Department of Cancer Molecular Sciences, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
* Author for correspondence (e-mail: stuart.decker{at}pfizer.com)
Accepted 25 July 2002
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Summary |
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Key words: 5' inositol phosphatase, SHIP2, Src kinase, Shc, Adhesion, Collagen I
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Introduction |
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Inositol phosphatases regulate the cellular levels of lipid second
messengers (Majerus et al.,
1999). A 3' inositol phosphatase PTEN/MMAC1 is frequently
inactivated in tumor cells leading to increased PI 3-kinase product,
phosphatidylinositol (3,4,5)-trisphosphate
[PtdIns(3,4,5)P3] resulting in activation of Akt/PKB. PTEN
also regulates integrin-mediated activation of extracellular signal regulated
kinase (ERK), interacts with FAK and inhibits adhesion, migration and invasion
processes (Di Cristofano and Pandolfi,
2000
). The 5' inositol phosphatases, SH2-containing inositol
5-phosphatase 1 and 2 (SHIP1 and SHIP2) specifically dephosphorylate
PtdIns(3,4,5)P3, and inositol (1,3,4,5)-tetrakiphosphate
[Ins(1,3,4,5)P4] on the D5 position of the inositol ring
(Erneux et al., 1998
). While
SHIP1 expression is restricted primarily to hematopoietic tissues, SHIP2
appears to be ubiquitous (Habib et al.,
1998
; Rohrschneider et al.,
2000
). Analogous to the negative regulation of growth factor and
antigen receptor-mediated signaling by SHIP1, some studies suggest that SHIP2
downregulates insulin and Fc
RIIB receptor signaling
(Muraille et al., 1999
;
Wada et al., 2001
). Targeted
deletion of SHIP2 in mice produced neonatal fatality attributed to
hypoglycemia and insulin hypersensitivity
(Clement et al., 2001
).
Overexpressed SHIP2 downregulated Akt activation and caused cell-cycle arrest
(Taylor et al., 2000
). In
addition, the same group reported that SHIP2 effectively utilizes
phosphatidylinositol (4,5)-biphosphate [PtdIns(4,5)P2] as
its substrate in addition to already reported
PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4
(Taylor et al., 2000
).
Besides an N-terminal SH2 domain, both SHIP1 and SHIP2 possess proline-rich
regions and NPXY motifs (two in SHIP1 and one in SHIP2) serving as potential
protein-protein interaction sites
(Rohrschneider et al., 2000).
SHIP2 also has a C-terminal SAM domain that is not present in SHIP1. Recently
we reported that an important regulator of adhesion and migration processes,
p130Cas, interacted with the SH2 domain of SHIP2
(Prasad et al., 2001
). In HeLa
cells, SHIP2 localized to focal contacts during attachment and to lamellipodia
in spreading cells. Wild-type SHIP2 promoted adhesion while catalytically
inactive SHIP2 inhibited spreading of HeLa cells. In this report, we further
characterize the involvement of SHIP2 in adhesion providing evidence that
SHIP2 tyrosine phosphorylation specifically occurs during cell attachment and
spreading on collagen I, and that phosphorylation is mediated through
activation of Src family kinases.
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Materials and Methods |
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Cell culture
HeLa, SH-SY5Y, Madin Darby canine kidney (MDCK) and 293T cells were
routinely cultured in DMEM (with high glucose, pyridoxine hydrochloride,
l-glutamine and without sodium pyruvate) containing 10% FBS. Culturing and
induction of differentiation of 3T3-L1 adipocytes was done as described
previously (Habib et al.,
1998). Transient transfections of HeLa cells were carried out
using Lipofectamine-Plus reagent (Invitrogen) according to the manufacturer's
instructions. Briefly, cells were cultured in 60 mm dishes 18-20 hours before
transfection to obtain 30-40% confluency at the time of transfection. 2 µg
DNA, 5 µl Plus reagent and 8 µl lipofectamine were used per 60 mm dish.
Transfections were carried out for 5 hours at 37°C. Experiments were
carried out 48 hours post-transfection. For cells cultured in 100 mm dishes,
7.5 µg DNA, 15 µl `Plus' reagent and 25 µl lipofectamine were used.
293T cells were transfected by a modified CaPO4 method
(Stratagene).
Construction of expression vectors encoding epitope-tagged SHIP2
cDNAs encoding full length SHIP2 with a FLAG epitope at the C-terminus
(SHIP2-FLAG) were cloned into the pcDNA3 mammalian expression vector.
Site-directed mutagenesis was used to replace the tyrosines with
phenylalanines. These constructs were tagged with the FLAG epitope at the
C-terminus.
Procedure for coating polystyrene (bacterial) petri dishes
Rat-tail collagen I was resuspended in 0.1 M acetic acid to 1 mg/ml
concentration with stirring for 1-3 hours at room temperature (RT). Collagen I
solution was stored at 4°C and pre-warmed to 37°C prior to coating.
Non-tissue culture treated (bacterial) polystyrene plates were coated in
phosphate buffered saline (PBS, 4.0 ml per dish) containing collagen I at
indicated concentrations with lids open in the laminar-flow hood for 1 hour.
Excess collagen solution was removed, washed twice with PBS and blocked in PBS
containing 1% bovine serum albumin (BSA) in the hood with lids open for 30
minutes. Plates were washed twice more with PBS prior to usage. Coating of
dishes with fibronectin (5 µg/cm2), collagen IV (6
µg/cm2), laminin (2 µg/cm2) and poly-L-lysine
(0.01% solution, 0.5 ml/25 cm2) was carried out similarly.
Immunoprecipitation and western blot analyses
HeLa cells cultured under various conditions were processed as follows. For
adherent (Ad) samples, confluent cells in 100 mm tissue dishes were serum
starved for 3 hours in DMEM containing 0.5% BSA, washed once with cold PBS and
scraped in HNTG lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1% Triton
X-100, 10% glycerol, 1 mM EGTA, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM
sodium fluoride, 0.2 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor
cocktail from Boehringer Mannheim). Detached cell lysates (D) were prepared as
follows. Cell monolayers were treated with 1x trypsin-EDTA (Invitrogen)
for 3 minutes. Trypsin was inactivated by soybean trypsin inhibitor (1 mg/ml
in DMEM containing 0.25% BSA). Cells were then centrifuged for 3 minutes at 50
g in a tabletop centrifuge and washed once with PBS prior to
lysis in HNTG buffer. For samples from re-attaching cells, serum starved cells
detached as above were re-plated in DMEM/0.5% BSA for indicated intervals on
bacterial petri dishes coated with either collagen I or other attachment
factors as indicated. At the indicated intervals, cells were gently washed
once with cold PBS and adherent cells were scraped in HNTG lysis buffer.
Medium and the PBS wash containing non-adherent cells (if any) were
centrifuged and the resulting cell pellet was combined with the lysate from
adherent cells. Immunoprecipitations from equal amounts of proteins and
western blots were carried out as described previously
(Prasad et al., 2000). For
SHIP2-Shc co-immunoprecipitation experiments (shown in
Fig. 8), NP-40 lysis buffer (20
mM Tris-HCl, pH 7.5, 1% NP-40, 10% glycerol, 25 mM NaCl, 1 mM PMSF, 1 mM EGTA,
1 mM EDTA, 0.2 mM sodium orthovanadate and protease inhibitor cocktail from
Boehringer Mannheim) was used. After immunoprecipitations with the indicated
antibodies and protein A/G agarose, immune complexes were washed three times
with NP-40 wash buffer (the same as NP-40 lysis buffer but containing 75 mM
NaCl). Samples were then boiled with SDS-sample buffer prior to
electrophoresis.
|
In vitro phosphorylation assays
FLAG-tagged wild-type SHIP2 or YY-FF (986-987) mutant proteins were
expressed in 293T cells by transient transfection. 48 hours post-transfection,
cells were lysed in HNTG buffer and FLAG-tagged proteins were purified using
anti-FLAG (M2) antibody and protein A/G plus agarose. Purified wild-type and
YY-FF mutant SHIP2 bound to protein A/G beads, were washed once with kinase
assay buffer (10 mM Hepes pH 7.5, 10 mM MgCl2, 10 mM
MnCl2, 1 mM DTT, 1 mM EGTA and 0.1 mM sodium orthovanadate) and
resuspended in assay buffer. Beads were then incubated with 5 units of
recombinant Src kinase (Upstate Biotech) and 200 µM ATP for 10 minutes at
30°C. Reactions were stopped by adding ice-cold assay buffer. Beads were
washed twice with assay buffer and resuspended in SDS-sample buffer. SDS-PAGE
and western analyses of the samples were carried out as described above.
Immunofluorescence staining
HeLa cells, cultured in 12-well dishes, were transiently transfected with
expression constructs of FLAG-tagged wild-type SHIP2 or YY-FF (986-987) mutant
expression constructs. 48 hour post-transfection, cells were trypsinized and
re-plated for 1 hour on chamber slides coated with collagen I (6
µg/cm2 for 1 hour) or fibronectin (5 µg/cm2 for 1
hour) followed by anti-FLAG (M2) immunofluorescence staining as previously
described (Prasad et al.,
2001). Prior to the final washes, cells were counterstained with
phalloidin-TRITC (1 µg/ml) for 15 minutes. Finally, cells were washed five
times in PBS followed by confocal microscopy.
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Results |
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Src inhibitors block SHIP2 tyrosine phosphorylation
Activation of the cytoplasmic tyrosine kinases, Src and FAK, is an early
event during cell attachment to ECM proteins. Several focal
adhesion-associated proteins including FAK are substrates of Src and
inhibition of Src activation prevents cell adhesion, spreading and migration
in several cell types (Cary et al.,
1999; Jones et al.,
2000
; Parise et al.,
2000
; Schwartz,
2001
). Therefore, we tested a possible role for Src family kinases
in collagen-I-induced SHIP2 tyrosine phosphorylation. To this end, we made use
of specific inhibitors of Src family kinases developed in a separate study
(Kraker et al., 2000
). When
plated on a collagen-I-coated surface in the presence of three different
Src-specific inhibitor compounds, PD173956 (#56), PD173958 (#58) and PD180970
(#70) at 1 µM concentration, tyrosine phosphorylation of SHIP2 was
completely blocked (Fig. 2;
Fig. 3A). The inhibitory effect
of compound PD173956 was seen in a dose-dependent manner starting at 50 nM
(Fig. 3B). Similar results were
also obtained with PD180970 (data not shown). Collagen-I-specific SHIP2
tyrosine phosphorylation was also observed in SH-SY5Y, a neuroblastoma cell
line, and in Madin Darby canine kidney (MDCK) cells. In SY5Y cells, background
levels of SHIP2 tyrosine phosphorylation were lower when cells were cultured
on plastic but the collagen-I-induced effect was robust
(Fig. 4A). In MDCK cells, cells
adherent on plastic displayed strong tyrosine phosphorylation of SHIP2, which
was decreased upon detachment and restored during attachment on collagen I
(Fig. 4B). It was noted that
the extent of SHIP2 modification may vary in a cell-type-dependent manner. In
both SH-SY5Y and MDCK cell types, collagen-I-dependent tyrosine
phosphorylation was completely blocked in the presence of 1 µM Src
inhibitor PD173956 (Fig.
4A,B).
|
|
Src kinases do not mediate EGF- or insulin-mediated tyrosine
phosphorylation of SHIP2
In HeLa cells, the Src inhibitors did not alter EGF-induced SHIP2 tyrosine
phosphorylation (Fig. 5A).
IGF-1 modestly induced SHIP2 tyrosine phosphorylation and the effect of Src
inhibitors appeared to be partial. In 3T3-L1 adipocytes, pretreatment with Src
inhibitor compounds PD173956 and PD180970 failed to prevent SHIP2 tyrosine
phosphorylation from occurring in response to insulin
(Fig. 5B).
|
Tyrosines 986-987 are important Src phosphorylation sites
To identify specific tyrosine residues phosphorylated in response to the
activation of Src, we examined tyrosine phosphorylation of SHIP2 mutants in
which tyrosines conforming to potential phosphorylation site were replaced by
phenylalanines. Expression constructs for FLAG-tagged wild-type or SHIP2
mutants were transiently transfected into HeLa cells. Anti-FLAG
immunoprecipitates from these cells demonstrated that the YY (986-987) double
mutant (YY-FF) in the NPAYY motif was weakly phosphorylated upon re-plating on
collagen I. The same effect was seen upon EGF treatment, indicating that the
tyrosines 986-987 can also be phosphorylated by a tyrosine kinase unrelated to
Src (Fig. 6A). SHIP2 proteins
with mutations in three other tyrosines that were potential phosphorylation
sites showed little change in tyrosine phosphorylation when compared with
wild-type SHIP2 (Fig. 6B). In
vitro phosphorylation assays using purified Src kinase indicated that Src
could directly phosphorylate SHIP2. These experiments also showed that the
986-987 YY-FF mutation effectively reduced tyrosine phosphorylation of SHIP2
by purified Src kinase (Fig.
6C). While YY 986-987 sites appear to be the major sites for Src
phosphorylation, it is also possible that phosphorylation at these sites may
be required for the subsequent phosphorylation of SHIP2 at additional
sites.
|
Exogenous Src kinase stimulates tyrosine phosphorylation of SHIP2 in
vivo
In transient transfection experiments shown in
Fig. 7A, a constitutively
active form of Src induced strong tyrosine phosphorylation of SHIP2.
Conversely, a dominant-negative form of Src kinase reduced collagen-I-induced
SHIP2 tyrosine phosphorylation by about 33-50%
(Fig. 7C). Three separate
experiments consistently produced this effect in experiments where the
transfection efficiency was approximately 30-40%.
|
Cell spreading on collagen I induces SHIP2 association with Shc via
the NPXY motif
The NPXY motif serves as an interaction site for the
phosphotyrosine-binding (PTB) domain present in signaling proteins. One such
PTB-domain-containing protein, Shc, was previously shown to associate with
SHIP2 upon stimulation with growth factors
(Habib et al., 1998). We
detected weak phosphotyrosine-containing protein bands co-precipitating with
SHIP2 that were migrating similarly to Shc proteins at a molecular mass of
55 kDa upon cell spreading on collagen I
(Fig. 5). We tested whether
SHIP2-Shc association was induced during cell adhesion on collagen I.
Co-immunoprecipitation experiments revealed induction of specific association
between SHIP2 and Shc proteins when detached cells were allowed to spread on
collagen I (Fig. 8A). Of the
three Shc forms, p52Shc was the more prominent species that co-precipitated
with SHIP2. SHIP2-Shc association was detected in both anti-SHIP2 and anti-Shc
immunoprecipitations (Fig.
8B,D). The SHIP2-Shc interaction induced by collagen I was
sensitive to the Src inhibitor PD180970 at 1 µM concentration
(Fig. 8B,D), Moreover,
coprecipitation of Shc proteins with SHIP2 occurred only when cells were
plated on collagen I but not on fibronectin or poly-L-lysine
(Fig. 8B,D). Mutations in the
NPAYY motif of SHIP2, YY-FF (986-987), abrogated collagen-I-induced SHIP2-Shc
interaction (Fig. 8C). A
protein species of approximately 50-55 kDa, corresponding to the heavy chain
of immunoglobulins, appeared in all the samples that migrated at the same
distance as p52Shc protein.
Phosphorylation of NPAYY motif of SHIP2 regulates lamellipodia
formation
We then wished to test the role of tyrosine phosphorylation of SHIP2 in
cell spreading. When cells expressing YY-FF (986-987) mutant SHIP2 were
allowed to spread on collagen I, marked deregulation of lamellipodia formation
was apparent compared with wild-type SHIP2-expressing cells
(Fig. 9). Wild-type SHIP2 was
distributed to lamellipodia of spreading cells
(Fig. 9A) as previously
described (Prasad et al.,
2001). A large number of cells also displayed focal contacts and
lamellipodia staining exclusively, with little cytoplasmic staining (not
shown). Both untransfected (data not shown) and wild-type SHIP2-expressing
cells spread circumferentially in a uniform fashion with broad lamellipodia
extensions (Fig. 9B,D). Cells
expressing YY-FF (986-987) mutant displayed multiple narrower membrane
protrusions with actin spikes at the extremities
(Fig. 9C,D). The effect was
seen in cells expressing high amounts of YY-FF mutant (brightly stained) and
not in cells expressing low levels (lightly stained). On fibronectin, cells
expressing wild-type or YY-FF mutant SHIP2 presented a more homogeneous
cytoplasmic staining with less intense lamellipodia localization
(Fig. 9E,G). In addition, YY-FF
mutant did not cause lamellipodia abnormalities
(Fig. 9G,H). No overt spreading
anomalies were observed in YY-FF mutants expressing cells allowed to spread on
laminin as well (data not shown). Taken together, these results indicate a
role for Src-mediated tyrosine phosphorylation of SHIP2 in cell spreading and
implicate SHIP2 as part of a signaling pathway downstream of Src kinases in
adhesion signaling induced by collagen I.
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Discussion |
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The results described here indicate for the first time that tyrosine
phosphorylation of SHIP2 lies downstream of Src kinases during cell adhesion.
In vitro phosphorylation experiments further suggest that SHIP2 could be a
direct substrate of Src (Fig.
6C). However, we recognize the fact that purified recombinant
kinases could potentially phosphorylate non-physiological substrates under
such conditions. It is plausible that SHIP2 may be phosphorylated in vivo by
another unrelated tyrosine kinase downstream of activated Src such as c-Abl,
FAK or Pyk2 (Cary et al., 1999;
Plattner et al., 1999
).
Integrin activation may cause recruitment of SHIP2 to focal contacts and
lamellipodia through SH2-mediated interaction with phosphorylated p130Cas
(Prasad et al., 2001
), where
SHIP2 could be phosphorylated.
Although it is still unclear how tyrosine phosphorylation might regulate
SHIP2 activity, proper localization of SHIP2, mediated in part by SH2-mediated
interactions, appears to be a pre-requisite for this modification
[(Taylor et al., 2000) (N.P.
and S.J.D., unpublished)]. Tyrosine phosphorylation of SHIP2, may initiate or
stabilize its interaction with other yet unidentified signaling molecules.
Data presented here support this notion as Src regulates the interaction
between SHIP2 and Shc adapter protein mediated through the NPAYY motif of
SHIP2. Such interactions may be critical in confining the function of SHIP2 to
specific sub-cellular sites. SHIP2 localization to areas where Src and
Src-substrates reside further underscores its relevance
(Dyson et al., 2001
;
Prasad et al., 2001
).
Interestingly, SHIP2 is constitutively tyrosine phosphorylated and associated
with Shc in Rous-sarcoma-virus-transformed fibroblasts and in BCR-ABL-positive
chronic myelogenous leukemia (CML) cells
(Habib et al., 1998
;
Wisniewski et al., 1999
). The
YY-FF (986-987) SHIP2 mutant displayed deregulation of lamellipodia formation
indicating an important role for interactions mediated through this motif in
cell spreading. A role for Shc in cell motility and regulation of actin
remodeling is well documented (Collins et
al., 1999
; Gu et al.,
1999
; Mauro et al.,
1999
). Therefore, it is possible that disruption of SHIP2-Shc
interaction may have significant impact on actin reorganization as shown by
irregular membrane protrusions and actin spikes in cells expressing the YY-FF
mutant.
Src is a critical component in adhesion signaling. Src kinase localizes to
focal adhesions in fibroblasts and to cell-cell junctions in epithelial cells
(Fincham et al., 1996;
Owens et al., 2000
). Src plays
an important role in turnover of focal adhesions and regulates cell motility
(Fincham and Frame, 1998
). In
epithelial cells, activated Src induces disassembly of cell-cell adhesions and
promotes ECM-dependent invasion (Owens et
al., 2000
). As SHIP2 appears to play an important role in cell
adhesion and spreading (Dyson et al.,
2001
; Prasad et al.,
2001
), phosphorylation of SHIP2 regulated by Src kinases
represents a molecular mechanism linking activation of tyrosine kinases
associated with integrin signaling to phospholipid metabolism. In support of
such a notion, some reports suggest that SHIP1 activity could be regulated
through tyrosine phosphorylation by Src family kinases following its
relocation to cytoskeleton (Gardai et al.,
2002
; Giuriato et al.,
2000
; Lamkin et al.,
1997
).
Recently, SHIP2 was shown to interact with an actin-binding protein filamin
and to regulate membranous actin (Dyson et
al., 2001). Filamin serves as a scaffold for RalA, RhoA, Rac and
Cdc42 proteins (Ohta et al.,
1999
). Rho family small GTPases are central regulators of cell
adhesion, spreading and migration and are activated by guanine nucleotide
exchange factors, GEFs (Ridley,
2001
). Many GEFs contain phosphoinositol-binding
pleckstrin-homology (PH) domains and are consequently regulated by
phosphotidylinositol metabolites. SHIP2 upon localization to focal contacts or
to lamellipodia, in conjunction with PI 3-kinase, could cause dynamic waves of
lipid second messengers, in turn regulating the activation of guanine
nucleotide exchange factors for Rho family proteins in a reversible and
dynamic fashion. Such dynamic waves of PtdIns metabolites' synthesis and
degradation during cell spreading and migration has been demonstrated
(Haugh et al., 2000
). By
contrast, SHIP2 activity could also regulate PtdIns(4,5)P2
levels (Taylor et al., 2000
).
PtdIns(4,5)P2, by virtue of its interaction with several
actin-binding proteins, plays a critical role in regulation of actin
remodeling (Czech, 2000
;
Takenawa and Itoh, 2001
).
Taken together, these studies describe a novel pathway involving Src kinases
and SHIP2 in the regulation of cytoskeleton, cell adhesion and motility.
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Acknowledgments |
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References |
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---|
Bachelot, C., Rameh, L., Parsons, T. and Cantley, L. C. (1996). Association of phosphatidylinositol 3-kinase, via the SH2 domains of p85, with focal adhesion kinase in polyoma middle t-transformed fibroblasts. Biochim. Biophy. Acta 1311,45 -52.[Medline]
Cary, L. A., Han, D. C. and Guan, J. L. (1999). Integrin-mediated signal transduction pathways. Histol. Histopathol. 14,1001 -1009.[Medline]
Clement, S., Krause, U., Desmedt, F., Tanti, J. F., Behrends, J., Pesesse, X., Sasaki, T., Penninger, J., Doherty, M., Malaisse, W. et al. (2001). The lipid phosphatase SHIP2 controls insulin sensitivity. Nature 409,92 -97.[CrossRef][Medline]
Collins, L. R., Ricketts, W. A., Yeh, L. and Cheresh, D.
(1999). Bifurcation of cell migratory and proliferative signaling
by the adaptor protein Shc. J. Cell Biol.
147,1561
-1568.
Corvera, S. and Czech, M. P. (1998). Direct targets of phosphoinositide 3-kinase products in membrane traffic and signal transduction. Trends Cell Biol. 8, 442-446.[CrossRef][Medline]
Czech, M. P. (2000). PIP2 and PIP3: complex roles at the cell surface. Cell 100,603 -606.[Medline]
Di Cristofano, A. and Pandolfi, P. P. (2000). The multiple roles of PTEN in tumor suppression. Cell 100,387 -390.[Medline]
Dyson, J. M., O'Malley, C. J., Becanovic, J., Munday, A. D.,
Berndt, M. C., Coghill, I. D., Nandurkar, H. H., Ooms, L. M. and Mitchell, C.
A. (2001). The SH2-containing inositol polyphosphate
5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.
J. Cell Biol. 155,1065
-1079.
Erneux, C., Govaerts, C., Communi, D. and Pesesse, X. (1998). The diversity and possible functions of the inositol polyphosphate 5-phosphatases. Biochim. Biophys. Acta 1436,185 -199.[Medline]
Fincham, V. J. and Frame, M. C. (1998). The
catalytic activity of Src is dispensable for translocation to focal adhesions
but controls the turnover of these structures during cell motility.
EMBO J. 17,81
-92.
Fincham, V. J., Unlu, M., Brunton, V. G., Pitts, J. D., Wyke, J. A. and Frame, M. C. (1996). Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins. J. Cell Biol. 135,1551 -1564.[Abstract]
Fukui, Y. and Hanafusa, H. (1989). Phosphatidylinositol kinase activity associates with viral p60src protein. Mol. Cell. Biol. 9,1651 -1658.[Medline]
Gardai, S., Whitlock, B. B., Helgason, C., Ambruso, D., Fadok,
V., Bratton, D. and Henson, P. M. (2002). Activation of SHIP
by NADPH oxidase-stimulated Lyn leads to enhanced apoptosis in neutrophils.
J. Biol. Chem. 277,5236
-5246.
Giuriato, S., Bodin, S., Erneux, C., Woscholski, R., Plantavid, M., Chap, H. and Payrastre, B. (2000). pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of alphaIIbbeta3 integrin in human platelets. Biochem. J. 348,107 -112.[CrossRef][Medline]
Gu, J., Tamura, M., Pankov, R., Danen, E. H., Takino, T.,
Matsumoto, K. and Yamada, K. M. (1999). Shc and FAK
differentially regulate cell motility and directionality modulated by PTEN.
J. Cell Biol. 146,389
-403.
Habib, T., Hejna, J. A., Moses, R. E. and Decker, S. J.
(1998). Growth factors and insulin stimulate tyrosine
phosphorylation of the 51C/SHIP2 protein. J. Biol.
Chem. 273,18605
-18609.
Haugh, J. M., Codazzi, F., Teruel, M. and Meyer, T.
(2000). Spatial sensing in fibroblasts mediated by 3'
phosphoinositides. J. Cell Biol.
151,1269
-1280.
Ji, P. and Haimovich, B. (1999). Integrin alpha IIb beta 3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PI 3-kinase. Biochim. Biophys. Acta 1448,543 -552.[Medline]
Jones, R. J., Brunton, V. G. and Frame, M. C. (2000). Adhesion-linked kinases in cancer; emphasis on src, focal adhesion kinase and PI 3-kinase. Eur. J. Cancer 36,1595 -1606.[CrossRef][Medline]
Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P. H.
and Downward, J. (1997). Matrix adhesion and Ras
transformation both activate a phosphoinositide 3-OH kinase and protein kinase
B/Akt cellular survival pathway. EMBO J.
16,2783
-2793.
King, W. G., Mattaliano, M. D., Chan, T. O., Tsichlis, P. N. and Brugge, J. S. (1997). Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. Mol. Cell. Biol. 17,4406 -4418.[Abstract]
Klein, C. E., Dressel, D., Steinmayer, T., Mauch, C., Eckes, B., Krieg, T., Bankert, R. B. and Weber, L. (1991). Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils. J. Cell Biol. 115,1427 -1436.[Abstract]
Kraker, A. J., Hartl, B. G., Amar, A. M., Barvian, M. R., Showalter, H. D. and Moore, C. W. (2000). Biochemical and cellular effects of c-Src kinase-selective pyrido[2, 3-d]pyrimidine tyrosine kinase inhibitors. Biochem. Pharmacol. 60,885 -898.[CrossRef][Medline]
Lamkin, T. D., Walk, S. F., Liu, L., Damen, J. E., Krystal, G.
and Ravichandran, K. S. (1997). Shc interaction with Src
homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the
Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP.
J. Biol. Chem. 272,10396
-10401.
Li, E., Stupack, D. G., Brown, S. L., Klemke, R., Schlaepfer, D.
D. and Nemerow, G. R. (2000). Association of p130CAS with
phosphatidylinositol-3-OH kinase mediates adenovirus cell entry. J.
Biol. Chem. 275,14729
-14735.
Majerus, P. W., Kisseleva, M. V. and Norris, F. A.
(1999). The role of phosphatases in inositol signaling reactions.
J. Biol. Chem. 274,10669
-10672.
Mauro, L., Sisci, D., Bartucci, M., Salerno, M., Kim, J., Tam, T., Guvakova, M. A., Ando, S. and Surmacz, E. (1999). SHC-alpha5betal integrin interactions regulate breast cancer cell adhesion and motility. Exp. Cell Res. 252,439 -448.[CrossRef][Medline]
Muraille, E., Pesesse, X., Kuntz, C. and Erneux, C. (1999). Distribution of the src-homology-2-domain-containing inositol 5-phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells. Biochem. J. 342,697 -705.[CrossRef][Medline]
Ohta, Y., Suzuki, N., Nakamura, S., Hartwig, J. H. and Stossel,
T. P. (1999). The small GTPase RalA targets filamin to induce
filopodia. Proc. Natl. Acad. Sci. USA
96,2122
-2128.
Owens, D. W., McLean, G. W., Wyke, A. W., Paraskeva, C.,
Parkinson, E. K., Frame, M. C. and Brunton, V. G. (2000). The
catalytic activity of the Src family kinases is required to disrupt
cadherin-dependent cell-cell contacts. Mol. Biol. Cell
11, 51-64.
Parise, L. V., Lee, J. and Juliano, R. L. (2000). New aspects of integrin signaling in cancer. Semin. Cancer Biol. 10,407 -414.[CrossRef][Medline]
Plattner, R., Kadlec, L., DeMali, K. A., Kazlauskas, A. and
Pendergast, A. M. (1999). c-Abl is activated by growth
factors and Src family kinases and has a role in the cellular response to
PDGF. Genes Dev. 13,2400
-2411.
Prasad, N., Topping, R. S., Zhou, D. and Decker, S. J. (2000). Oxidative stress and vanadate induce tyrosine phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). Biochemistry 39,6929 -6935.[CrossRef][Medline]
Prasad, N., Topping, R. S. and Decker, S. J.
(2001). SH2-containing inositol 5'-phosphatase SHIP2
associates with the p130(Cas) adapter protein and regulates cellular adhesion
and spreading. Mol. Cell. Biol.
21,1416
-1428.
Ravanti, L., Heino, J., Lopez-Otin, C. and Kahari, V. M.
(1999). Induction of collagenase-3 (MMP-13) expression in human
skin fibroblasts by three-dimensional collagen is mediated by p38
mitogen-activated protein kinase. J. Biol. Chem.
274,2446
-2455.
Ridley, A. J. (2001). Rho family proteins: coordinating cell responses. Trends Cell Biol. 11,471 -477.[CrossRef][Medline]
Riikonen, T., Westermarck, J., Koivisto, L., Broberg, A.,
Kahari, V. M. and Heino, J. (1995). Integrin alpha 2 beta 1
is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene
expression. J. Biol. Chem.
270,13548
-13552.
Rohrschneider, L. R., Fuller, J. F., Wolf, I., Liu, Y. and
Lucas, D. M. (2000). Structure, function, and biology of SHIP
proteins. Genes Dev. 14,505
-520.
Schwartz, M. A. (2001). Integrin signaling revisited. Trends Cell Biol. 11,466 -470.[CrossRef][Medline]
Takenawa, T. and Itoh, T. (2001). Phosphoinositides, key molecules for regulation of actin cytoskeletal organization and membrane traffic from the plasma membrane. Biochim. Biophys. Acta 1533,190 -206.[Medline]
Taylor, V., Wong, M., Brandts, C., Reilly, L., Dean, N. M.,
Cowsert, L. M., Moodie, S. and Stokoe, D. (2000). 5'
phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell
cycle arrest in glioblastoma cells. Mol. Cell. Biol.
20,6860
-6871.
Wada, T., Sasaoka, T., Funaki, M., Hori, H., Murakami, S.,
Ishiki, M., Haruta, T., Asano, T., Ogawa, W., Ishihara, H. et al.
(2001). Overexpression of SH2-containing inositol phosphatase 2
results in negative regulation of insulin-induced metabolic actions in 3T3-L1
adipocytes via its 5'-phosphatase catalytic activity. Mol.
Cell. Biol. 21,1633
-1646.
Watton, S. J. and Downward, J. (1999). Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction. Curr. Biol. 9, 43-436.[CrossRef][Medline]
Wisniewski, D., Strife, A., Swendeman, S., Erdjument-Bromage,
H., Geromanos, S., Kavanaugh, W. M., Tempst, P. and Clarkson, B.
(1999). A novel SH2-containing phosphatidylinositol
3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine
phosphorylated and associated with src homologous and collagen gene (SHC) in
chronic myelogenous leukemia progenitor cells. Blood
93,2707
-2720.
Zheng, D. Q., Woodard, A. S., Tallini, G. and Languino, L.
R. (2000). Substrate specificity of alpha(v)beta(3)
integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway
activation. J. Biol. Chem.
275,24565
-24574.