisense oligonucleotides to HPK1 suppressed Epo-dependent cell growth
and Epo-induced erythroid differentiation. We therefore conclude that
Epo induces activation of both HPK1 and HS1, whereas cellular stresses
activate only HS1, and that the HPK1-JNK/SAPK pathway is involved in
Epo-induced growth and differentiation signals.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MITOGEN-ACTIVATED protein (MAP) kinases
form a large family of serine-threonine protein kinases conserved
through evolution.1,2 In mammalian cells, four distinct MAP
kinase cascades have been identified: extracellular signal-regulated
kinases (ERK),3,4 c-Jun amino-terminal kinases (JNK) or
stress-activated protein kinases (SAPK),5,6 p38 MAP kinase
(p38) or cytokine suppressive anti-inflammatory drug binding
protein,7,8 and Erk5/BMK.9,10 The classical MAP
kinases (ERK1 and ERK2) are activated by a variety of cell growth and
differentiation stimuli2,11,12 and play a critical role in
mitogenic signaling.13 The p38 and JNK/SAPK cascades are
primarily activated by various environmental stresses (osmotic shock,
UV radiation, heat shock, x-ray radiation, hydrogen peroxide, and
protein synthesis inhibitors) and by proinflammatory cytokines such as
tumor necrosis factor- and interleukin-1
(IL-1).5-8,14-21 Cellular stresses and proinflammatory
cytokines induce apoptotic cell death21; thus, it has been
suggested that JNK/SAPK and p38 have a function in apoptotic
signals.22,23 Various hematopoietic cytokines,
interleukins, and colony-stimulating factors regulating hematopoietic
cell growth, survival, and differentiation were also found to activate
JNK/SAPK and p38.24-28 Furthermore, we recently found that
JNK/SAPK and p38 play a crucial role in erythropoietin (Epo)-induced
erythroid differentiation as well as Epo-dependent cell
growth.29 Crawley et al30 reported that p38
acts in IL-2- and IL-7-driven T-cell proliferation. Very recently it
was reported that p38 plays a critical role in neuronal differentiation
of PC12 cells.31 Thus, JNK/SAPK and p38 appear to act not
only in apoptotic signaling, but also in mitogenic and differentiation processes.
Hematopoietic progenitor kinase-1 (HPK1) is a member of a family of
mammalian Ste20-related kinases.32,33 Initially identified by subtractive hybridization in hematopoietic progenitor cells and
subsequently shown to be predominantly expressed in hematopoietic cells,32 HPK1 bears extensive homology to the Ste20
homologues GC-kinase,34 KHS,35 and
GLK.36 Transfection studies in nonhematopoietic cells
identified HPK1 as a specific activator of the JNK/SAPK pathway.32,33 Within the kinase cascade leading to its
activation JNK/SAPK is immediately preceded by the upstream kinases
MKK4/SEK137 and MKK7/SEK2,38-40 which
themselves can be phosphorylated and activated by different kinases
including MEKK1,41 MLK2/3,42,43 or
TPL-2.44 All members of the GC-kinase family of Ste20
homologues are capable of activating the JNK/SAPK cascade in
transfection models. While kinase activity has been demonstrated to be
a prerequisite for that function, the exact mechanism of activation of
subsequent pathway elements remains elusive. At all levels of the
JNK/SAPK kinase cascade either multiple kinases or different homologues of a given kinase have been shown to be active. While the existence of
multiple input elements enhances the possible levels of regulation of
MAPK pathways and contributes elements of developmental and tissue
specificity, it also complicates their analysis. The specificity and
precise biological function of the various upstream kinases involved in
JNK/SAPK activation in hematopoiesis remains currently unclear. Several
pathways leading to JNK/SAPK activation by HPK1 have been postulated,
involving the three different MAPK kinase kinases, MLK3,32
MEKK133 and TAK1.45 However, no direct involvement of HPK1 in the activation of the JNK/SAPK cascade in
hematopoietic cells has been demonstrated to date. Furthermore, it has
not been examined whether environmental stresses can activate HPK1, and
natural upstream agonists causing HPK1 activation have yet to be
identified. The identification of upstream agonists and interacting
proteins is therefore an important step towards a better understanding
of the biology of HPK1 and related Ste20 homologous kinases.
Hematopoietic cell-specific protein 1 (HS1) is one of the major
substrates of the Src family protein tyrosine kinases (Lyn, Blk, Fyn,
or Lck)46-48 and Syk/Zap-70 kinases.46,49,50
HS1 is highly tyrosine-phosphorylated during B-cell antigen receptor (BCR)-mediated signaling,46,47 and its phosphorylation is
synergistically enhanced by the Src family and Syk/Zap-70 kinases
associated with BCR.46,49,50 Phosphorylation of HS1 was
also reported to be induced by IL-5 treatment of B cells51
and Fc RI cross-linking of mast cells.52 Studies using
HS1-deficient mice53 or HS1-deficient mutant WEHI-231 cell
lines54 indicated that HS1 may function not only in B-cell
proliferation, but also in apoptotic events after BCR cross-linking. It
was recently found that tyrosine phosphorylation of HS1 is actually
required for BCR-induced apoptosis.46 However, the precise
role of HS1 in cell growth and apoptosis has not been determined.
We examined a possible involvement of HPK1 in Epo receptor-mediated and
environmental stress-induced JNK/SAPK activation in FD-EPO cells, which
grow in response to Epo,25 and in SKT6 cells, which can be
differentiated to hemoglobinized cells in response to
Epo.55 We found that Epo, but not environmental stresses, induced rapid and transient activation of HPK1, whereas both Epo and
environmental stresses induced activation of JNK/SAPK. A screen for
potential HPK1 binding proteins identified HS1, which we found constitutively associated with HPK1 in hematopoietic cells. HS1 is
tyrosine-phosphorylated in response to both Epo stimulation and
environmental stresses. The HPK1-JNK/SAPK pathway and the HS1-mediated
signaling pathway might share common regulatory elements. We discuss
here possible functions of the HPK1-JNK/SAPK and HPK1-HS1 pathways in
Epo receptor-mediated and cellular stress-induced signal transduction.
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MATERIALS AND METHODS |
Reagents.
A polyclonal rabbit antiserum against a HPK1 peptide
(#5),32 rabbit antiserum against mouse GST-HS1 (amino acid
residues 57 to 233),53 and a mouse monoclonal antibody
(2D20) specific to human HS1 (amino acid residues 306 to
320)46 were described previously. Antibodies against mouse
JNK1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Antiphosphotyrosine antibody, 4G10, was purchased from Upstate
Biotechnology (Lake Placid, NY). Human Epo (2.6 × 105
U/mg) was a gift from Kirin Brewery (Tokyo, Japan). GST-c-Jun was
prepared as reported.25 Phosphothioester oligonucleotides (S-oligos) were prepared and purified by BEX (Tokyo, Japan).
Cell culture.
Epo-dependent FD-EPO cells25 were maintained in RPMI-1640
medium supplemented with 10% fetal calf serum and 1 U/mL of human Epo.
Epo-responsive mouse erythroleukemia SKT6 cells have been described55 and were maintained in Ham F-12 medium
supplemented with 10% fetal calf serum. SKT6 cell proliferation is
independent of Epo and is neither enhanced nor impaired by the presence
of Epo. SKT6 cells were induced to differentiate by the addition of 0.5 U/mL of recombinant human Epo, followed by incubation for 4.5 days, and
hemoglobin-positive cells were stained by 0.05% 2,7-diaminofluorene
(DAF).29
In vitro HPK1 kinase assay.
FD-EPO cells were starved in RPMI 1640 medium containing 0.4% fetal
calf serum, 0.125 mg/mL of transferrin, and 0.01% bovine serum albumin
without Epo for 12 hours and were restimulated with or without 1 U/mL
of Epo for up to 60 minutes. SKT6 cells were stimulated with Epo
without starvation. To test for osmo-stress and heat-shock responses,
FD-EPO cells or SKT6 cells were incubated in the medium containing 0.35 mol/L NaCl for 30 minutes or incubated at 42°C for 1 hour. Cells
were lysed in a lysis buffer (50 mmol/L Tris, pH 7.5, 0.5% Nonidet
P-40, 150 mmol/L NaCl, 100 mmol/L sodium fluoride, 10 mmol/L sodium
pyrophosphate, 1 mmol/L EDTA, 2 mmol/L Pefabloc, 10 ng/mL leupeptin,
and 10 ng/mL aprotinin). Insoluble material was then removed by
centrifugation and the precleared cell lysate was incubated with
anti-HPK1 (#5)-specific antibody at 4°C for 2 hours.
Immunocomplexes were bound to protein A-Sepharose at 4°C for 1 hour, washed 5 times with TNE (140 mmol/L NaCl, 50 mmol/L Tris-HCl, pH
8.0, 5 mmol/L EDTA) containing 1% Nonidet P-40, twice with TNE, and
once with KB (50 mmol/L Tris-HCl, pH 7.5, 8 mmol/L MgCl2, 2 mmol/L MnCl2, 1 mmol/L dithiothreitol
[DTT]). Kinase reactions were performed in 30 µL of KB
at 30°C for 20 minutes in the presence of 10 µCi of
[ -32P]ATP. The reactions were terminated by mixing
with Laemmli sample buffer and boiling. The samples were resolved by
7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and autoradiographed.
Colt- cDNA library screen.
A Colt (cloning of ligand targets) library screen was performed as
published by Sparks et al.56 Briefly, a 15-day mouse embryo
cDNA library in EXlox (Novagen, Madison, WI) was screened following
the manufacturer's protocol at approximately 50,000 plaques/15-cm
dish. Six to 8 hours after plating, plaque size was determined and
isopropyl -D-thiogalactopyranoside-soaked filters, which had previously been air-dried, were applied. After overnight incubation at 37°C, the filters were removed and washed in
phosphate-buffered saline (PBS) containing 0.1% Triton X-100 until no
bacterial debris was left. Filters were then blocked in PBS containing
2.5% bovine serum albumin (BSA) for 30 minutes at ambient temperature.
A biotin-labeled peptide (APFENIPPPLPPKPKFR) comprising the second
proline-rich/SH3-ligand motif of the HPK1 central portion was
synthesized and purified by high-performance liquid chromatography
(HPLC). To generate multivalent complexes, 60 pmol of
peptide was incubated in 100 µL PBS with 2 µg streptavidin-coupled
alkaline phosphatase (Bio-Rad, Hercules, CA). Excess
biotin binding sites were blocked by addition of 125 µg biotin and 5 minutes of incubation. The whole reaction mixture was then dissolved in
3 mL PBS containing 2.5% BSA and added to a preblocked filter. After
incubation for 1 hour at room temperature, the filters were washed
three times with PBS containing 0.1% Triton X-100 and then developed
by the addition of 10 mL buffer (100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, 50 mmol/L MgCl2) containing 44 µL nitroblue
tetrazolium chloride (75 mg/mL 70% dimethylformamide) and 33 µL
5-bromo-4-chloro-3-indoyl-phosphate-p-toluidine salt (50 mg/mL
in dimethylformamide). At optimal signal strength, the chromogenic
buffer was removed and the reaction was stopped by the addition of 50 mmol/L HCl. Positive plaques were then collected, phage-eluted, and
rescreened until homogeneous preparations were obtained.
Immunoprecipitation and immunoblotting.
FD-EPO cells were starved in RPMI 1640 medium containing 0.4% fetal
calf serum, 0.125 mg/mL transferrin, and 0.01% BSA without Epo for 12 hours and restimulated with or without 1 U/mL Epo for up to 3 hours.
SKT6 cells were stimulated with Epo without starvation. In some cases,
the cells were incubated in the medium containing 0.35 mol/L NaCl for
30 minutes or incubated at 42°C for 1 hour. The stimulated and
unstimulated cells were immediately lysed in a lysis buffer (50 mmol/L
Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 150 mmol/L NaCl, 100 mmol/L
sodium fluoride, 10 mmol/L sodium pyrophosphate, 1 mmol/L EDTA, 2 mmol/L Pefabloc, 10 ng/mL leupeptin, and 10 ng/mL aprotinin). Insoluble
material was then removed by centrifugation and the cell lysate was
incubated with anti-HS1-specific rabbit antibody at 4°C for 2 hours. The immunocomplexes were then bound to protein A-Sepharose at
4°C for 1 hour. The beads were washed 5 times with lysis buffer
containing 0.1 % Nonidet P-40 before being boiled in Laemmli sample
buffer. Samples were fractionated in 7.5% SDS-PAGE and
electrotransferred to ECL membrane (Amersham, Little Chalfont,
UK). The membrane was blocked in 5% BSA in TBS-T (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, and 0.5% Tween 20) and
incubated with anti-HPK1 antibody (#5), anti-HS1 antibody (2D20), or
antiphosphotyrosine antibody (4G10) for 2 hours. After washing 3 times
with TBS-T, the membrane was incubated with antimouse or antirabbit
IgG-conjugated horseradish peroxidase antibody, and the antibody
complexes were visualized by an ECL system (Amersham).
Effect of S-oligonucleotides.
Various concentrations of antisense, sense, or scrambled
S-oligonucleotides derived from the HPK1 mRNA sequence were mixed in
the culture medium before the addition of 0.5 U/mL Epo. The S-oligonucleotides in the medium were replenished after 24 hours. The
antisense HPK1 S-oligonucleotide used was
5'-AGGGTCCACGACGTCCATCC-3'. As a control, the corresponding
sense S-oligonucleotides and scrambled S-oligonucleotides were used.
Cell proliferation of FD-EPO cells was measured by a colorimetric assay
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT; Sigma, St Louis, MO), originally developed by
Mosmann.57 Exponentially growing cells (exactly 3 × 104) were plated on microtiter plates in 100 µL of
culture medium in the presence or absence of Epo and various
concentrations of antisense, sense, or scrambled S-oligonucleotides.
After incubation at 37°C for 3 days, 10 µL of 5 mg/mL MTT in PBS
was added to each well and incubated at 37°C for an additional 4 hours; the colorimetric reaction was stopped by the addition of 100 µL of 0.04 mol/L HCl in isopropanol and thorough mixing. Optical
densities were measured using a microplate reader with a test
wavelength of 570 nm and a reference wavelength of 630 nm.
SKT6 cells treated with S-ologonucleotides were cultured in the
presence of 0.5 U/mL Epo for 4.5 days, and hemoglobin-positive cells
were stained by DAF.
| |
RESULTS |
Epo but not osmotic shock or heat shock activates HPK1.
To identify upstream regulators of HPK1, we tested for a possible
activation of HPK1 in response to Epo stimulation in the Epo-dependent
FD-EPO cells and Epo-responsive SKT6 cells. Using an HPK1-specific
polyclonal antipeptide rabbit serum (HPK1 #5), HPK1 was
immunoprecipitated from FD-EPO or SKT6 cells before and at various
times after Epo stimulation. The activity of HPK1 kinase was then
determined by its ability to autophosphorylate in vitro in the presence
of [-32P]ATP and Mg2+. A distinct increase
in HPK1 in vitro autophosphorylation in response to Epo was observed in
both FD-EPO cells (Fig 1A) and SKT6 cells
(Fig 1B). In both cell lines, autophosphorylation levels rapidly
increased as early as 3 minutes after Epo stimulation (lane 2) and
reached their maximum about 5 minutes after stimulation (lane 3).
Within the next 30 minutes, HPK1 kinase activity decreased, and 1 hour
after stimulation only basal level activity was detectable (lanes 4 through 6). Half of the HPK1 immunoprecipitates used for kinase assays
were probed with anti-HPK1 antibody to confirm that there were equal
amounts of HPK1 in these samples (Fig 1A and B, bottom). These results
indicate that HPK1 is rapidly and transiently activated in FD-EPO cells
as well as in SKT6 cells after Epo-receptor engagement.
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| Fig 1.
Epo but not osmotic shock or heat shock lead to
activation of HPK1 kinase activity. (A) In vitro HPK1
autophosphorylation in Epo-dependent FD-EPO cells (A) and in
Epo-responsive SKT6 cells (B). HPK1 was immunoprecipitated using a
HPK1-specific antibody after Epo stimulation at the indicated time
points (lanes 1 through 6) or after 0.35 mol/L NaCl treatment for 30 minutes (lane 7) or heat shock at 42°C for 30 minutes (lane 8). In
vitro kinase assays were performed in the presence of
[-32P]ATP. The arrow indicates the autophosphorylated
HPK1, and the band below is nonspecific.
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Various environmental stresses are known to induce activation of the
JNK/SAPK and p38 kinase pathways.5-8,14-21 We therefore investigated a possible activation of HPK1 in response to osmotic shock
and heat shock in FD-EPO and SKT6 cells. However, HPK1 kinase activity
was neither affected by exposure of FD-EPO or SKT6 cells to 0.35 mol/L
NaCl for 30 minutes (lane 7) nor by a heat shock at 42°C for 30 minutes (lane 8). In contrast, JNK1 was fully activated under the same
conditions as described previously.25 Thus, neither in
FD-EPO cells nor in SKT6 cells is HPK1 activity regulated by environmental stresses. HPK1 is therefore not likely to mediate JNK/SAPK activation in response to stressful stimuli.
In parallel to HPK1 activation in response to Epo, a JNK/SAPK in vitro
kinase assay showed that JNK1 was clearly activated by both Epo
stimulation and environmental stresses, as reported previously.25 The JNK1 activation profile in response to
Epo stimulation was somewhat delayed compared with that of HPK1.
Maximal HPK1 activity was observed after 5 minutes (Fig 1A and B),
whereas JNK1 activity peaked only 15 minutes after Epo stimulation.
These results suggest that Epo-receptor engagement but not
environmental stress induces activation of HPK1, which may in turn lead
to activation of JNK/SAPK in hematopoietic FD-EPO and SKT6 cells, as
previously described for transfected nonhematopoietic
cells.32,33
Identification of HPK1 interacting proteins.
To further define the Epo-dependent HPK1 signaling pathway in FD-EPO
cells, we attempted to identify proteins that physically interact with
HPK1. HPK1 consists of an N-terminally located kinase domain, followed
by a central region harboring 4 proline-rich motifs and a long
presumably regulatory C-terminal tail. Three of the 4 proline-rich
regions have previously been shown to be capable to act as SH3 domain
ligands.58 As recently demonstrated by Sparks et
al,56 peptides modeled after such SH3 ligands can be used
to identify their target SH3 domains with relatively limited cross-reactivity. Of the 4 proline-rich regions in HPK1 (P1 through P4), we selected proline-rich region P2 as a bait for our Colt (cloning
of ligand targets) screen. In previous experiments, P2 had displayed
the strongest SH3 domain affinity.58
Screening of a 14-day mouse embryonic cDNA library led to the
isolation of 12 cDNAs, all of which encoded SH3 domain containing proteins (Table 1). Two pairs of isolates
were derived from a shared progenitor-phage each, so that the screen
yielded in total 10 independent isolates. For each cDNA, we identified
a corresponding entry in the data sequence base. Table 1 summarizes the
identities of all 12 cDNAs. Somewhat surprisingly, drebrin, a neuronal
actin binding protein, was most highly represented within the HPK1
interacting proteins. When we aligned the amino acid sequences of the
different SH3-domains of the identified candidate proteins, we noticed
an unusually high level of homology between the drebrin, cortactin, and
HS1 SH3 domains (Fig 2).
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| Fig 2.
Sequence alignment of 5 potential HPK1 interacting
proteins. Residues identical between the individual proteins and murine
HS1 are shaded. Characteristic residues found in a large number of SH3
domains are indicated by hollow letters.
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HS1 is constitutively associated with HPK1 and is
tyrosine-phosphorylated in response to Epo and to osmotic shock.
HS1 has been described to be expressed in hematopoietic cells, drebrin
is specifically expressed in neurons, and cortactin is not expressed in
hematopoietic cells except megakaryocytes/platelets.59-61 We found HS1 but not cortactin or drebrin expressed in FD-EPO cells and
SKT6 cells (data not shown). We therefore tested for a possible
interaction between HS1 and HPK1 in FD-EPO cells
(Fig 3A) and SKT6 cells (Fig 3B) before and
after Epo stimulation as well as in cells that had been exposed to
osmotic shock. HS1 was immunoprecipitated with an anti-HS1-specific
rabbit antibody, and the immunocomplexes were separated by SDS-PAGE and
probed with anti-HPK1 antiserum #5. HPK1 was found to be constitutively associated with HS1, independent of stimulation of the Epo receptor (Fig 3A and B, upper panels, lanes 1 and 2) in both cell lines. Association levels were also insensitive to osmotic shock (Fig 3A and
B, upper panels, lanes 1 and 3) in both cell lines. Similar result was
obtained after exposure of cells to heat shock (data not shown). Equal
amounts of immunoprecipitated HS1 were demonstrated by immunoblotting
(Fig 3A and B, lower panels). We failed to detect HS1 in anti-HPK1
immunoprecipitates, most likely due to interference of the anti-HPK1
serum #5 with HS1 binding. Anti-HPK1 serum #5 is directed against a
peptide derived from the proline-rich central part of HPK1. Our results
suggest that HPK1 constitutively interacts with HS1 and thereby forms a
possible link between the JNK/SAPK and HS1 signaling cascades.
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| Fig 3.
HS1 constitutively associates with HPK1. Binding of HPK1
and HS1 in FD-EPO cells (A) or in SKT6 cells (B) before (lane 1) and
after (lane 2) Epo stimulation or osmotic shock (lane 3). Anti-HS1
immunoprecipitates were probed with anti-HPK1 antibody (upper panels)
or anti-HS1 antibody (lower panels). Arrows indicate HS1.
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The same filters shown in Fig 3A and B were reprobed with the
antiphosphotyrosine-specific antibody 4G10. We found that both Epo and
osmotic shock induced tyrosine phosphorylation of HS1 in FD-EPO cells
(Fig 4A) and SKT6 cells (Fig 4B). In
contrast, we failed to detect tyrosine-phosphorylation of HPK1. These
results clearly indicate that HS1 can be tyrosine-phosphorylated in
response to Epo stimulation and osmotic shock.
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| Fig 4.
HS1 is tyrosine-phosphorylated in response to Epo and
osmotic shock. The same filters shown in Fig 3 were reprobed with the
antiphosphotyrosine antibody 4G10. The arrow indicates the
tyrosine-phosphorylated HS1.
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HPK1 activation is involved in Epo-dependent cell growth.
We studied the possible involvement of HPK1 in Epo-dependent FD-EPO
cell growth by application of an antisense S-oligonucleotide strategy.
In the presence of antisense S-oligonuleotides to HPK1 mRNA,
Epo-dependent cell growth was significantly inhibited in a
dose-dependent manner (Fig 5A,
 ). In contrast, the scrambled S-oligonucleotide (Fig 5A,  )
and sense S-oligonucleotide (Fig 5A,  ) had little effect. Immunoblot
analysis of HPK1 demonstrated a clear reduction in HPK1 expression
levels in the presence of antisense S-oligonucleotides (Fig 5A, below).
Our results argue in favor of an involvement of Epo-induced HPK1
activation, which in turn contributes to activation of JNK/SAPK pathway
in Epo-dependent cell proliferation of FD-EPO cells.
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| Fig 5.
Antisense oligonucleotide-mediated abrogation of HPK1
expression in FD-EPO cells and SKT6 cells leads to reduced
Epo-dependent proliferation and inhibition of erythroid
differentiation. Various concentrations (0 to 30 µmol/L) of HPK1
antisense S-oligonucleotides (), HPK1 sense S-oligonucleotides
(), or scrambled S-oligonucleotides () were mixed with FD-EPO
cells (A) or with SKT6 cells (B) in the presence of Epo. Cellular
proliferation was measured using a MTT assay. The hemoglobinized cells
were stained with DAF, and the percentage of hemoglobinized cells
without oligonucleotides is shown as 100%. Values shown are the means
of six experiments.
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HPK1 activation is involved in Epo-induced erythroid differentiation.
Similarly, we examined the possible involvement of HPK1 in Epo-induced
hemoglobinization of SKT6 cells. In the presence of antisense
S-oligonuleotides to HPK1 mRNA, Epo-induced erythroid differentiation
was significantly inhibited in a dose-dependent manner (Fig 5B, ),
whereas scrambled S-oligonucleotide (Fig 5B, ) and sense
S-oligonucleotide (Fig 5B, ) had little effect. Immunoblot analysis
of HPK1 showed a clear reduction in HPK1 expression by antisense
S-oligonucleotides (Fig 5B, below). Taken together, these results
indicate that the HPK1-JNK/SAPK pathway, at least in part, plays a role
in Epo-dependent cell growth and differentiation.
| |
DISCUSSION |
Various hematopoietic cytokines, interleukins, and colony-stimulating
factors regulating hematopoietic cell growth, survival, and
differentiation were recently found to activate the JNK/SAPK, p38, and
ERK kinases.24-30 However, the role of these three distinct MAP kinases in hematopoiesis has not yet been determined in detail. We
show here that HPK1 is activated by Epo but not by environmental stresses in FD-EPO and SKT6 cells. This finding makes an involvement of
HPK1 as an upstream element mediating the Epo-induced activation of the
JNK/SAPK cascade likely. Interestingly, in the same cell lines, HPK1
does not seem to be involved in environmental stress-induced JNK/SAPK
activation. Potentially, HPK1 could also function as an upstream kinase
in other hematopoietic cytokine-induced pathways leading to JNK/SAPK
activation.24,25,27 Environmental stresses induce the
production of ceramide as a second messenger,62 which in
turn stimulates activation of TAK1,63 which may function as
a MAPK kinase kinase within the JNK/SAPK cascade. Based on the capacity
of dominant negative versions of TAK1 to inhibit HPK1-induced JNK/SAPK
activation in transfection experiments, TAK1 was implicated as a HPK1
downstream element. Our results implicate HPK1 in growth and/or
differentiation factor-stimulated JNK/SAPK activation rather than
stress-induced JNK/SAPK activation. Discovery and identification of the
JNK/SAPK interacting protein JIP1 as a mammalian Ste5 analogous
scaffolding protein defines a possible HPK1 downstream signaling
complex, comprising the MLK-family, MKK7, and JNK/SAPK.64
Although the precise mechanism of subsequent activation within the
triple kinase cascade MLK-MKK-JNK/SAPK is known to be phosphorylation
of regulatory threonine and tyrosine residues in kinase subdomain VIII,
it is not clear how Ste20 homologues activate their downstream
effectors mechanistically. Given the dependence of JNK/SAPK activation
on kinase activity of the respective upstream Ste20 homologue, it is
reasonable to assume, however, that kinase activity reflects activity
as an upstream pathway activator. We identified here Epo as a natural
agonist of HPK1 activation and found that HPK1-JNK/SAPK pathway plays a
role in Epo-induced cell growth and differentiation.
The elements that link HPK1 to the engaged Epo receptor remain unknown.
We have previously shown that after ectopic expression the adapter
molecule Grb2 can link HPK1 to tyrosine kinases like Src family kinases
or the EGF receptor.58 To pursue the quest for upstream
elements, we screened for additional HPK1 interaction proteins using a
peptide derived from the central proline-rich region P2 of HPK1. We
found the neuronal actin binding protein drebrin to be highly
represented in our screen for HPK1 binding SH3 domains. This result may
simply reflect a bias of the screening procedure or a representational
bias of the cDNA library, because a published screen using similar
proline-rich peptides of the class II type and an identical cDNA
library yielded identical SH3 domains.56 However, given the
expression of HPK1 in mouse embryos and neonates, we cannot exclude the
possibility that drebrin may be a valid HPK1 interaction partner at
those developmental stages. Nevertheless, we interpret the remarkably
high homology between the SH3 domains of drebrin, cortactin, and HS1 as
an indication of specificity of our screen. We present evidence here
that, at endogenous expression levels, HPK1 is constitutively
associated with HS1.
It has been reported that HS1 is one of the major substrates of the Src
family kinases.46-48 One of the Src family members, Lyn,
has been reported to physically associate with the Epo receptor and to
phosphorylate Stat5 as well as the Epo receptor65; in addition, Lyn is essential for erythroid differentiation.66 Thus, we examined the possible scenario that Lyn physically interacts with the Epo receptor and phosphorylates HS1 in response to Epo receptor engagement. However, at endogenous expression levels, we
failed to detect the interaction between Lyn and HS1 independent of Epo
stimulation or osmotic shock, although Lyn was constitutively tyrosine-phosphorylated (data not shown). Thus, the tyrosine kinase that phosphorylates HS1 after Epo stimulation and osmotic shock remains
to be identified. Furthermore, two phosphorylated HS1 bands were
detected in Fig 4B. This might indicate that there exists a
posttranslationally modified or an alternatively spliced form of HS1 or
a very closedly related HS1 gene product in SKT6 cells. This point also
has to be clarified.
We demonstrate here that both Epo and environmental stresses induce
tyrosine-phosphorylation of HS1. Although the functional significance
of tyrosine phosphorylation of HS1 in Epo-induced signaling remains to
be understood, HS1 is likely to be involved in erythroid proliferation
and differentiation rather than apoptosis. In contrast, in osmotic
shock-induced signaling, HS1 phosphorylation may play an important role
in signaling cascades leading to apoptotic cell death as observed in
BCR-induced apoptosis.
| |
ACKNOWLEDGMENT |
The authors thank Dr Tony Pawson for his interest and continuous
support and Noriko Takahashi and Junko Iita for technical assistance.
| |
FOOTNOTES |
Submitted September 22, 1998; accepted January 11, 1999.
Y.N. and F.K. contributed equally to this work.
Supported in part by a Special Grant for Promotion of Research from The
Institute of Physical and Chemical Research (RIKEN) and by grants from
the Ministry of Education, Science and Culture of Japan, the Science
and Technology Agency of Japan, the Uehara Memorial Foundation, and the
Suzuken Memorial Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kazuo Todokoro, PhD, Tsukuba Life Science
Center, The Institute of Physical and Chemical Research (RIKEN), 3-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan; e-mail:
todokoro{at}rtc.riken.go.jp.
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ABSTRACT
INTRODUCTION