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INTRODUCTION |
SHIP1 is a 145-kDa SH2-containing inositol phosphatase which
selectively hydrolyzes the 5'-phosphate from inositol
1,3,4,5-tetraphosphate (Ins(1,3,4,5)P4)1
and phosphatidylinositol 3,4,5-trisphosphate
(PtdIns(3,4,5)P3) (1, 2). SHIP2 is a more widely expressed
PtdIns(3,4,5)P3-specific 5'-phosphatase related to SHIP1
(3, 4). In addition, a smaller spliced form of SHIP1 has been
identified (5). SHIP1 and SHIP2 are transiently tyrosine phosphorylated
by growth factor stimulation and by activation of immunoregulatory
receptors (1, 6-9).
SHIP1 functions in part by modifying a signaling pathway that is
initiated by activation of phosphatidylinositol 3-kinase (PI3K) (10,
11), a lipid kinase with pleiotropic effects (12). SHIP1 would be
expected to metabolize the PI3K lipid product
PtdIns(3,4,5)P3 to phosphatidylinositol 3,4-bisphosphate
(PtdIns(3,4)P2). However, it is not entirely clear at this
time how such changes in phosphatidylinositol metabolism mediate
biological effects. Mice with a disruption of the SHIP1 gene
fail to thrive and develop a myeloproliferative disorder with extensive
infiltration of myeloid cells in the lung (13). Also, marrow progenitor
cells of these mice are hyper-responsive to hematopoietic growth
factors (13) and chemokines (14). In cell line models, SHIP1 negatively
regulates growth, differentiation, or migration, and it may have an
important role in apoptosis (2, 15-17). The enzyme activity of SHIP1
has not been shown to change after receptor activation, suggesting that
relocation of SHIP1 to the cell membrane may be critical for signaling
(18). Therefore, transient interaction of SHIP1 with signaling
complexes associated with transmembrane receptors or
membrane-associated proteins is likely to be important in regulating
its function.
In addition to its enzymatic activity as a regulator of bioactive
phospholipids, SHIP1 can also function as an adaptor protein. SHIP1 was
originally identified as a SHC-binding protein, an interaction later
shown to be mediated by the SH2 domain of SHIP1 (15), and by the
protein tyrosine-binding (PTB) domain of SHC (2, 19). GRB2 competes
with SHIP1 for SH2 binding to SHC (15, 20) or binds to a C-terminal
proline-rich region in SHIP1 through its SH3 domains (1). Another
prominent SHIP1-binding protein is the SH2 containing tyrosine
phosphatase SHP-2 (21, 22). Deleting the SH2 domain of SHIP1 impairs
apoptotic activity and prevents tyrosine phosphorylation (15). It is
therefore likely that SHIP1 may be involved in the regulation of
several distinct signaling pathways.
We have previously demonstrated that expression of SHIP1 is drastically
reduced in cells transformed by the BCR/ABL oncogene (17). BCR/ABL is
generated by the t(9,22) (q34;q11) Philadelphia chromosome (Ph)
translocation and is the transforming protein in chronic myelogenous
leukemia (23). One feature of primary chronic myelogenous
leukemia cells is altered adhesion to fibronectin and hypermotility
(24, 25). We have shown that re-expression of SHIP1 in
BCR/ABL-transformed cells reduces spontaneous Transwell migration (17).
The exact mechanism whereby SHIP1 regulates migration in normal and
transformed cells is unknown.
In this study, we have used a BCR/ABL-transformed Ba/F3 cell line with
inducible SHIP1 expression as a model system to investigate the
signaling activities of SHIP1. We demonstrate that Tyr917
and Tyr1020 in SHIP1 are important for the effects of SHIP1
on migration, and serve as binding sites for DOK1, a signaling protein
previously linked to the regulation of migration (26). In addition, we show that the SHIP1·DOK1 complex contains P13K and the unique adapter protein CRKL, also previously linked to migration in
hematopoietic cells (27). We propose that SHIP1 regulates migration
through the DOK1·CRKL·PI3K complex, and that the loss of SHIP1
expression in BCR/ABL-transformed cells results in further activation
of migration. Further studies will define the exact role of downstream targets of DOK1 and CRKL as well as their role in regulating migration.
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MATERIALS AND METHODS |
Cell Culture--
The murine hematopoietic line Ba/F3 was grown
in RPMI 1640 with 10% (v/v) fetal calf serum and 10% (v/v) WEHI-3B
conditioned medium (as a source of murine IL-3). Ba/F3 cell lines
transfected with a BCR/ABL cDNA (Ba/F3.p210), a TEL/ABL cDNA
(Ba/F3.TEL/ABL), and a v-Abl cDNA (Ba/F3.vAbl) were grown in RPMI
1640 with 10% (v/v) fetal calf serum. Ba/F3 cells stably transfected
with a plasmid containing the reverse tetracycline-controlled
transactivator (Ton.BaF.1, obtained from G. Q. Daley, MIT,
Cambridge, MA) were transfected with a BCR/ABL cDNA
(Ba/F3.p210.TetON) and cultured under the same conditions as the
Ba/F3.p210 cell line. In some experiments Ba/F3 cells were deprived of
growth factors for 18 h in RPMI 1640 medium containing 0.5% (w/v)
bovine serum albumin.
Preparation of Cellular Lysates and
Immunoprecipitation--
Cells were washed once in phosphate-buffered
saline and lysed in buffer containing 50 mM Tris (pH 8.0),
150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v)
deoxycholic acid, 0.1% (w/v) SDS, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 1 mM sodium orthovanadate, and 40 µg/ml leupeptin at
108 cells/ml buffer. Cells were incubated on ice for 20 min
and suspended vigorously every 5 min. Insoluble particles were
precipitated by centrifugation for 15 min at 12,000 × g. Lysates were subjected to immunoprecipitation or directly
analyzed through immunoblotting. Proteins were immunoprecipitated from
cellular lysates by incubation with the primary antibody and Protein A-
or Protein G-Sepharose beads in lysis buffer for 2 or 18 h at
4 °C and finally washed three times with buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% (v/v)
Nonidet P-40, 100 mM NaF, and 10% (v/v) glycerol.
Immunoblotting (Western Blotting) and Antibodies--
Proteins
were separated under reducing conditions by SDS-7.5% PAGE and
electrophoretically transferred to ImmobilonTM
polyvinylidene difluoride (Millipore, Bedford, MA) in buffer containing
25 mM Tris, 192 mM glycine, and 20% (v/v)
methanol at 4 °C. The membrane was blocked for 1 h at 25 °C
or 18 h at 4 °C in 5% (w/v) fat-free dry milk powder in TBS
(10 mM Tris-HCl (pH 8.0), 150 mM NaCl). The
membrane was washed three times for 5 min in TBST (0.1% Tween 20 in
TBS), incubated with the primary antibody for 2 h at 25 °C or
18 h at 4 °C, washed three times for 5 min in TBST, and
incubated in the secondary HRP-coupled antibody (anti-mouse and
anti-rabbit, Amersham Pharmacia Biotech, 1:5000 dilution in TBST;
anti-goat, Santa Cruz Laboratories, 1:10000 dilution in 5% (w/v)
fat-free dry-milk powder in TBST) for 1 h. HRP activity was
detected using HRP substrates (Enhanced Luminol Reagent-kit for
immunoblotting, PerkinElmer Life Sciences, Boston, MA) and X-Omat Blue
XB-1-film (Kodak, Rochester, NY). Tyrosine-phosphorylated proteins were
detected using the monoclonal antibody 4G10 (kindly provided by Dr. B. Druker, Oregon Health Science University, Portland, OR). Mouse
monoclonal antibodies against SHIP1 (clone P1C1), DOK1 (Santa Cruz
Biotechnology, Santa Cruz, CA), ABL (clone 3F12), and CRKL (clone 3-5, for immunoblotting only) were used for immunoblotting or
immunoprecipitation. Polyclonal rabbit antisera against
p85PI3K (UBI, Lake Placid, NY), CRKL (Santa Cruz
Biotechnology, for immunoprecipitations only), and CBL (Santa Cruz
Biotechnology) were used for immunoblotting or immunoprecipitation.
Protein Overlay Assay (Far Western
Blotting)--
Immunoprecipitated proteins were transferred after
SDS-PAGE to nitrocellulose membranes. The membrane was blocked for
18 h at 4 °C with 5% nonfat dry milk in PB-T (0.1% Tween 20 in 25 mM sodium phosphate solution (pH 7.2). The membrane
was then washed in PB-T two times for 10 min at 25 °C, followed by
incubation in GST fusion protein solution (60 µg of GST fusion
protein in binding buffer (5% (v/v) 0.5 M sodium phosphate
solution, 150 mM NaCl, 0.1% (v/v) Tween 20, 2.5 mM EDTA (pH 5.0), 20 mM NaF, 1 mM
Na3VO4, 1% (w/v) fat-free dry milk powder, 1 mM dithiothreitol, 20 µg/ml aprotinin, and 40 µg/ml
leupeptin)) for 2 h at 25 °C. After washing in PB-T six times
for 10 min at 25 °C, the membrane was incubated in anti-GST
monoclonal antibody (Santa Cruz Biotechnology; 1:500 in 40 ml binding
buffer) for 2 h at 25 °C. Membrane was washed again in PB-T six
times for 10 min at 25 °C, followed by a 1-h incubation in
HRP-coupled anti-mouse IgG antibody (1:5000 in binding buffer) at
25 °C. The membrane was then washed in PB-T 6 times for 10 min at
25 °C. HRP activity was detected using HRP substrates (Enhanced
Luminol Reagent-kit for Western blotting, PerkinElmer Life Sciences)
and X-Omat Blue XB-1-film (Kodak). Bacterial expression vectors for GST
fusion proteins of the murine SHIP SH2 domain, the murine DOK PTB
domain, the CRKL SH2, and the CRKL SH3 domain were used. The GST fusion
proteins were expressed in Escherichia coli by
isopropyl-1-thio-
-D-galactopyranoside induction and
isolated from sonicated bacterial lysates using glutathione-Sepharose
beads (Amersham Pharmacia Biotech, Piscataway, NJ) according to the
manufacturer's directions.
Expression Constructs and Transient
Expression--
Site-directed mutagenesis was performed on the
pBluescript-SHIP1 plasmid (2) using the QuikChange Site-directed
Mutagenesis Kit (Stratagene, La Jolla, CA) according to the
manufacturer's directions. Complementary overlapping oligonucleotides
were synthesized for altering Tyr917 to Phe and
Tyr1020 to Phe. Relevant regions were sequenced to confirm
successful mutagenesis. The murine SHIP1 wild type and mutant cDNA
was subcloned into the EcoRI site of the pTRE expression
vector (CLONTECH Laboratories, Palo Alto, CA). The
pTRE-SHIP1 expression construct was used for transfection into
Ba/F3.p210.TetON cells. For Transwell migration experiments
pTRE-SHIP1 constructs were co-transfected with an enhanced green
fluorescence protein (EGFP) expression construct (EGFP-C1,
CLONTECH Laboratories). EGFP positive cells were
sorted 1 day after transfection using a Coulter Epics Altra or Elite flow cytometer (Coulter Corp. Miami, FL). SHIP1 expression was induced
1 day after transfection by treatment with 1 µg/ml doxycycline.
Transwell Migration Assay--
The lower chamber of a Transwell
plate (8-µm pore size polycarbonate membrane, Corning Costar Corp.,
Cambridge, MA) was filled with 600 µl of starvation media (0.5%
(w/v) bovine serum albumin in RPMI 1640). Cells were counted using a
Coulter particle counter (Coulter Counter Z2, Beckman Coulter,
Fullerton, CA) and resuspended at 2 × 106
cells/ml in starvation media. 100 µl of this cell suspension was
transferred to the upper chamber. The medium contained either doxycycline (1 µg/ml) or no stimulus in the control samples. After 2.5 h, cells in the lower compartment were resuspended and counted using a Coulter particle counter. The spontaneous Transwell migration of cells was expressed as a "migration index" (number of migrating cells treated with doxycycline divided by the number of migrating cells
left untreated). The standard error of the mean was calculated from the
migration indices of independently performed experiments. The
statistical significance of the data was analyzed using the Student's
t test.
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RESULTS |
SHIP1 Forms Complexes with Multiple Proteins in BCR/ABL Transformed
Ba/F3 Cells Re-expressing SHIP1--
We and others have previously
shown that SHIP1 is a negative regulator of cell migration (14, 17).
However, none of the known signaling pathways associated with SHIP1
have been directly linked to this process. Furthermore, we have shown
that loss of SHIP1 expression in BCR/ABL-transformed cells enhances
migration (17). To investigate the molecular mechanisms that regulate migration in these cells, we looked for novel SHIP1-associated signaling complexes in BCR/ABL-transformed cells. To visualize SHIP1-containing complexes, it was helpful to overexpress SHIP1 in
BCR/ABL-transformed cells. A doxycycline inducible expression system
was used to increase SHIP1 expression by severalfold in Ba/F3.p210.pTRE-SHIP cells compared with the untreated cells or the
Ba/F3.p210.pTRE cells (Fig. 1A,
left panel). However, there was slightly increased expression of
SHIP1 in untreated Ba/F3.p210.pTRE-SHIP cells compared with untreated
Ba/F3.p210.pTRE cells, likely indicating that the promotor is leaky.
Re-expression of the SHIP1 protein in Ba/F3.p210 cells led to the
co-immunoprecipitation of additional tyrosine-phosphorylated proteins
with an apparent molecular mass of 210, 190, 140, 120, and 50-70 kDa.
Association of tyrosine-phosphorylated proteins with SHIP1 of
comparable molecular mass could also be observed in similar experiments
with the parental Ba/F3.p210 cells when the blot was exposed for a long
time (22).

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Fig. 1.
SHIP1 and DOK1 form a complex in Ba/F3.p210
cells re-expressing SHIP1. A-C, SHIP1 expression was induced
by doxycycline treatment of Ba/F3.p210.pTRE-SHIP cells. Lysates of
untreated cells ( ) or cells treated for 18 h with 1 µg/ml
doxycycline (+) were used for precipitations. A, lysates of
20 × 106 Ba/F3.p210.pTRE and Ba/F3.p210.pTRE-SHIP
cells were incubated with anti-SHIP1 and anti-DOK1 antibodies and
SHIP1, DOK1, or tyrosine-phosphorylated proteins were detected by
immunoblotting (I.B.). B, lysates of 20 × 106 Ba/F3.p210.pTRE-SHIP cells were incubated with 5 µg
of GST and GST fusion protein of the SHIP1 SH2 or the DOK1 PTB domain
immobilized on glutathione beads. Co-precipitation of ABL, SHIP1, or
DOK1 was detected by immunoblotting (I.B.). C,
lysates of 20 × 106 Ba/F3.p210.pTRE-SHIP cells were
immunoprecipitated with antibodies against SHIP1, DOK1, and BCR/ABL as
indicated. Proteins were separated by SDS-PAGE and transferred to
nitrocellulose membrane. Specific direct binding of GST and a GST
fusion protein of the DOK1 PTB domain or the SHIP1 SH2 domain to
proteins in the immunoprecipitates was detected in a protein overlay
assay. The molecular mass of the proteins is indicated in kDa on the
left of each figure. D, Ba/F3.p210.TetON cell
were transfected with the empty vector (pTRE), a vector containing
full-length SHIP1 (pTRE-SHIP), or SHIP1 containing a Y917F
(pTRE-SHIP.Y917F) or a Y1020F (pTRE-SHIP.Y1020F) point mutation. SHIP1
protein was induced 1 day after transfectionly by doxycycline treatment
for 24 h. SHIP1 expression was detected by immunoblotting
(I.B.) in whole cell lysate (2.5 × 105
cells) or in precipitations using a GST-DOK1 PTB domain fusion protein
and lysates of 25 × 106 cells. The molecular mass of
the proteins is as indicated in kDa on the left of each
figure.
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SHIP1 Binds to p62DOK1 in BCR/ABL-transformed
Cells--
The 62-kDa tyrosine-phosphorylated protein that
co-immunoprecipitated with SHIP1 was identified as DOK1 (Fig. 1A,
left bottom panel). These results were confirmed by
immunoprecipitation with anti-DOK1 followed by immunoblotting with
anti-SHIP1 using lysates of Ba/F3.p210 cells re-expressing SHIP1 (Fig.
1A, right panel). We also found PI3K co-precipitating with
SHIP1 in doxycycline-treated Ba/F3.p210.pTRE-SHIP1 (Fig. 1A, left
bottom panel). In contrast, there was only a very small amount of
PI3K found to be constitutively associated with DOK1 when the blot was
exposed for a long time (data not shown), indicating that inducible
interaction of PI3K with SHIP1 was not mediated through interaction
with DOK1. Immunoprecipitations of SHIP1 and DOK1 showed overlapping
phosphotyrosine patterns, suggesting that both proteins were in the
same signaling complex in these cells.
The above results suggest the potential formation of a multimeric
signaling complex including SHIP1 and DOK1. Since SHIP1 has one SH2
domain and DOK1 has a PTB domain, we sought to determine the mechanism
of binding of SHIP1 to DOK1 using GST fusion proteins containing the
phosphotyrosine interaction domains of each protein. Similar to the
previous experiments, we used lysates of untreated and
doxycycline-treated Ba/F3.p210.pTRE-SHIP and Ba/F3.p210.pTRE cells to
test the interaction of SHIP1 with DOK1 (Fig. 1B). The SH2
domain of SHIP1 precipitated both SHIP1 and DOK1 from lysates of
doxycycline-treated Ba/F3.p210.pTRE-SHIP and a small amount of DOK1
from untreated cells. The BCR/ABL oncoprotein itself was found to
precipitate with the SHIP1 SH2 domain in untreated as well as in
doxycycline-treated Ba/F3.p210.pTRE and Ba/F3.p210.pTRE-SHIP cells. In
contrast, the PTB domain of DOK1 precipitated SHIP1 and a small amount
of BCR/ABL only from lysates of doxycycline-treated Ba/F3.p210.pTRE-SHIP cells. Neither BCR/ABL, SHIP1, nor DOK1 were found
to bind to GST alone.
The in vitro GST fusion protein precipitations with SHIP1
and DOK1 did not indicate whether binding of the SH2 or PTB domains was
direct or indirect. A protein overlay assay was used to identify direct
in vitro interactions. Cellular lysates from untreated and
doxycycline-treated Ba/F3.p210.pTRE-SHIP cells were used for immunoprecipitations with anti-SHIP1 and anti-DOK1 antibodies. GST
protein alone did not bind to SHIP1 or DOK1 proteins in
immunoprecipitations (Fig. 1C, top left panel). Direct
binding of a single 145-kDa protein band in SHIP1 immunoprecipitates
using the GST-DOK1-PTB protein as a probe was found in Ba/F3.p210 cells
re-expressing SHIP1 (Fig. 1C, top right panel). A weak
interaction between the SHIP1 SH2 domain and DOK1 was detected in SHIP1
overexpressing cells, but the SHIP1-SH2 domain did not bind to SHIP1
itself (Fig. 1C, bottom right panel). We also observed
direct in vitro binding of the SHIP1 SH2 domain to BCR/ABL
that was increased in SHIP1 over-expressing cells. These data suggest
that overexpression of SHIP1 protects the dephosphorylation of DOK1 and
BCR/ABL on a site that is important for binding the SHIP1-SH2 domain.
Thus, DOK1 is linked through its PTB domain to SHIP1, whereas the SH2 domain of SHIP1 is only involved in a weak interaction with DOK1.
Since SHIP1 has two binding sites for the PTB domain of SHC, we also
tested if these sites regulate binding to the DOK1-PTB domain.
Full-length SHIP1 and the SHC-binding mutants of SHIP1 containing the
Y917F and Y1020F substitutions in the SHC-binding site (28) were
expressed in Ba/F3.p210.TetON cells. The cells were treated with
doxycycline to induce SHIP1 expression. Cells transfected with the
SHIP1 containing vectors expressed high levels of SHIP1 compared with
cells transfected with the empty vector (Fig. 1D, left
panel). Using the DOK1-PTB domain, a significant amount of SHIP1
was found to precipitate from cells re-expressing SHIP1, but not from
cells transfected with the empty vector (Fig. 1D, right
panel). The amount of SHIP1 tyrosine mutants precipitating with
the DOK1-PTB domain was reduced significantly when compared with wild
type SHIP1. These data are consistent with previous findings
demonstrating that optimal binding of the SHC PTB domain to SHIP1 is
reduced but not abolished by mutating either tyrosine (28).
SHIP1 and CRKL Are Associated in BCR/ABL-transformed Ba/F3 Cells
Re-expressing SHIP1--
Since DOK1 has previously been shown to
co-precipitate with CRKL in BCR/ABL-transformed cells (29), we also
asked if CRKL was found in the complex with SHIP1. Ba/F3.p210.pTRE and
Ba/F3.p210.pTRE-SHIP cells were either left untreated or treated with
doxycycline and CRKL protein was immunoprecipitated from whole cell
lysate. SHIP1 was found to co-precipitate with CRKL only when
re-expressed in Ba/F3.p210.pTRE-SHIP (Fig.
2A). We also found PI3K in
this complex which we had previously demonstrated to bind
constitutively to the CRKL SH3 domain (30). SHIP1 was not found in a
complex with CRKL in Ba/F3.p210.pTRE or unstimulated Ba/F3 cells (not
shown). Next, the molecular interactions of CRKL with SHIP1 using GST fusion proteins of the SHIP-SH2, CRKL-SH2, or CRKL-SH3 domains were
determined. We did not observe significant co-precipitation of CRKL in
lysates of Ba/F3.p210 cells (not shown). In contrast, SHIP1
co-precipitated with the CRKL-SH2 domain when re-expressed in
Ba/F3.p210.pTRE-SHIP cells (Fig. 2B). As a control, we also showed binding of the CRKL-SH3 domain to the p85 regulatory subunit of
PI3K (30) and binding of the CRKL-SH2 domain to p120CBL
(31). Re-expression of SHIP1 did not significantly alter either interaction. Since SHIP1 contains Tyr-X-X-Pro
motifs, potential binding sites for the CRKL SH2 domain, we also
determined if there was direct in vitro interaction between
SHIP1 and CRKL. GST or a GST-CRKL fusion proteins were used for protein
overlay experiments with CRKL and CBL immunoprecipitations of untreated
and doxycycline-treated Ba/F3.p210.pTRE-SHIP cells. The CRKL-SH2 domain
was found to bind in SHIP1 immunoprecipitations to a 145-kDa protein in
cells re-expressing SHIP1 (Fig. 2C). In addition, the CRKL
SH2 domain was found to bind to proteins with an apparent molecular
mass of 55 and 70 kDa in SHIP1 immunoprecipitations. As a positive
control, the CRKL SH2 domain bound to CBL in CBL immunoprecipitations.
These data suggest that there was direct in vitro binding of
CRKL through its SH2 domain to SHIP1.

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Fig. 2.
SHIP1 and CRKL form a complex in Ba/F3.p210
cells re-expressing SHIP1. A-C, SHIP1 expression was
induced by doxycycline treatment of Ba/F3.p210.pTRE-SHIP cells. Lysates
of untreated cells ( ) or cells treated for 18 h with 1 µg/ml
doxycycline (+) were used for precipitations. The molecular mass of the
proteins is indicated in kDa on the left of each figure.
A, lysates of 20 × 106
Ba/F3.p210.pTRE-SHIP and Ba/F3.p210.pTRE-SHIP cells were incubated with
anti-CRKL antibodies and SHIP, CRKL, or p85PI3K (PI3K)
protein detected by immunoblotting (I.B.). B,
lysates of 20 × 106 Ba/F3.p210.pTRE-SHIP cells were
incubated with 5 µg of GST and GST fusion protein of the CRKL SH2 or
the CRKL SH3 domain immobilized on glutathione beads. Co-precipitation
of SHIP, CBL, or p85PI3K (PI3K) was detected by
immunoblotting (I.B.). C, lysates of 20 × 106 Ba/F3.p210.pTRE-SHIP cells were immunoprecipitated with
antibodies against SHIP1 and CBL as indicated. Proteins were separated
by SDS-PAGE, and transferred to polyvinylidene difluoride membrane.
Specific direct binding of GST and a GST fusion protein of the CRKL SH2
domain to proteins in the immunoprecipitates was detected in a protein
overlay assay.
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SHIP1 Binds to DOK1 and CRKL in Ba/F3 Cells Transformed by TEL/ABL
and v-Abl--
We next investigated the potential interaction of SHIP1
with DOK1 and CRKL in cells that are transformed by activated forms of
Abl, different from BCR/ABL, including TEL/ABL and v-Abl. Cells transformed by activated forms of ABL have increased levels of tyrosine-phosphorylated proteins compared with untransformed cells. Both Ba/F3.TEL/ABL- and Ba/F3.v-Abl-transformed cells had higher levels
of SHIP1 compared with Ba/F3.p210 cells (Fig.
3A). Transformation of Ba/F3
cells by TEL/ABL and v-Abl induces growth factor independence and
requires ABL kinase activity. We have shown before that treatment of
Ba/F3 cells transformed by activated ABL kinases with the ABL kinase
inhibitor STI571 results in re-expression of SHIP and returns these
cells to growth factor dependence. Consistent with these findings, we
found that STI571 treatment reduced the Transwell migration of TEL/ABL
cells by 51.1% (n = 3) and v-ABL cells by 47.9%
(n = 3).

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Fig. 3.
SHIP1 associates with CRKL and DOK1 in cells
stimulated transformed by v-ABL and TEL/ABL. A and
B, lysates of Ba/F3, Ba/F3.p210, Ba/F3.TEL-ABL and
Ba/F3.v-Abl were used to detect tyrosine-phosphorylated proteins
(p-Tyr) by immunoblotting (I.B.). The blots were
stripped and reprobed with antibodies against SHIP1,
p85PI3K (PI3K), DOK1, or CRKL. The molecular mass of the
proteins is indicated in kDa on the left of each figure.
A, total cell lysates of 2.5 × 105 cells
were used for immunoblotting. B, SHIP1 was
immunoprecipitated from cell lysates of 20 × 106
cells. TEL/ABL and v-Abl are indicated by arrows.
C, DOK1 and CRKL were immunoprecipitated (IP)
from cell lysates of 20 × 106 cells as
indicated.
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Tyrosine phosphorylation of the 145-kDa protein SHIP1 was increased in
cells transformed by TEL/ABL and v-Abl compared with Ba/F3 cells (Fig.
3B). In some experiments, we also detected low level
tyrosine phosphorylation of SHIP1 in growth factor-deprived Ba/F3
cells. In addition, SHIP1 co-precipitated with major
tyrosine-phosphorylated proteins with an apparent molecular mass of
170, 120, and 50-70 kDa in TEL/ABL-transformed cells and 150, 120, and
50-70 kDa in v-Abl-transformed cells. ABL immunoblotting also
demonstrated that c-ABL and the oncoproteins TEL/ABL and v-Abl were
found in this complex.
Next, we also tested if DOK1 and CRKL were in a complex with SHIP1 in
TEL/ABL and v-Abl-transformed cells. Consistent with the previous
results, we found co-precipation of the 145-kDa phosphotyrosine protein
SHIP1 with DOK1 (Fig. 3C, left panel). The amount of SHIP1 associated with both proteins was increased in the transformed cell
lines compared with the untransformed Ba/F3 cells and it correlated to
the amount of SHIP1 expressed in either Ba/F3.TEL/ABL or Ba/F3.v-Abl
cells. However, we found only a very small amount of SHIP1 associated
with CRKL in v-Abl but not TEL-ABL-transformed cells (Fig. 3C,
right panel).
Tyrosines 917 and 1020 in SHIP1 Regulate Spontaneous Transwell
Migration--
BCR/ABL-transformed Ba/F3 cells demonstrate a
significant level of spontaneous migration which can be reduced by
re-expression of SHIP1. Using the above described doxycycline inducible
expression system, we co-transfected SHIP1 and EGFP expression vectors
and sorted for EGFP positive cells 24 h after transfection. SHIP1 and SHIP1 mutant levels were increased severalfold after doxycycline treatment in transiently transfected cells (Fig.
4A). The effect on Transwell
migration of EGFP sorted wild type SHIP, SHIP-Y917F, and SHIP-Y1020F
mutant transfected cells was investigated after doxycycline treatment
and compared with untreated cells. We had previously shown that
doxycycline treatment alone does not alter Transwell migration (17).
The migration index of 0.74 (n = 4, p < 0.01) in cells transiently expressing wild type SHIP1 demonstrates a
significant decrease in spontaneous migration (Fig. 4B).
However, the decrease was smaller than the previously described
decrease in stably transfected cell lines after SHIP1 expression (17). In contrast, SHIP-Y917F and SHIP-Y1020F expression in Ba/F3.p210.TetON cells induced a small but not significant decrease in Transwell migration. The mean of the migration indices was found to be 0.88 for
SHIP-Y917F (n = 4, p = 0.1) and 0.94 for SHIP-Y1020F (n = 3, p = 0.2)
expressing cells. These data suggest that Tyr917 and
Tyr1020 in SHIP1 are likely to be involved in regulating
SHIP1-dependent migration.

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Fig. 4.
Tyr917 and Tyr1020 in
SHIP1 regulate spontaneous Transwell migration. A
and B, Ba/F3.p210.TetON cells transfected with a wild type
SHIP1 expression vector (pTRE-SHIP) and vectors containing the SHIP1
mutants Y917F (pTRE-SHIP.Y917F) or Y1020F (pTRE-SHIP.Y1020F) were
either left untreated ( ) or treated with doxycycline (+) and used for
immunoblotting (I.B.) or Transwell migration. A,
expression of wild type SHIP1 and the two SHIP1 mutants was detected in
total cell lysates (2.5 × 105 cells) by
immunoblotting (I.B.) with anti-SHIP1 antibodies. The blot
was stripped and reprobed with anti-PI3K antibodies. The molecular mass
of the proteins is indicated in kDa on the left of the
figure. B, cells were used for Transwell migration assays
and the number of viable cells in the lower chamber was determined
after 2.5 h. The change in migration of four independent
experiments was calculated as a migration index. The error
bars indicate the standard error of the mean. The p
values were calculated and a significant decrease (p < 0.01) indicated by an asterisk (*).
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DISCUSSION |
Expression of the phosphatidylinositol-5-phosphatase SHIP1 is
rapidly and reversibly down-regulated by BCR/ABL and this requires ABL
kinase activity (17). Down-regulation of SHIP1 by BCR/ABL is of
particular significance because disruption of the SHIP1 gene
by gene targeting results in a myeloproliferative disorder in mice
(13). We have shown that re-expression of SHIP1 in BCR/ABL-transformed Ba/F3 cells reduces hypermotility (17), a characteristic of BCR/ABL
transformation (25). However, the mechanism whereby SHIP1 expression
regulates migration is unknown.
In this report, we demonstrate the presence of protein complexes that
contain SHIP1, DOK1, and CRKL in a murine BCR/ABL-transformed hematopoietic cell line. CRKL is constitutively associated with PI3K
which is also recruited to this complex. Expression of SHIP1 is reduced
in cell lines transformed by BCR/ABL, and the remaining SHIP1 is
heavily tyrosine phosphorylated (22). In untransformed cells SHIP1,
CRKL, and DOK1 are not spontaneously tyrosine phosphorylated and do not
co-immunoprecipitate, demonstrating that this complex is altered by
BCR/ABL. DOK1 and CRKL are also constitutively tyrosine phosphorylated
in BCR/ABL-transformed cells and the interaction with SHIP1 is
increased considerably following re-expression of SHIP1.
DOK1 (for downstream of kinase) was originally identified as a
tyrosine-phosphorylated protein in cells transformed by oncogenic tyrosine kinases. The DOK1 cDNA was cloned from cells transformed by activated forms of ABL and identified as the 62-kDa Ras
GTPase-activating protein (RasGAP)-associated protein (32, 33). DOK1
belongs to a family of related proteins that also includes DOK2 (34) and DOK3 (35). DOK1 has been found to be tyrosine phosphorylated in
response to steel factor (32, 36), epidermal growth factor (37), or
insulin (26, 38) and other stimuli. DOK1 contains a pleckstrin homology
domain that facilitates interaction with phosphoinositides and a
PTB domain that is likely to interact with
Asn-Pro-Xxx-phospho-Tyr motifs (39, 40). The major phosphotyrosine site
in DOK1 enables recruitment of SH2-containing proteins such as NCK
which has been found to bind to DOK1 in response to insulin stimulation
(26). We have demonstrated direct in vitro binding of DOK1
to SHIP1 through the DOK1 PTB domain. Interestingly, DOK3 was also
recently found in a signaling complex with SHIP1 (35). During
preparation of this article others also reported that DOK1 is found in
a signaling complex with SHIP1 (41, 42). The exact function of DOK1 is
unknown but it has been demonstrated that tyrosine phosphorylation of
DOK1 regulates its binding to RasGAP (32, 33, 43). Tyrosine
phosphorylation of DOK1 is also likely to be required for its
inhibitory effect on RasGAP activity (43). Overexpression of DOK1
enhances insulin-induced cell migration and requires that the
pleckstrin homology domain and the major phosphotyrosine site
(Tyr361, murine sequence) of DOK1 be intact (26). This is
of interest, since we show interaction of SHIP1 and DOK1 in
BCR/ABL-transformed cells, suggesting that both molecules act in the
same signaling pathway. We have demonstrated that SHIP1 regulates
migration (17), and here demonstrate that Tyr917 and
Tyr1020, major regulatory sites for this effect, also
regulate binding to DOK1. Overexpression of SHIP1 in the Ph+ cell line
K562 has been shown to decrease synthesis of hemoglobin protein and
-globin mRNA in response to hemin, an inducer of erythroid
differentiation (44). This process was also inhibited by mutating
Tyr1020 to phenylalanine. Interestingly, this
phosphotyrosine in SHIP1 is also a binding site for the adapter protein
SHC and it is therefore likely that SHC and DOK1 compete for binding to
SHIP1 (28). Whereas DOK1 is thought to be a negative regulator of RAS
activation (43), SHC is believed to be involved in activation of Ras by interacting with the GRB2·SOS complex (45). It is uncertain if
activation of Ras is sufficient to regulate Transwell migration in
these cells. Nevertheless, it has been suggested that RAS can affect
migration and motility in different in vitro models
(46-49).
The other adapter protein found in a complex with SHIP1 was CRKL, an
SH2/SH3 domain containing phosphotyrosine protein. CRKL was originally
described as a major tyrosine-phosphorylated protein in stable phase
chronic myelogenous leukemia neutrophils. Cloning of the CRKL cDNA
revealed that it belonged to the CRK family of adapter proteins that
also includes v-CRK, CRK-I, and CRK-II (50-52). CRKL and CRK have been
described to be involved in oncogenic and normal signaling and both can
interact constitutively or transiently with various signaling proteins
(53). For example, CRKL can bind to tyrosine-phosphorylated HEF1 after
integrin ligation through its SH2 domain (54) or CRKL forms a
constitutive complex with c-ABL through its SH3 domain (31). Here we
demonstrate that the CRKL SH2 domain can bind directly to SHIP1, likely
through a phospho-Tyr-Xxx-Xxx-Pro site within SHIP1 (53). It is
possible that one function of CRKL in this complex is to recruit other signaling proteins through its SH3 domain into the proximity of SHIP1.
Tyrosine phosphorylation and interaction of SHIP1 with signaling
proteins is expected to alter its accumulation at the cell membrane and
relocationg SHIP1 to the membrane has been implicated in the regulation
of its enzyme activity (18). We have previously shown that CRKL is
linked through its SH3 domain to PI3K itself (30) and here we show that
PI3K is also found in a complex with SHIP1. SHIP1 and CRKL were also
shown to be in a signaling complex after Fc-
receptor (CD89)
ligation in U937 cells (55). In normal cells, CRKL may be involved in
the regulation of bioactive phospholipid levels by interacting with
SHIP1 and PI3K. Overexpression of CRKL increases fibronectin-induced
Transwell migration of Ba/F3 cells and this also required the CRKL SH2
domain (27).
The role of the SHIP·CRKL·PI3K complex in BCR/ABL transformation is
of interest, since there is striking evidence that PI3K is important
for transformation by BCR/ABL (56). It will therefore also be important
to evaluate the role of CRKL in the regulation of PI3K or SHIP1. An
important question in signaling through PI3K is how specificity is
obtained, since PI3K is involved in the generation of several different
bioactive 3-phosphorylated phospholipids that regulated different
functions and interact with different proteins (12). It is possible
that CRKL brings PI3K to the proximity of SHIP1 at the cell membrane to
generate certain levels of PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 and therefore generate a transient and
defined signaling focal point of bioactive phospholipids. Such a
colocalization of PI3K with a specific phosphatase to a subcellular
compartment could well mediate specificity in PI3K signaling and lead
to the activation of defined signaling pathways. Nevertheless, it is also possible that CRKL recruits other signaling molecules to SHIP1 and
regulates a function in addition to migration.
We also found the formation of a SHIP1·DOK1·CRKL complex in cells
transformed by the constitutively activated ABL tyrosine kinase
oncogene v-ABL, indicating that this complex is not unique to BCR/ABL
transformation. In TEL/ABL-transformed cells, the levels of SHIP1
protein were significantly reduced and we could not detect co-immunoprecipitation of CRKL with the SHIP1·DOK1 complex. The TEL/ABL oncogene is the result of a rare translocation that results in
the fusion of the ETS family transcription factor gene TEL with c-ABL
and is associated with acute lymphocytic leukemia and acute
myelogeneous leukemia (57, 58). v-Abl is the transforming protein in
the Abelson murine leukemia virus (59, 60). It is striking that both
TEL/ABL- and v-Abl-transformed cell lines have higher levels of SHIP1
compared with BCR/ABL-transformed cells. This would suggest that high
levels of SHIP1 and formation of the SHIP1·DOK1·CRKL signaling
complex alone is unlikely to inhibit cell growth in these cell line
models. It is more likely that this signaling complex is involved in
certain aspects of transformation such as migration.
We have demonstrated here and previously that overexpression of SHIP1
in untransformed and re-expression of SHIP1 in BCR/ABL-transformed Ba/F3 cells led to a decrease in spontaneous Transwell migration. Altered migration may lead to premature release of cells from the
marrow as well as accumulation in the blood and we are testing this
hypothesis. Since SHIP1, DOK1, and CRKL are believed to have important
signaling roles related to cell migration (17, 26, 27), and these
results overall suggest that the functions of these signaling proteins
are linked. Each of the proteins in the complex is tyrosine
phosphorylated, additional interactions with other signaling proteins
could be directed through additional SH2 or PTB domain interactions and
involve one or more adapter proteins. Identification of downstream
targets of either protein will help to further understand the function
of SHIP1 and contribution of this signaling complex to transformation.