From the First Department of Internal Medicine, 2Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine Kumamoto 860-0811, Japan
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ABSTRACT |
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The RON receptor-type tyrosine kinase, a member
of the hepatocyte growth factor receptor family, is a receptor for
macrophage-stimulating protein (MSP). Recently, we observed that MSP
induces morphological changes in interleukin
(IL)-3-dependent Ba/F3 cells ectopically expressing RON. We
show here that stimulation of those cells with either MSP or IL-3
increases tyrosine phosphorylation of proteins of 130, 110, 90, 62, and
58 kDa and induces similar morphological changes, accompanied by unique
nuclear shape and redistribution of F-actin. A tyrosine kinase
inhibitor, genistein, blocked both the increase in tyrosine
phosphorylation and morphological changes. Upon stimulation with either
MSP or IL-3, prominent tyrosine-phosphorylated pp90 was similarly
co-immunoprecipitated with the common Receptor-type tyrosine kinases
(RTKs)1 play a critical role
in pleiotropic cell functions such as proliferation and differentiation (1, 2). All RTKs are composed of three major domains, an extracellular
ligand binding domain, a single membrane-spanning domain, and a
cytoplasmic domain that contains a tyrosine kinase catalytic domain (3,
4). After ligand binding, RTKs undergo dimerization leading to
activation of their intrinsic tyrosine kinase activity and
autophosphorylation (5). The tyrosine-phosphorylated regions of the
receptor function as high-affinity binding sites for proteins
containing Src homology 2 and phosphotyrosine-binding domains, which
transduce recep tor-generated signals upon phosphorylation (1,
2, 6).
The RON receptor-type tyrosine kinase is a member of a subfamily of RTK
that includes the c-Met hepatocyte growth factor receptor and c-Sea
(7). The human RON gene was cloned from human keratinocytes (8), and
the mouse homolog, formerly known as STK (stem cell-derived tyrosine kinase), was derived from mouse
hematopoietic stem cells (9). RON, as well as the hepatocyte growth
factor receptor, is synthesized as a single-chain precursor, then
cleaved to a mature disulfide-linked heterodimer composed of an
extracellular MSP is an 80-kDa serum protein that belongs to a family characterized
by the presence of a highly conserved triple disulfide loop structure
(Kringle domain). The family includes prothrombin, plasminogen,
urokinase, and hepatocyte growth factor (14-17). MSP was shown to
induce murine resident peritoneal macrophages to become responsive to
chemoattractant C5a (18). MSP has multiple biological effects. In
murine resident peritoneal macrophages it induces cell spreading and
migration (18), direct chemotaxis (19), stimulates ingestion of
complement-coated erythrocytes (15), and inhibits endotoxin- or
cytokine-induced expression of inducible nitric oxide synthase mRNA
(20). MSP also plays roles in chemotaxis of keratinocytes (21), bone
resorption in osteoclasts (22), and in ciliary motility in bronchial
epithelial cells (23).
It has been established that the IL-3 receptor shares a common Recently, we found that stimulation with MSP induces cell growth
through phosphorylation of two C-terminal tyrosine residues in the
multifunctional docking site of RON (31). Stimulation with MSP also
induces cell shape changes (11). To gain further insight into signal
transduction events required for morphological changes and cell growth,
we have compared MSP- and IL-3-induced tyrosine phosphorylation in
IL-3-dependent Ba/F3 cells that ectopically express RON on
the cell surface. Binding of MSP to RON activates Reagents and Antibodies--
Recombinant human MSP and
recombinant mouse IL-3 were kindly provided by Drs. M. Hagiya and T. Takehara (Toyobo Co. Ltd, Shiga, Japan) and Dr. T. Sudo (Toray
Industries, Inc., Kamakura, Japan), respectively. A rabbit polyclonal
and a biotinylated rat monoclonal antibody against RON recognize the
intracellular C-terminal region and the extracellular domain,
respectively (11). Anti-phosphotyrosine (4G10 and FITC-conjugated 4G10)
and anti-mouse JAK2 were obtained from Upstate Biotechnology, Inc.
(Lake Placid, NY). Anti-mouse Plasmids and Cells--
cDNAs for murine full-length RON and
RON-F1330/F1337, carrying combined mutations of tyrosine 1330 and 1337 to phenylalanine, were described previously (31). The murine
IL-3-dependent pro-B cell line Ba/F3 stably expressed with
either wild-type RON or RON-F1330/F1337 was cultured in serum-free
medium containing 100 units/ml IL-3, as described (31).
Morphological Changes--
Wild-type RON- or
RON-F1330/F1337-expressing Ba/F3 cells were deprived of IL-3 for
12 h before stimulation. Cells were adjusted to 1.6 × 105 cells/ml in RPMI 1640 medium supplemented with 25 mM HEPES, pH 7.2, and 0.1% bovine serum albumin (BSA),
preincubated in the presence of 1 mM
Na3VO4 for 5 min, and then stimulated with 100 ng/ml MSP or 100 units/ml IL-3 in the presence of 1 mM
Na3VO4 for the indicated periods at 30 °C.
Treatment of cells with Na3VO4 specifically
enhanced MSP- or IL-3-induced morphological changes. Morphological
changes were quantitated by counting the number of cells characterized
by distortion, bending, stretching, and branching of the cell body
under a phase-contrast microscope in three randomly selected high-power
fields (magnification, ×200). Cells having filopodia and lamellipodia
around the cell periphery were not counted. For treatment with various
inhibitors, cells (1.6 × 105 cells/ml) were
pretreated with various concentrations of genistein dissolved in 0.25%
Me2SO, 20 µg/ml actinomycin D, or 10 µg/ml cycloheximide in 0.5% ethanol at 37 °C for 30 min, and then
stimulated with MSP or IL-3 as described above. Murine bone marrow
cells were obtained from C57BL/6 mice and long-term cultured in
Immunofluorescence--
Cells (1.6 × 105
cells/ml) were stimulated with MSP or IL-3 as described above. The
cells were fixed with 3.7% paraformaldehyde at room temperature for 20 min. After washing, the cells were permeabilized with
phosphate-buffered saline containing 3% BSA and 0.1% saponin for 20 min, as described (32), and then stained with rhodamine-phalloidin for
F-actin or propidium iodide for nuclei. For detection of colocalization
of tyrosine-phosphorylated proteins and RON, cells were fixed as above,
blocked with phosphate-buffered saline containing 3% BSA for 20 min,
and then stained with biotinylated anti-RON for 1 h. After
permeabilized with saponin, the cells were stained with FITC-conjugated
anti-phosphotyrosine antibody for 1 h and Texas red-conjugated
streptavidin for 30 min. To prevent dephosphorylation, 10 mM Na3VO4 was included throughout
the staining procedure, as described (33). To detect colocalization of
RON and Cell Migration Assay--
50 µl of cell suspension (1 × 106 cells/ml in RPMI 1640 medium supplemented with 25 mM HEPES, pH 7.2, and 0.1% BSA) in triplicate were placed
in the top wells of a 48-well microchemotaxis chamber (NeuroProbe Inc.,
Cabin John, MD). The lower wells, separated by a 5-µm pore
polycarbonate membrane, were filled with 30 µl of 100 ng/ml MSP, 100 units/ml IL-3, or medium alone. After incubation at 37 °C for 3 h, cells migrating through the membrane were fixed and stained with
May-Giemsa. The cells were counted under a microscope in five randomly
selected high-power fields (magnification, ×400). Results were
expressed as cell number/5 high-power fields (5 hpf).
Detection of Tyrosine Phosphorylation by Western Blotting and
Immunoprecipitation--
Cells (2 × 107 cells/ml)
were stimulated in the presence of 1 mM
Na3VO4 at 30 °C as described above, unless
stated. The cells were washed with HEPES-buffered saline (50 mM HEPES, pH 7.2, 150 mM NaCl and 10 mM Na3VO4) at 4 °C, and then
lysed with Triton X-100 lysis buffer (50 mM HEPES, pH 7.4, 1% Triton X-100, 4 mM EDTA, 100 mM NaF, and 10 mM Na3VO4) containing 50 µg/ml
aprotinin, 200 µM leupeptin, 50 µM
pepstatin A, and 2 mM phenylmethylsulfonyl fluoride at
4 °C. Lysates were subjected to SDS-polyacrylamide gel
electrophoresis and electrotransferred onto polyvinylidene difluoride
membranes. Immunodetection was performed by enhanced chemiluminescence,
as described (31, 34, 35). Immunoprecipitations were performed with
anti-RON, anti- In Vitro Kinase Assay--
Protein G-Sepharose beads (Pharmacia)
were precoated with both anti-RON reactive to the epitope in the
extracellular domain of RON and anti- Morphological Changes Induced by MSP or IL-3--
It is often
observed that exposure of bone marrow cells and
IL-3-dependent cell lines to IL-3 leads to changes in cell
morphology and cell growth. Fig.
1A shows an example of cell
shape changes of IL-3-dependent long-term cultured bone
marrow cells. Our previous finding (11) indicates that MSP stimulation
induces morphological changes in the IL-3-dependent pro-B
cell line Ba/F3 ectopically expressing wild-type RON. We therefore
compared the effects of MSP and IL-3 on cell morphology in these cells.
MSP and IL-3 were found to induce similar morphological changes. A
decrease in incubation temperature from 37 to 30 °C and addition of
Na3VO4, a tyrosine phosphatase inhibitor,
increased the proportion of cells morphologically changed (Fig.
1B, upper panels). Morphological changes were
detected at 15 min and peaked at ~60 min of stimulation (data not
shown). Phosphorylation of two C-terminal tyrosine residues, Tyr-1330 and Tyr-1337, in the multifunctional docking site of RON plays a
critical role in RON-mediated signal transduction (31). Surprisingly, cells expressing RON-F1330/F1337, which carried mutations of tyrosine 1330 and 1337 to phenylalanine, responded to MSP with striking changes
of morphology (lower panels).
To characterize MSP- or IL-3-induced shape changes, cells were stained
with propidium iodide for nuclei and rhodamine-phalloidin for F-actin,
and analyzed by Nomarski differential-interference-contrast and
confocal fluorescence microscopy. Fig. 1C shows that
stimulation with MSP or IL-3 induced drastic morphological changes
accompanied by unique nuclear shape changes and redistribution and an
increase in F-actin. Most cells showed uropod-like structures. Note
that lobulated nuclei were preferentially observed upon MSP
stimulation, suggesting that the activity of MSP is stronger than that
of IL-3. These results indicate that MSP and IL-3 induce similar shape changes, and that MSP-induced shape changes are not mediated through the multifunctional docking site of RON.
Blockade of MSP- or IL-3-induced Shape Changes by
Genistein--
Tyrosine phosphorylation is involved in MSP and IL-3
signaling. We examined whether genistein, a tyrosine kinase inhibitor, blocked morphological changes. Treatment of wild-type RON-expressing cells with genistein inhibited both MSP- and IL-3-induced morphological changes, whereas genistein alone had no effect on morphology and viability (Fig. 2, A and
B). Another tyrosine kinase inhibitor, herbimycin A (20 µg/ml), also blocked morphological changes (data not shown).
Morphological changes were not inhibited by actinomycin D (ActD), an
RNA synthesis inhibitor, or cycloheximide (CHX), a protein
synthesis inhibitor (Fig. 2B, a). MSP was more
potent than IL-3 in both cell types (Fig. 2B, a
and b), and the proportion of cells morphologically changed
in RON-F1330/F1337-expressing cells was larger than that observed in
wild-type RON-expressing cells (data not shown). In
RON-F1330/F1337-expressing cells, morphological changes were also
blocked by genistein (Fig. 2B, b). These results indicate that tyrosine phosphorylation is required for morphological changes.
Tyrosine Phosphorylation of Cellular Proteins Induced by MSP or
IL-3--
To determine whether MSP or IL-3 induced tyrosine
phosphorylation of cellular proteins, Western blotting was performed
using the anti-phosphotyrosine mAb 4G10 (Fig.
3). Stimulation of wild-type RON-expressing cells with MSP increased tyrosine phosphorylation of RON
at 145 kDa and of proteins at 130, 110, 90, 62, and 58 kDa (Fig.
3A). A similar increase in tyrosine phosphorylation of
proteins at 130, 110, 90, 62, and 58 kDa was observed upon stimulation
with IL-3. Tyrosine phosphorylation of RON per se was not
induced by stimulation with IL-3. Addition of
Na3VO4 to cell suspensions throughout
stimulation periods and the decrease in reaction temperature from
37 °C (Fig. 3A, lanes 4-6) to 30 °C
(lanes 1-3) enhanced increases in the level of MSP- or
IL-3-induced tyrosine phosphorylation probably due to inhibition of
rapid dephosphorylation. These results are consistent with the
morphological observations (Figs. 1 and 2) and suggest that
RON-mediated signaling is closely related to the IL-3-induced signaling
through tyrosine phosphorylation.
In addition, increased levels of tyrosine phosphorylation were apparent
5 min after stimulation, peaked at 15 min, and sustained for at least
60 min of stimulation (data not shown). These kinetics suggest that
MSP- or IL-3-induced tyrosine phosphorylation precedes morphological
changes. Furthermore, the levels of tyrosine phosphorylation induced by
MSP were generally higher than those induced by IL-3, consistent with
observations that MSP was more potent than IL-3 in promoting
morphological changes (Fig. 2). pp90 was a prominent tyrosine-phosphorylated protein consisting of four subspecies (pp86,
pp87, pp91, and pp94) (Fig. 3B, upper panel).
Fig. 3B (lower panel) represents schematic
locations of pp90 subspecies.
Interaction of RON with the Common
The common
IL-3-induced receptor aggregation initiates autophosphorylation and
activation of JAK2 tyrosine kinase, and activated JAK2 in turn
phosphorylates
To examine whether RON kinase could directly tyrosine-phosphorylate
Interaction among RON,
RON-mediated tyrosine phosphorylation of Cell Growth and Migration--
IL-3 induced a strong proliferative
response in wild-type RON-expressing cells, whereas MSP-stimulated
cells displayed modest growth responses or prolonged survival (Fig.
7). However, in
RON-F1330/F1337-expressing cells, treatment with MSP or medium alone
did not induce cell growth, whereas IL-3 stimulation induced a strong
proliferative response (data not shown). These results are consistent
with our previous demonstration that MSP stimulation results in modest growth responses (31).
MSP is reported to induce chemotactic responses of murine peritoneal
macrophages and keratinocytes (19, 21). IL-3 has also been shown to act
as a chemotactic factor for eosinophils (37). To examine the effects of
MSP and IL-3 on chemotaxis, cell migration assays were performed. MSP
and IL-3 induced cell migration toward respective stimulants. After
3 h of stimulation, numbers of migrating cells increased
approximately 4-fold (205 ± 10 cells/5 hpf) and 2.8-fold
(132 ± 25 cells/5 hpf) in response to MSP and IL-3, respectively,
compared with medium alone (47 ± 23 cells/5 hpf). Cell migration
to MSP or IL-3 was inhibited by 50 µg/ml genistein (control, 17 ± 4 cells/5 hpf; MSP, 27 ± 1 cells/5 hpf; IL-3, 21 ± 6 cells/5 hpf). Similar results were obtained in
RON-F1330/F1337-expressing cells (data not shown). These results
suggest that RON-mediated tyrosine phosphorylation of In this study, we use IL-3-dependent cells ectopically
expressing RON to characterize distinct pathways involved in cell
growth and changes in morphology seen following IL-3 and MSP stimulation.
Our model for the mechanism of IL-3 receptor-mediated morphological
changes is shown in Fig. 8. In
unstimulated cells (Fig. 8A), ectopically expressed RON
kinase localizes with the IL-3 receptor chain of IL-3 receptor
(
c). Unlike IL-3, stimulation with MSP increased tyrosine phosphorylation of
c without activation of
JAK2, resulting in morphological changes with modest cell growth.
Confocal immunofluorescence analyses showed colocalization of RON,
c, and tyrosine-phosphorylated proteins. In
vitro kinase assays revealed that autophosphorylated RON
phosphorylated
c. These results suggest that the
signaling pathway for morphological changes through
c
and its associated protein pp90 is distinct from the pathway for cell
growth in the IL-3 signal transduction system.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain and a transmembrane
chain with intrinsic
tyrosine kinase activity (10, 11). RON has been identified as a
receptor for macrophage-stimulating protein (MSP) and binding of MSP to
RON stimulates autophosphorylation of RON to transmit
signals intracellularly (10-13).
(
c) subunit with the IL-5 receptor and the
granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor and
that each receptor is composed of a unique
subunit and the
c subunit (24-26). Each
subunit specifically
exhibits low-affinity binding to IL-3, IL-5, or GM-CSF, whereas the
c subunit lacks direct ligand binding but confers high
affinity binding to the
subunit. Binding of IL-3 to its receptor
complex induces activation of the JAK2 tyrosine kinase. In turn,
autophosphorylated JAK2 kinase activates
c to recruit
various signaling molecules to the tyrosine-phosphorylated
c and triggers signal transduction events mediated by
the cytoplasmic domain of
c (27-30).
c
without activation of JAK2 to produce morphological changes. Our
findings suggest that IL-3 stimulation activates two distinct signaling
pathways, one involved in morphological changes and the other in cell growth.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain common to IL-3, IL-5, and
GM-CSF receptors (
c), and Texas red-conjugated
streptavidin were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA) and PharMingen (San Diego, CA), respectively. Horseradish
peroxidase-conjugated F(ab')2 fragments of anti-mouse Ig
and of anti-rabbit Ig (Amersham Pharmacia Biotech) and
rhodamine-phalloidin (Molecular Probe, Eugene, OR) were also used.
-minimum essential medium containing 10% fetal bovine serum in the
presence of 100 units/ml IL-3 for 14 days. Cells were deprived of IL-3 for 7 h and then stimulated with 100 units/ml IL-3 for 1 h at 37 °C.
c, cells were fixed as above, blocked with
phosphate-buffered saline containing 3% BSA for 20 min, and then
stained with biotinylated rat anti-RON for 1 h, followed by
staining with Texas red-conjugated streptavidin for 30 min. After
permeabilization with saponin, cells were stained with
anti-
c for 30 min, followed by staining with
FITC-conjugated anti-rabbit IgG for 30 min. FITC-anti-rabbit IgG was
not cross-reactive with the rat anti-RON antibody. Confocal and
Nomarski differential-interference-contrast images were obtained using
a Fluoview laser scanning microscope (Olympus, Tokyo, Japan). Z-series
sections were recorded at 0.5-µm intervals and merged. To ensure no
bleed-through from the fluorescein signal into the red channel,
fluorescein and Texas red fluorochromes were independently excited at
488 nm and 568 nm, respectively. Emission signals were detected at
between 510 and 550 nm for fluorescein, more than 585 nm for Texas red,
and more than 610 nm for propidium iodide.
c, and anti-JAK2, and the immune
complexes were washed with Triton X-100 lysis buffer at 4 °C and
analyzed as described (31, 34, 35).
c antibodies.
First,
c was immunoprecipitated from the Triton X-100
lysate of parental Ba/F3 cells on the precoated beads. After washing
with Triton X-100 lysis buffer, RIPA buffer (50 mM HEPES,
pH 7.4, 1% Triton X-100, 4 mM EDTA, 1% deoxycholate, 0.1% SDS, 10 mM Na3VO4), and
Triton X-100 lysis buffer containing 1 M NaCl, the beads
were incubated with equal amounts of Triton X-100 lysates of either
COS-7 cells or COS-7 cells transiently transfected with RON. RON kinase
expressed in COS-7 cells was highly autophosphorylated without exposure
to MSP, probably due to aggregation of abundantly expressed RON. After
washing as above, equal amounts of each immunoprecipitate were
subjected to in vitro kinase assays. The immunoprecipitates
were suspended with 70 µl of kinase buffer (50 mM HEPES,
pH 7.4, 0.1% Triton X-100, 5 mM MgCl2, and 5 mM MnCl2) containing 0.2 mM
Na3VO4 and 100 µM ATP. After
incubation at 30 °C for the indicated periods, reactions were
terminated by addition of 2× SDS sample buffer and boiling for 3 min.
SDS-polyacrylamide gel electrophoresis and immunodetections were
performed as described above. Phosphorylated bands detected with
anti-pTyr (4G10) were quantified with a densitometer (ATTO, Tokyo).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Morphological changes accompanied by F-actin
reorganization upon stimulation with MSP or IL-3. A,
phase-contrast micrographs of IL-3-dependent long-term
cultured bone marrow cells. Cells were cultured in the presence of 100 units/ml IL-3 at 37 °C (a). The cells became round after
IL-3 deprivation for 7 h (b) and were restimulated with
100 units/ml IL-3 for 1 h (c). Arrowheads
indicate cells morphologically changed. Bar, 20 µm. B,
phase-contrast micrographs of either wild-type RON- or
RON-F1330/F1337-expressing Ba/F3 cells. Cells deprived of IL-3 for
12 h were stimulated with 100 ng/ml MSP (b and
e) or 100 units/ml IL-3 (c and f) in
the presence of 1 mM Na3VO4 for
1 h at 30 °C after preincubation with 1 mM
Na3VO4 for 5 min at 30 °C as described under
"Experimental Procedures." Bar, 20 µm. C, Nomarski
differential-interference-contrast and con-focal fluorescence
microscopic analyses of either wild-type RON- or
RON-F1330/F1337-expressing Ba/F3 cells. After stimulation with 100 ng/ml MSP, 100 units/ml IL-3, or medium alone as above, cells were
stained with propidium iodide for nuclei and rhodamine-phalloidin for
F-actin. Bar, 5 µm.
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Fig. 2.
Effect of genistein on MSP- or IL-3-induced
morphological changes. A, phase-contrast micrographs of
wild-type RON-expressing cells. Cells deprived of IL-3 for 12 h
were pretreated with Me2SO alone (upper panels)
or 50 µg/ml genistein (lower panels) for 30 min and then
stimulated with MSP or IL-3 in the presence of Me2SO or
genistein as described in Fig. 1. Bar, 20 µm. B,
quantification of morphological changes. Wild-type RON-expressing cells
(a) were stimulated with MSP (hatched bars), IL-3
(stippled bars), or medium alone (open bars) for
1 h as described in Fig. 1. The number of cells morphologically
changed in the presence of the indicated concentrations of genistein
(µg/ml), 20 µg/ml actinomycin D (ActD) and 10 µg/ml
cycloheximide (CHX) were examined in three randomly selected
high-power fields (magnification, ×200). The results represent the
mean ± S.D. from three different experiments (%).
RON-F1330/F1337-expressing cells (b) were pretreated with
Me2SO alone or 50 µg/ml genistein for 30 min and then
stimulated for 30 min as above. The results represent the mean ± S.D. from three different experiments (%).
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Fig. 3.
Tyrosine phosphorylation of cellular proteins
induced by MSP or IL-3. A, Western blots of equal
amounts of Triton X-100 lysates from wild-type RON-expressing cells
stimulated with MSP or IL-3 at 30 °C (lanes 1-3) and
37 °C (lanes 4-6) for 15 min in the presence of 1 mM Na3VO4 as described under
"Experimental Procedures," probed with anti-pTyr (4G10)
(upper panels) and reprobed with rabbit anti-RON
(lower panels). In the RON blot, the upper band represents
the cytoplasmic single-chain precursor of RON. B, the
upper panel shows pp90 (pp86, pp87, pp91, and pp94) upon
stimulation with medium alone (lane 1), MSP (lane
2), and IL-3 (lane 3); the lower panel shows
schematic locations of pp90 subspecies (pp86, pp87, pp91, and
pp94).
Chain of IL-3 Receptor
(
c)--
Because treatment with genistein inhibited
MSP- or IL-3-induced morphological changes (Fig. 2), we examined
whether genistein indeed inhibited MSP- or IL-3-induced tyrosine
phosphorylation in both wild-type RON- (Fig.
4, A-C) and
RON-F1330/F1337-expressing cells (E-G). Tyrosine
phosphorylation was similarly induced in both cell types, and treatment
with genistein inhibited increases in tyrosine phosphorylation of RON,
pp130, pp110, pp90, pp62, and pp58 (Fig. 4, A and
E). Inhibition of RON autophosphorylation (B and
F) indicates that RON kinase activity is repressed by
genistein.
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Fig. 4.
MSP-induced tyrosine phosphorylation of
c. Panels A and
E, Western blots of equal amounts of Triton X-100 lysates
from either wild-type RON- or RON-F1330/F1337-expressing cells, probed
with anti-pTyr. Cells deprived of IL-3 for 12 h were pretreated
with Me2SO alone (lanes 1-3) or 50 µg/ml
genistein (lanes 4-6), and then stimulated with MSP or IL-3
as described in Fig. 2. Panels B-D and
F-H, Western blots of RON,
c, and
JAK2 immunoprecipitates from the Triton X-100 lysates, probed with
anti-pTyr and reprobed with respective antibodies.
chain of IL-3 receptor (
c) is known to
play a pivotal role in signal transduction mediated by the IL-3
receptor system (27). Because
c appeared to correspond
to pp130 on anti-
c blots (data not shown; see Figs.
3A and 4, A and E), we therefore examined whether
c is involved in RON-mediated
signaling. Fig. 4, C and G show by
immunoprecipitation that MSP, like IL-3, induced an increase in
tyrosine phosphorylation of the
c doublet. The levels of
c tyrosine phosphorylation induced by MSP were generally higher than those induced by IL-3. In addition, MSP- or IL-3-induced tyrosine phosphorylation of
c was inhibited by genistein
(Fig. 4, C and G, lanes 4-6).
c to recruit various signaling molecules (36). We analyzed an increase in tyrosine phosphorylation of JAK2. Fig.
4, D and H show that MSP, unlike IL-3, was unable
to increase tyrosine phosphorylation of JAK2, suggesting that
activation of JAK2 is not required for RON-mediated signaling.
c, an in vitro kinase assay was performed
with RON kinase immunoprecipitated from RON-transfected COS-7 cells.
Fig. 5, A and B
show that tyrosine phosphorylation of
c was clearly
detected at 5 min (Fig. 5A, lane 5), and
increased at 30 min (lane 6), whereas an increase in
tyrosine phosphorylation of
c was not detected at 0 min
(lane 4).
c was not tyrosine-phosphorylated without RON kinase (lanes 1-3). These results suggest that
RON kinase could directly tyrosine-phosphorylate
c.
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Fig. 5.
Tyrosine phosphorylation of
c by RON kinase in
vitro. A,
c
immunoprecipitates were incubated with (lanes 4-6) or
without (lanes 1-3) RON kinase in reaction buffer
containing 100 µM ATP. Samples were taken at the
indicated times. Western blot of each sample was probed with anti-pTyr
(upper panel) and reprobed with anti-
c
(middle panel) and anti-RON (lower panel).
B, phosphorylated bands of
c detected with
anti-pTyr were quantified by densitography. The levels of tyrosine
phosphorylation at each incubation time (5 and 30 min) relative to that
of the control (0 min) are shown.
c, and pp90--
To examine
whether
c could associate with tyrosine-phosphorylated
proteins, co-immunoprecipitation analyses were performed with Triton
X-100 lysates obtained from wild-type RON-expressing cells. We found
that
c was specifically co-immunoprecipitated with pp90
but not with pp62 and pp58 (Fig.
6A; data not shown). Three
pp90 subspecies, pp87, pp91, and pp94, were co-immunoprecipitated with
MSP- or IL-3-activated
c, whereas pp86 was associated
with control
c. The degree of association of pp87, pp91,
and pp94 with
c corresponded to that of tyrosine
phosphorylation of
c (see Fig. 6, shorter exposure).
Neither pp86, pp87, pp91, nor pp94 was identified as STAT5, Vav, the
p85 subunit of phosphatidylinositol 3-kinase, or ezrin (data not
shown). These results suggest that tyrosine-phosphorylated
c forms a novel complex with pp90.
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Fig. 6.
Association of
c with pp90 and colocalization of
c with RON. Panel
A, Western blot of
c immunoprecipitates from
Triton X-100 lysates of wild-type RON-expressing cells, probed with
anti-pTyr (upper panels) and anti-
c
(lower panel). Cells were stimulated with medium alone
(lanes 1 and 1'), MSP (lanes 2 and
2'), and IL-3 (lanes 3 and 3'), as
described in Fig. 1. A shorter exposure of the film is shown on the
right. Asterisks indicate the positions of pp90
subspecies. Panels B and C, wild-type
RON-expressing cells were stimulated with medium alone (upper
panels) and MSP (lower panels) for 1 h as
described in Fig. 1. After fixation, cells were double-stained with
anti-pTyr (B) or anti-
c (C) (green
channel), together with anti-RON (B and C)
(red channel), as described under "Experimental
Procedures" and analyzed by Nomarski
differentiation-interference-contrast and confocal immunofluorescence
microscopy. The right panels show superimposed images of
green and red channels. Arrowheads
indicate the areas of colocalization of RON with
tyrosine-phosphorylated proteins and of RON with
c. Bar,
5 µm.
c suggests that
RON,
c, and tyrosine-phosphorylated proteins could be
colocalized. To test this, confocal immunofluorescence microscopic
analyses were performed. Fig. 6B (upper panels)
shows a uniform distribution of tyrosine-phosphorylated proteins and
RON in control cells. On the other hand, cells stimulated with MSP
showed the redistribution and colocalization of RON and
tyrosine-phosphorylated proteins as well as increases in tyrosine
phosphorylation of cellular proteins. Colocalization of RON and
tyrosine-phosphorylated proteins was prominently observed in regions
containing many granules and uropod-like structures (Fig.
6B, arrowheads, lower panels). As
shown in Fig. 6C, control cells showed colocalized
c and RON in a uniform distribution. Interestingly,
cells stimulated with MSP showed the redistribution and colocalization
of RON and
c. The pattern of colocalization of
c with RON is similar to that of tyrosine-phosphorylated
proteins and RON (arrowheads, lower panels),
consistent with redistribution of F-actin upon stimulation (Fig.
1C). These results suggest that RON,
c, and
tyrosine-phosphorylated proteins are colocalized particularly in
uropod-like regions of stimulated cells.
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Fig. 7.
Cell growth. After IL-3 deprivation for
12 h, wild-type RON-expressing Ba/F3 cells were stimulated with
MSP or IL-3 as described in Fig. 1. Viable cells were counted by trypan
blue exclusion, and the data represent the mean ± S.D. from three
different experiments.
c
underlying morphological changes contributes to migration but not to
cell growth.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
c. Neither JAK2
kinase that constitutively associates with
c nor RON
kinase is activated in the absence of IL-3 or MSP. When cells are
stimulated with IL-3 (Fig. 8B), JAK2 is activated to
phosphorylate both
c at multiple sites and recruited
STAT5 to trigger cell growth. Moreover, activation of JAK2 increases tyrosine phosphorylation of several proteins including pp90, which associates with
c. When cells are stimulated with MSP
(Fig. 8C), RON kinase activated by autophosphorylation
triggers increased tyrosine phosphorylation of
c and
several proteins including pp90, resulting in morphological changes. In
contrast to IL-3, MSP stimulation does not activate JAK2 or promote
strong proliferative responses. Modest cell growth induced by MSP
results from phosphorylation of two tyrosine residues in the
multifunctional docking site of RON. pp90 is located downstream of
activated JAK2 in the IL-3 signal transduction pathway. Activated RON
kinase presumably phosphorylates p90 without activation of JAK2; this
path leads to
c-mediated morphological changes but not
to proliferation.
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Fig. 8.
A model for two distinct signaling pathways
via c. Panel
A, ectopically expressed RON tyrosine kinase localizes with
c in unstimulated cells. Panel B, when IL-3
binds to its receptor that consists of
and
c chains,
JAK2 tyrosine kinase is activated to phosphorylate
c and
STAT5 triggering cell growth. In addition, stimulation with IL-3 leads
to morphological changes through tyrosine phosphorylation of
c and its associated protein pp90. Panel
C, when MSP binds to its receptor RON, RON kinase is
activated through autophosphorylation. Subsequently, activated RON
phosphorylates
c and pp90 without activation of JAK2,
leading to morphological changes. In contrast to IL-3, MSP stimulation
does not activate JAK2 and induces modest cell growth probably due to
tyrosine phosphorylation of two tyrosine residues in the
multifunctional docking site of RON.
It has been well established that tyrosine phosphorylation of
c is crucial for IL-3 signal transduction (24-30, 36,
38, 39). Binding of IL-3 to its receptor induces activation of JAK2 tyrosine kinase and triggers tyrosine phosphorylation of the
cytoplasmic domain of
c phosphorylation sites, resulting
in recruitment of signaling proteins, such as STAT5 and c-Myc.
Stimulation of the c-Kit receptor-type tyrosine kinase with the Kit
ligand phosphorylates and activates the erythropoietin receptor, which
associates with JAK2 (40). The activated
subunit of the IL-6
receptor and the ErbB2 receptor-type tyrosine kinase interact directly
with each other (41). The results presented here provide another example of cross-talk between a receptor-type tyrosine kinase and a
signal-transducing subunit/domain of the cytokine receptor family.
Based on our observations of crosstalk between RON and
c
in cell shape changes, we hypothesize that ectopically expressed RON
preferentially phosphorylates some of tyrosine residues on
c that can be phosphorylated by activated JAK2 following
stimulation with IL-3. Identification of the
c tyrosine
residues responsible for morphological changes will help further our
understanding of this signaling pathway.
We recently showed that two C-terminal tyrosine residues, Tyr-1330 and
Tyr-1337, in the multifunctional docking site of RON that are conserved
among the hepatocyte growth factor family receptors are principally
responsible for the signal transduction involved in cell growth (31).
After MSP binds to RON, signaling molecules such as Shc, phospholipase
C, phosphatidylinositol 3-kinase, Grb2, and others are recruited to
the two C-terminal tyrosine phosphorylation sites. We therefore
compared morphological changes and tyrosine phosphorylation in
wild-type RON-expressing cells with those in RON-F1330/F1337-expressing
cells. Surprisingly, the F1330/1337 mutation enhanced the morphological
responses, indicating that the multifunctional docking site is not
required. However, the kinase activity of RON is required for
morphological changes.
Intriguingly, treatment of cells with Na3VO4 at 30 °C during stimulation periods greatly augments not only the morphological responses but also the level of tyrosine phosphorylation of cellular proteins, because this treatment prevents tyrosine-phosphorylated proteins from rapid dephosphorylation by a yet unidentified protein-tyrosine phosphatase(s). We have evidence that activity of a protein-tyrosine phosphatase(s) is tightly associated with RON (data not shown), although the site of interaction of RON with the phosphatase(s) is not known. It is possible that the F1330/1337 mutation enhances activity of a protein-tyrosine phosphatase(s) by an unknown mechanism. Moreover, treatment with the protein-tyrosine kinase inhibitors, genistein and herbimycin A, completely blocks the morphological changes observed in both wild-type RON-expressing or RON-F1330/F1337-expressing cells. The delicate balance between tyrosine phosphorylation and dephosphorylation of specific proteins plays an important role in MSP- or IL-3-induced morphological changes.
pp90 consists of 4 subspecies, pp86, pp87, pp91, and pp94. pp86 is
associated with c in the unstimulated state. Upon
stimulation with MSP or IL-3, pp87, pp91, and pp94 are
tyrosine-phosphorylated and associated with
c, and the
levels of tyrosine phosphorylation of these proteins correspond to
those of tyrosine phosphorylation of
c. In addition,
using sucrose density gradient fractionation analyses we found that
pp90 was localized in the higher density lysate fractions at ~400 kDa
(data not shown), consistent with the results that pp90 can form a
protein complex with
c. Treatment of cells with MSP
relocates RON,
c, and tyrosine-phosphorylated proteins
to uropod-like structures where F-actin is redistributed and
concentrated. These results suggest that phosphorylation of pp90 could
control distribution and reorganization of actin filaments. Despite
efforts to purify pp90, at present we are unable to immunoprecipitate pp90 with the anti-phosphotyrosine antibody 4G10, probably because phosphotyrosine residues are not exposed to the surface of the protein
complex. Although we do not know that the migration of pp90 subspecies
on SDS-polyacrylamide gels stems from one molecule having four
different phosphorylation states or from four distinct molecules,
identification of pp90 will help us more fully understand how
activation of
c initiates morphological changes.
Previous data with LyD9 IL-3-dependent pro-B cells, which
ectopically express the IL-2 receptor, and FDC.P-1
IL-3-dependent hematopoietic precursor cells show that
stimulation of the former with IL-3 or IL-2 and the latter with IL-3
induces tyrosine phosphorylation of an ~90-kDa protein accompanied by
early cell shape changes and later cell growth (42). It is possible
that the IL-2 receptor, which associates with JAK1 and JAK3, also
interacts with c. Given that morphological
change-related pp90 is induced in IL-3-dependent Ba/F3
cells, we hypothesize that a tyrosine-phosphorylated protein at ~90
kDa seen in other IL-3-dependent LyD9 and FDC.P-1 cells is
identical to pp90 seen in Ba/F3 cells.
In conclusion, we have elucidated a novel IL-3 signaling pathway
involving pp90, which associates with c, and leading to morphological changes and subsequent cell migration. Our findings demonstrate that the signaling events of this pathway are distinct from
those required for cell growth. Cell migration is accompanied by
induction of morphological changes (43). To move through small openings
in various tissues, cells must be able to change their shapes
drastically. Because the receptors for IL-3, IL-5, and GM-CSF each
consist of a ligand-binding
chain associated with a common signal
transducer
c chain (24-27), it is reasonable to predict
that, in addition to IL-3 in immature hematopoietic cells, IL-5 and
GM-CSF can induce pp90 required for morphological changes of
eosinophils and neutrophils/macrophages, respectively.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Michio Hagiya and Toyohiro Takehara (Toyobo Co. Ltd) and Dr. Tetsuo Sudo (Toray Industries, Inc) for generously providing MSP and IL-3.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.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.
To whom correspondence should be addressed: Dept. of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422-8526, Japan. Tel.: 81-54-264-5703; Fax: 81-54-264-5705.
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ABBREVIATIONS |
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The abbreviations used are: RTKs, receptor-type tyrosine kinases; MSP, macrophage-stimulating protein; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleukin; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; hpf, high-power fields.
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