From the Institut für Biochemie, OE 4310, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30623 Hannover and § Roche Diagnostics GmbH, Pharma Research Penzberg, Nonnenwald 2, D-82372 Penzberg, Germany
Received for publication, October 12, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Hepatocyte growth factor (HGF)/scatter
factor is a multifunctional cytokine that induces mitogenesis,
motility, and morphogenesis in epithelial, endothelial, and neuronal
cells. The receptor for HGF/scatter factor was identified as c-Met
tyrosine kinase, and activation of the receptor induces multiple
signaling cascades. To gain further insight into c-Met-mediated
multiple events at a molecular level, we isolated several signaling
molecules including a novel binding partner of c-Met, SH2
domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot
analysis revealed that SHIP-1 is expressed in the epithelial cell
line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at
phosphotyrosine 1356 at the multifunctional docking site. Because a
number of signaling molecules such as Grb2, phosphatidylinositol
3-kinase, and Gab1 bind to the multifunctional docking site, we further
performed an in vitro competition study using glutathione
S-transferase- or His-tagged signaling molecules with c-Met
tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1
bound preferentially over phosphatidylinositol 3-kinase. Surprisingly,
MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis
within 2 days after HGF treatment, whereas wild-type MDCK cells showed
tubulogenesis only after 6 days following treatment without altering
cell scattering or cell growth potency. Furthermore, overexpression of
a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated
branching tubulogenesis.
Met tyrosine kinase is the receptor for hepatocyte growth factor
(HGF)1/scatter factor (1, 2),
and signaling via the binding of this receptor to a ligand has been
shown to affect a wide range of biological activities, including
angiogenesis (3, 4), cellular motility (5), growth (6, 7), invasion
(8-10), and morphological differentiation including neuronal branching (11-14). c-Met also plays a role in normal embryological development (15-17), tissue regeneration (18), and wound healing (19). Furthermore, HGF also cooperates with nerve growth factor to enhance axonal outgrowth from cultured dorsal root ganglion neurons (14).
Binding of the growth factors causes activation of their inherent
receptor tyrosine kinases leading to autophosphorylation of the
cytoplasmic domains at multiple tyrosine residues. The newly formed
phosphotyrosines constitute binding sites for the Src homology 2 (SH2)
domain- and/or phosphotyrosine binding domain-containing cytoplasmic
proteins. Met tyrosine kinase was reported to interact with several
substrates including the growth factor receptor-bound protein (Grb) 2 (20), STAT3 (21), the p85 subunit of phosphatidylinositol (PI)
3-kinase (22), Shc (23), phospholipase C- The underlying mechanisms of these multiple molecular events, however,
are still poorly understood. In the present study we identified
SH2-containing inositol 5-phosphatase (SHIP) 1 as a novel binding
partner of c-Met by yeast two-hybrid screening using a rat brain
library. SHIP-1 binds to one of the tyrosine residues at the multiple
substrate binding site, 1356pYVNV, which is also the
binding site for Grb2. Interestingly, the YVNV motif is identical to
the common binding site of Shc to SHIP and Grb2. Furthermore, we show
that PI 3-kinase was not able to bind c-Met in the presence of
Grb2, Gab1, or SHIP-1. Overexpression of SHIP-1 in MDCK cells
drastically enhanced the branching potency of c-Met without affecting
the mitogenic and scattering potency. Furthermore, overexpression of a
mutant SHIP-1 lacking catalytic activity impaired HGF-mediated
branching tubulogenesis.
Plasmid Constructions, Transfection, and Yeast Two-hybrid
Screening--
The construction of LexA fusion genes encoding the
cytoplasmic domains of c-Met, c-Fms, TrkA, insulin receptor, and c-Kit downstream of LexA and the expression in
Saccharomyces cerevisiae strain L40 were
described previously (24, 31, 32). Met mutants containing
tyrosine/phenylalanine replacements have been described (24). A single
colony, selected for expression of the LexA fusion protein, was tested
for autophosphorylation and used for transformation with the VP16
cDNA library derived from an 8-day rat brain (33). GST·Grb2, GST·SHIP, GST·Gab1, GST·PI 3-kinase fusion
proteins were generated in the pGex system (Amersham Pharmacia
Biotech). His-tagged SHIP, Grb2, and PI 3-kinase were generated
in pQE30 (Qiagen, Hilden, Germany). Myc-tagged wild-type and mutant
SHIP-1 were generated using pcDNA3.1Myc-His (Invitrogen, Carlsbad, CA).
Binding Assay Using the Two-hybrid System--
The qualitative
and quantitative evaluations of various two-hybrid protein/protein
interactions were described previously (31).
Cells and Antibodies--
MDCK cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% FCS. HGF was from
Sigma. Scattering and branching tubulogenesis assays were
performed as described by Gual et al. (30).
Monoclonal antibodies against phosphotyrosine (4G10) and c-Met were
from Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal antibody
against Myc and rabbit IgG against c-Met and SHIP-1 were from Santa
Cruz Biotechnology (Santa Cruz, CA).
Tyrosine Kinase Assays and Immunoblotting--
These assays were
performed as published (31, 34).
Coimmunoprecipitation Study--
After incubation for 8 h
with medium containing 0.02% FCS, MDCK cells were stimulated with HGF
(60 ng/ml) for 5, 10, or 30 min. Cells were extracted with lysis buffer
containing 50 mM HEPES, pH 7.5, 150 mM NaCl,
1% Triton X-100, 1% trasylol, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 400 µM sodium
orthovanadate. One aliquot of each clarified lysate (5 × 106 cells) was incubated for 16 h at 4 °C with
anti-SHIP or anti-Met antibody preabsorbed on Staphylococcus
aureus Cowan I strain. After washing, materials were analyzed by
SDS-PAGE and by immunoblot analysis using the appropriate antibodies.
Binding of Cellular Proteins to GST Fusion Proteins and
His-tagged Proteins--
GST fusion proteins and His-tagged proteins
were produced as recommended by the manufacturer. Purified GST fusion
proteins were bound for 1 h at 4 °C to glutathione-agarose
beads (40 µl slurry; Amersham Pharmacia Biotech) suspended in the
binding buffer (50 mM HEPES, pH 7.5, 150 mM
NaCl, 1% Triton X-100, 1% trasylol, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 20 mg/ml bovine serum albumin,
and 200 µM sodium orthovanadate). After
incubation with 32P-labeled autophosphorylated c-Met
overnight, beads were washed five times with binding buffer, and
pellets were analyzed by SDS-PAGE.
SHIP-1 Associates with c-Met Tyrosine Kinase--
To identify
neuronal proteins that specifically interact with the cytoplasmic
domain of c-Met, we employed a yeast two-hybrid screening using an 8 day-old rat brain library (24, 33, 35). A total of 800 cDNA clones
of various length were obtained, together encoding seven different
proteins. Five of these proteins were previously identified as
c-Met-binding proteins including Gab1, PI 3-kinase, Grb2, PLC
SHIP-1 was originally identified as a signaling molecule in
cytokine-stimulated hematopoietic cells such as macrophage
colony-stimulating factor-stimulated cells (36) and has been clearly
demonstrated to participate in the signaling pathways in hematopoietic
cell systems (37). To determine whether SHIP-1 is a relevant signaling molecule in other cell systems, we next analyzed SHIP-1 expression in
various cell lines such as colon carcinoma CACO-2; MDCK cells; two
human breast carcinoma cell lines, EFM-19 and EFM-192A; and Raji cells.
For a control, we applied a cell lysate derived from myeloid
projenitor cell line FDC-P1Mac11 cells (38). In agreement with a
previous report (36), SHIP-1 is expressed at high levels in
myeloid progenitor cell line FDC-P1Mac11 cells. In addition, SHIP-1 was also detected in the epithelial cell lines, MDCK, both human
breast carcinoma cell lines, and Raji cells (Fig.
1).
To demonstrate the protein/protein interaction between SHIP and c-Met
in vivo, MDCK cells were incubated for 8 h with medium containing 0.02% FCS, then stimulated with HGF (60 ng/ml) for 5, 10, and 30 min, and studied by immunoprecipitation. Aliquots of 5 × 106 cells from each preparation were lysed for
immunoprecipitations using the SHIP-specific or c-Met-specific
antibodies. Precipitated material was analyzed for the presence of
c-Met or SHIP by SDS-PAGE and Western blotting using SHIP-, c-Met-, or
phosphotyrosine (4G10)-specific antibodies (Fig.
2). Following HGF stimulation, tyrosine
phosphorylation of c-Met was observed throughout the experiment. Prior
to HGF stimulation (0 min), no c-Met was detectable in the
SHIP-specific immune complexes. In contrast, between 5 and 30 min after
HGF stimulation, c-Met was coprecipitated with SHIP (Fig. 2).
The Phosphorylated Tyrosine 1356 Provides the Binding Site for
SHIP-1--
To determine whether the SHIP-1-c-Met interaction relied
on the presence of particular phosphotyrosine residues of c-Met, we
employed a set of c-Met mutants in which established binding sites for
defined binding partners were destroyed (24). These mutants included
Y1349F (Y14F), Y1356F (Y15F), the double mutant Y14F/Y15F, and
the kinase negative mutant K1110A. We examined the binding of these
mutants with the SH2 domain of SHIP-1, PI 3-kinase, Grb2, Grb10,
PLC SHIP Binds to c-Met and c-Fms but Not to c-Kit, TrkA, or the
Insulin Receptor--
Signaling molecules that bind to the
multifunctional docking site such as PI 3-kinase, Shc, and PLC Hierarchical Binding of Grb2, Gab1, SHIP, and PI 3-Kinase to the
Multifunctional Docking Site of c-Met--
The multifunctional docking
site of c-Met provides binding sites for many substrates including PI
3-kinase, Grb2, Gab1, and SHIP. As shown in Fig. 3, PI 3-kinase and
Gab1 bind at both phosphotyrosine 1349 and phosphotyrosine 1356, whereas SHIP and Grb2 bind only at phosphotyrosine 1356. This
observation raises the question whether two of these molecules bind to
c-Met simultaneously or sequentially. To answer this question, we have
generated the GST·SH2 domain of Grb2 (Grb2(SH2)), p85 of PI 3-kinase
(PI 3-kinase(SH2)), and SHIP (SHIP(SH2)) fusion proteins and GST·MBD
of Gab1 (Gab1(MBD)), which were incubated with
tyrosine-autophosphorylated c-Met in the presence and absence of
His-tagged PI 3-kinase(SH2), Grb2(SH2), or SHIP(SH2). Firstly,
we compared the binding of c-Met to PI 3-kinase and SHIP. When equal
amounts of both molecules were incubated with c-Met, SHIP bound
predominantly to c-Met. Secondly, Grb2 bound to c-Met preferentially
over PI 3-kinase and Gab1 (Fig. 5).
Thirdly, SHIP did not compete with the c-Met-Gab1 interaction. Taken
together, these results indicate that when phosphotyrosine 1356 binds
to either Grb2 or SHIP, phosphotyrosine 1349 does not provide a binding
site for PI 3-kinase and that when Grb2 binds to phosphotyrosine 1356, even Gab1 does not bind to c-Met, suggesting that in vivo
these molecules may bind to a single molecule of c-Met
sequentially.
Overexpression of SHIP-1 in MDCK Cells Drastically Potentiated
Branching Tubulogenesis Induced by c-Met without Altering
HGF-stimulated Cell Proliferation and Scattering--
To investigate
the biological role of SHIP-1 in c-Met-mediated signaling, we
overexpressed the Myc-tagged SHIP-1 gene in MDCK cells and isolated 19 different clones, each constitutively expressing distinct levels of an
Myc-tagged SHIP-1. The two clones (clones 15 and 21) employed in these
studies expressed equally high levels of SHIP-1 (Fig.
6A). Using these mutant cell
lines together with the wild-type MDCK cells as control, we examined
cell dissociation (scattering), cell proliferation, and branching
tubulogenesis induced by HGF stimulation. Firstly, the scattering
effect of HGF to both transfectants was indistinguishable from that of
the wild type (Fig. 6B). Secondly, in agreement with data
obtained from the fibroblast system (36), the overexpression of SHIP-1 showed no influence on [3H]thymidine incorporation,
suggesting that overexpression of SHIP-1 did not affect cell growth of
MDCK cells (Fig. 6C). Finally, we tested the ability of
these cells to form tubules in semi-solid collagen. To our surprise,
both transfectants that overexpressed Myc-tagged SHIP started to form
branching tubules within 24 h after HGF treatment. Two days after
HGF stimulation, transfectants formed long branching tubules (Fig.
7), whereas wild-type MDCK cells formed
branching tubules only 6-7 days after HGF treatment (Fig. 7).
Furthermore, in agreement with previous data (43), even 7 days after
HGF treatment, about 70% of wild-type cells formed tubules; however,
almost all transfectants formed tubules. In the absence of HGF, none of
the transfectants formed any branching tubules throughout a time period
of 2 weeks (data not shown). Taken together, these observations suggest
that overexpression of SHIP clearly enhances HGF-mediated
tubulogenesis.
Overexpression of a Mutant SHIP-1 Lacking Catalytic Activity in
MDCK Cells Impaired HGF-mediated Branching Tubulogenesis--
As shown
above, SHIP-1 overexpression accelerated tubulogenesis. This fact
raised the question whether this phenotype results from enhanced
phosphoinositol phosphatase activity or from displacement of protein
binding to the receptor at the SHIP-1 binding site. To answer this
question, we next generated a mutant SHIP-1 lacking catalytic activity
by deleting amino acid residues 666-680 (44) using
pcDNA3.1Myc-His, and we overexpressed this mutant in MDCK cells. Twelve different clones were isolated, and the two clones (clones 1 and 4) employed in this study expressed equally high levels
of mutant SHIP-1 (Fig. 8A).
Using these mutant cell lines together with the
pcDNA3.1Myc-His-transfected MDCK cells as control, we examined
branching tubulogenesis induced by the HGF stimulation. In agreement
with previous experiments using wild-type MDCK cells, the control
transfectant formed branching tubules within 7 days after the HGF
treatment. Two clones that overexpressed a mutant SHIP-1, however,
failed to form branching tubules over 9 days (Fig. 8B),
suggesting that the effects of SHIP-1 overexpression to accelerate
tubulogenesis are due to the enhanced phosphoinositide phosphatase
activity.
The results presented above can be summarized as follows.
Firstly, we have identified SHIP as a novel binding partner of c-Met. Secondly, SHIP binds to tyrosine 1356 at the multifunctional docking site of c-Met. Thirdly, we showed here that this site, which contains two phosphotyrosine residues, binds in vitro to only one
signaling molecule at a time. Fourthly, overexpression of SHIP
drastically enhanced the tubulogenesis potency of c-Met without
altering the c-Met-mediated cell scattering and cell proliferation.
Cells that overexpressed a mutant SHIP-1 lacking catalytic activity,
however, failed to form branching tubules in the presence of HGF,
suggesting that the effects of SHIP-1 overexpression to accelerate
tubulogenesis are due to the enhanced phosphoinositide phosphatase activity.
c-Met signaling is mediated by a multifunctional docking site
comprising two phosphotyrosines arranged in tandem (20). Most tyrosine
kinase receptors including c-Fms, c-Kit, and epidermal growth factor
receptor autophosphorylate at multiple sites that bind several
signaling molecules, suggesting that one receptor molecule is able to
simultaneously associate with multiple signaling molecules. In
addition, we showed here that the multifunctional docking site of c-Met
binds to only one signaling molecule and that when several effector
molecules are present simultaneously, one can observe in
vitro the hierarchical binding of proteins, such as Grb2, Gab1,
SHIP, and PI 3-kinase. Our competition assay reveals that PI 3-kinase
did not compete with c-Met-Grb2 and c-Met-SHIP interactions; however,
Grb2 competed with all of these interactions, suggesting that the
binding order of these effector molecules is crucial for the
c-Met-mediated multiple signal transduction. In vivo binding
of full-length molecules, however, may be dependent upon their
allosteric interactions. It is therefore also possible that multiple
complexes of signaling molecules are assembled to the activated
receptor. Furthermore, we have to take into account that the number and
the subcellular distribution of each of the signaling molecules
expressed in a given cell are different. For instance, when HGF-treated
cells are grown in a three-dimensional collagen matrix, cells form
tubules that have a lumen surrounded by well polarized epithelial
cells, with a smooth basal surface in contact with the collagen matrix
and an apical surface rich in microvilli that faces the lumen (11). The
subcellular localization of c-Met and substrates differs in these cells
from nonpolarized cells that are tested for cell scattering. For the
cell-scattering assay, cells were incubated in liquid culture medium
without collagen.
We show here that the SHIP-1 binding site, tyrosine 1356, is also a
binding site for Grb2 with the YXNX motif. Does
SHIP-1 regularly share the binding site with Grb2? In the case of
c-Fms, tyrosine 696 and tyrosine 921 provide the binding sites for Grb2 with the YXNX motif, 696YKNI and
921YTNL, respectively (31). Mutation of both sites,
however, did not exert an influence on the SHIP-1-c-Fms association,
indicating that none of these sites acts as a binding site for SHIP-1
(data not shown). These data suggest that the second or fourth flanking position is also important for binding. Interestingly, the SH2 domains
of SHIP and Grb2 also share the binding site 317pYVNV of
Shc (45), the sequence of which is identical to the 1356pYVNV of c-Met, indicating that YVNV is a consensus
sequence for the SHIP binding site (Table I). Interestingly, the point
mutation of asparagine 1358 into histidine abolished the branching
potential of c-Met (29).
A most striking observation of our studies regards the fact that
overexpression of SHIP-1 drastically enhanced the HGF-mediated branching tubulogenesis without altering cell growth and cell scattering in response to HGF (Figs. 6 and 7). Here, both transfectants that overexpressed Myc-tagged SHIP started to form branching tubules within 24 h after the HGF treatment. SHIP plays a role in inositol phosphate and phosphatidylinositol phosphate metabolism (46). SHIP-1
displays 5-phosphatase activity specifically with both phosphatidylinositol 3,4,5-trisphosphate and inositol
1,3,4,5-tetraphosphate as substrate. Phee et al. (47)
reported that enzymatic activity of SHIP is regulated by a plasma
membrane localization and, as a result of this regulation,
significant reduction in the cellular phosphatidylinositol
3,4,5-trisphosphate level was observed. Furthermore, a striking
correlation was observed between phosphatidylinositol 3,4-bisphosphate
production and tyrosine phosphorylation of SHIP-1, as well as its
relocation to the cytoskeleton upon thrombin stimulation in human blood
platelets (48). Here, phosphatidylinositol 3,4-bisphosphate may bind to
pleckstrin homolog domains of still undefined enzymes, which may
promote relocalization to the membrane and provide enzyme access to new
lipid substrates or regulatory kinases. Indeed, the serine/threonine
kinase B, also known as Akt has been shown to bind phosphatidylinositol
3,4-bisphosphate. In addition, the members of the protein
kinase C family, PKC
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(PLC
) (20), c-Src
(20), and Gab1 (24). Interestingly, all these proteins associate with
c-Met via a multiple substrate binding site. The c-Met-mediated
signaling was well studied in the epithelial cell system, MDCK cells.
On planar culture surfaces, sub-confluent MDCK cells normally form
coherent islands of relatively flattened cells. HGF has been shown to
alter this morphology by promoting the initial expansion of colonies
followed by dispersion of the cells that make up these colonies and an
increase in their motility (cell scattering) in liquid cell culture
medium (25). The activation of a number of tyrosine kinase receptors
including Neu, Ros epidermal growth factor receptor, and keratinocyte
growth factor receptor induced cell scattering in MDCK cells (26).
Royal and Park (27) reported that PI 3-kinase and Ras are required
for cell scattering. In addition, stimulation with HGF induces the
formation of tubular structures from MDCK cells grown in a
three-dimensional collagen matrix (11). For this, the epithelial cells
are grown for several days in collagen, in which they form cysts. When
HGF/scatter factor is added, individual cells dissociate and form
continuous tubules (28). It has been reported that a number of
signaling molecules including Gab1(24), Grb2 (29), PLC
(30), and
STAT3 (21) are required for this process.
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RESULTS
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ABSTRACT
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,
and c-Src. Furthermore, we detected the interaction with a novel
c-Met-interactive zinc finger-containing protein; however, the
biological significance of this association remains to be studied. In
addition to these molecules, we isolated SHIP-1 as a novel c-Met
binding partner.
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Fig. 1.
SHIP is expressed in MDCK, hematopoietic
cells, and human breast carcinoma cell lines. Cell lysates from
myeloid progenitor cell line FDC-P1Mac11, MDCK epithelial
cells, CACO-2 colon carcinoma cells (CaCo2), Raji human
Burkitt lymphoma cells, and two human breast carcinoma cells (EFM-19
and EFM-192A) (5 × 105 cells for each sample) were
analyzed by SDS-PAGE and immunoblotting using the anti-SHIP-1 rabbit
IgG.
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Fig. 2.
SHIP binds to c-Met tyrosine kinase.
MDCK cells were stimulated with HGF for 5, 10, and 30 min. Cell lysates
obtained from 5 × 106 cells were subjected to
immunoprecipitation (IP) using SHIP-1 or anti-c-Met
antibodies. Immunoprecipitates were analyzed by SDS-PAGE and
immunoblotting using the anti-Met, anti-SHIP, and anti-phosphotyrosine
antibodies. For a control, an aliquot of each lysate (5 × 104 cells) was analyzed by SDS-PAGE.
, and c-Src, the Met binding domain (MBD) of Gab1, and the
phosphotyrosine binding domain of Shc. As shown in Fig.
3, Grb2, Grb10, and SHIP-1 did not
associate with the Y15F mutant, suggesting that phosphotyrosine 1356 at the multifunctional docking site is the only binding site for these
proteins. On the other hand, PI 3-kinase, Gab1, PLC
, c-Src, and Shc
bind to both Y14F (Y1349F) and Y15F (Y1356F) (Fig. 3).
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Fig. 3.
Phosphotyrosine 1356 of c-Met is a binding
site of SHIP. Interaction of SHIP, PI 3-kinase, Grb2, and
Gab1 with c-Met mutants carrying tyrosine/phenylalanine or
lysine/alanine replacements as Y1349F (Y14F), Y1356F (Y15F), double
mutant Y14F/Y15F, and kinase negative mutant K1110A. These mutants were
coexpressed with the SH2 domain of SHIP, PI 3-kinase, Grb2, Grb10,
c-Src, or PLC , the MBD domain of Gab1, or the phosphotyrosine
binding domain of Shc, as VP16 fusion proteins in YRN974 (31). Aliquots
of 10,000 cells of four independent transformants were analyzed for
fluorescence intensity using a Becton Dickinson FACScan flow cytometer.
Values represent the means obtained in four independent
experiments.
associate with most receptor tyrosine kinases. Because SHIP-1 binds at
the same site in c-Met with these molecules, we performed similar
binding analyses with four additional activated receptor tyrosine
kinases including c-Fms, c-Kit, TrkA, and the insulin receptor. Western
blotting with anti-phosphotyrosine antibody clearly demonstrated that
these receptor moieties were phosphorylated on tyrosine in the yeast two-hybrid assay (data not shown). As expected, c-Fms binds to SHIP-1;
however, SHIP-1 did not bind other tyrosine kinase receptors such as
c-Kit, TrkA, or the insulin receptor (Fig.
4). In agreement with previous data
(39-41), PI 3-kinase binds to c-Fms, c-Kit, and the insulin receptor,
the sequences of which contain the typical consensus motif,
pYXXM, for the PI 3-kinase binding site (42). It is
noteworthy that both of the PI 3-kinase binding sites of c-Met,
tyrosine 1349 and 1356, and their following amino acid sequences are
different from this motif (Table I).
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Fig. 4.
SHIP binds to activated c-Met and macrophage
colony-stimulating factor receptor but not to c-Kit, TrkA, or the
insulin receptor. pBTM116 carrying cloned cDNAs encoding the
cytoplasmic domains of c-Met, c-Fms, c-Kit, TrkA, or the
insulin receptor (IR) were cotransformed with pVP16 carrying
the cDNA of the SH2 domains of SHIP, Grb2, PLC , or PI 3-kinase
or the MBD domain of Gab1. Ten thousand cells of four independent
transformants were analyzed for fluorescence intensity using a Becton
Dickinson FACScan flow cytometer.
pYVNV in c-Met and Shc binds to both SHIP and Grb2
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Fig. 5.
Hierarchical binding of Grb2, Gab1, SHIP, and
PI 3-kinase to the multiple substrate binding site of c-Met. GST
fusion proteins containing the SH2 domain of SHIP, Grb2, and p85 of PI
3-kinase and the MBD domain of Gab1 were purified from
Escherichia coli strain DH5 using glutathione-Sepharose.
The same proteins, but His-tagged, were purified from E. coli strain M15. GST fusion protein or GST (2 µg each) was bound
to glutathione-Sepharose beads and incubated with
32P-labeled c-Met, as obtained by an in vitro
kinase reaction with or without corresponding amounts of His-tagged
proteins. Following washing, bound protein was analyzed by SDS-PAGE
(B). For a control, aliquots were analyzed by SDS-PAGE and
staining with Coomassie Brilliant Blue (A).
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Fig. 6.
Overexpression of SHIP did not alter cell
growth and scattering in response to HGF. A, Myc-tagged
SHIP was transfected into MDCK cells. The established cell lines,
clones 15 and 21, were analyzed by immunoblot using anti-Myc antibody.
B, cells were stimulated with HGF (20 ng/ml) and chemically
fixed 24 h after HGF treatment. Cells were visualized by Giemsa
staining. C, the [3H]thymidine incorporation
in wild-type (wt) and SHIP-overexpressing MDCK (clones 15 and 21) cells in the presence of FCS or HGF. The
[3H]thymidine incorporation study was performed as
described previously (21). Sister cell cultures (8 × 103 cells per well) were starved for 24 h in medium
containing 0.1% FCS before stimulation with HGF.
[3H]Thymidine (100 µCi/ml) was added for 4 h.
Trichloroacetic acid-precipitable radioactivity was determined and
expressed as the mean value of four independent experiments.
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Fig. 7.
Overexpression of SHIP in MDCK cells
potentiates HGF-mediated branching tubulogenesis. Wild-type
(wt) and SHIP-overexpressing MDCK cell lines, clones 15 and
21, were grown in collagen for 5 days, and then medium containing HGF
(40 ng/ml) was added and changed every 2 days. After 2, 4, and 7 days,
the branching tubulogenesis response was evaluated. Photomicrographs by
Hoffman modulation contrast microphotometry show a representative
experiment that had been performed in triplicate.
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Fig. 8.
Overexpression of a mutant SHIP-1 lacking
catalytic activity in MDCK cells impaired HGF-mediated branching
tubulogenesis. A, MDCK cells transfected with
pcDNA3.1MycHis (Control) and mutant SHIP-1-overexpressing MDCK cell
lines, clones 1 and 4, lacking catalytic activity (SHIP 666-680)
(44), were analyzed by immunoblot using anti-Myc antibody.
B, cells were grown in collagen for 5 days, and then medium
containing HGF (40 ng/ml) was added and changed every 2 days. After 5, 7, and 9 days, the branching tubulogenesis response was evaluated.
Photomicrographs by Hoffman modulation contrast microphotometry show a
representative experiment that had been performed in triplicate.
SF, scatter factor.
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and PKC
, are activated by
phosphatidylinositol 3,4-bisphosphate (49). Interestingly, in the
platelet system, Hartwig et al. (50) reported that
phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing
and capping by human gelsolin in vitro, suggesting that the
product of SHIP-1 plays a role in the cytoskeleton rearrangement that
is important for morphogenesis. On the other hand, Maroun et
al. (51) reported that the ability of Gab1 to bind
phosphatidylinositol 3,4,5-trisphosphate is crucial for subcellular
localization of Gab1 and for efficient morphogenesis mediated by c-Met.
However, the same authors (43) reported that Gab1 phosphorylation
per se is not sufficient to induce branching tubulogenesis
and suggested that a Met-specific substrate, in addition to Gab1, is
required for branching tubulogenesis. Recently, it has been reported
that SHIP interacted with the protein inhibitor of activated STAT1,
PIAS1 (52). Another member of the STAT family, STAT3, was reported to
be an important molecule for c-Met-mediated branching tubulogenesis
(21). Here, SHIP-1 may also participate in the STAT pathway. Recently,
both amplification of PI 3-kinase and loss of function of a 3'-lipid
phosphatase, PTEN, have been shown in multiple human tumors (53-55),
demonstrating that the regulation of phosphatidylinositol
3,4,5-trisphosphate at the cellular membrane may be critical for the
control of multiple biological processes including tumorigenesis. At
the moment, the molecular mechanism of potentiation of HGF-mediated
branching by SHIP-1 overexpression remains unclear; however, inositol
phosphate and/or phosphatidylinositol phosphate metabolisms play a key
role in epithelial cell dissociation, reassociation, and formation of
continuous tubules.
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ACKNOWLEDGEMENTS |
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We thank Karsten Heidrich and Regina Wilms (Medizinische Hochschule Hannover) for technical assistance; Walter Birchmeier (Max-Delbruek Center of Molecular Medicine, Berlin, Germany) for generously providing LexA-Met and its mutants, LexA-insulin receptor, LexA-TrkA, and LexA-Kit cDNAs; Thomas Südhof (University of Texas) for the rat brain cDNA library; and Larry Rohrschneider (Fred Hutchinson Cancer Center, Seattle, WA) for providing the SHIP cDNA.
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FOOTNOTES |
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* This research was supported by the Deutsche Forschungsgemeinschaft (Ta-111/7/-1, 2), International Human Frontier Science Program, and Fonds der Chemischen Industrie (to H. N.).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.
Submitted as part of a Ph.D. thesis in University of Hannover.
¶ To whom correspondence should be addressed. Tel.: 0049-511-532-2857; Fax: 0049-511-532-2827; E-mail: Tamura.Teruko@MH- Hannover.de.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M009333200
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ABBREVIATIONS |
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The abbreviations used are:
HGF, hepatocyte growth factor;
SH2, Src homology 2;
Grb, growth factor
receptor-bound protein;
PI, phosphatidylinositol;
PLC, phospholipase
C-
;
MDCK, Madin-Darby canine kidney;
SHIP, SH2-containing inositol
5-phosphatase;
pY, phosphotyrosine;
GST, glutathione
S-transferase;
FCS, fetal calf serum;
SDS-PAGE, SDS-polyacrylamide gel electrophoresis;
MBD, Met binding domain..
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