Molecular and Cellular Biology, August 2001, p. 5082-5093, Vol. 21, No. 15
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.15.5082-5093.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
6
4
Integrin-Dependent Activation of Phosphoinositide 3-OH Kinase
and Promotion of Invasion
Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
Received 17 November 2000/Returned for modification 16 January 2001/Accepted 30 April 2001
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ABSTRACT |
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Expression of the
6
4 integrin increases the invasive
potential of carcinoma cells by a mechanism that involves activation of
phosphoinositide 3-OH kinase (PI3K). In the present study, we
investigated the signaling pathway by which the
6
4 integrin activates PI3K. Neither the
6 nor the
4 cytoplasmic domain
contains the consensus binding motif for PI3K, pYMXM, indicating that
additional proteins are likely to be involved in the activation of this
lipid kinase by the
6
4 integrin. We identified insulin receptor
substrate 1 (IRS-1) and IRS-2 as signaling intermediates in the
activation of PI3K by the
6
4 integrin. IRS-1 and IRS-2 are
cytoplasmic adapter proteins that do not contain intrinsic kinase
activity but rather function by recruiting proteins to surface
receptors, where they organize signaling complexes. Ligation of the
6
4 receptor promotes tyrosine phosphorylation of IRS-1 and IRS-2 and increases their association with PI3K, as determined by
coimmunoprecipitation. Moreover, we identified a tyrosine residue in
the cytoplasmic domain of the
4 subunit, Y1494, that is required for
6
4-dependent phosphorylation of IRS-2 and activation of PI3K in
response to receptor ligation. Most importantly, Y1494 is essential for
the ability of the
6
4 integrin to promote carcinoma invasion.
Taken together, these results imply a key role for the IRS proteins in
the
6
4-dependent promotion of carcinoma invasion.
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INTRODUCTION |
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Cell adhesion molecules play
an important role in normal epithelia, and changes in their expression
and function contribute to the progression of epithelial cells to
invasive, metastatic carcinoma. For example, cell-cell interactions in
many tumors are altered through a quantitative decrease in cadherin
expression, which reduces intercellular adhesion (5). This
disruption of cell-cell adhesion is permissive for increased cell
motility. Cell adhesion can also be modified through qualitative
changes in receptor function that promote the dynamic adhesion that is required for motile, invasive cells (31). In recent years,
a significant amount of evidence to suggest that the
6
4 integrin is a member of this category of adhesion receptors has accumulated. Specifically, the expression of this integrin receptor is maintained in
carcinoma cells but it functions in a manner distinct from its role in
normal epithelial cells (55). The involvement of the
4
integrin subunit in carcinoma cell biology was initially suggested by
its identification as a tumor-related antigen expressed in metastatic
cancer (22). Since then, many studies have reported a
strong association of
4 expression with solid-tumor progression. For
example, the
4 subunit is not expressed in the normal thyroid but
its expression correlates with the progression to invasive thyroid
carcinoma (62). The
4 subunit is also expressed in androgen receptor-negative invasive prostate carcinomas and at the
leading edges of invading gastric carcinomas (6, 75). Moreover, expression of the
4 subunit correlates with a poor prognosis in patients with squamous cell, breast, and colon carcinomas (23, 71, 82). These correlative data have been supported more recently by functional studies that have provided mechanistic insight into how the
6
4 integrin contributes to tumor
progression. In previous studies, we demonstrated that the
6
4
integrin can increase the invasive potential of breast carcinoma cells,
a finding that has been confirmed for other cell types as well
(12, 21, 64, 67). Furthermore, expression of the
6
4
receptor increases the survival of p53 mutant carcinoma cells (2,
74). Given that invasion and survival are two critical functions
of metastatic cells, it is important to understand in more detail the
mechanism of action of the
6
4 integrin in tumor cells.
In normal epithelia, the
6
4 integrin functions as a receptor for
the laminin family of extracellular matrix proteins and mediates the
stable attachment of epithelial cells to the underlying basement
membrane (7, 41). Many studies, including those involving
knockout of the
4 subunit, have substantiated the importance of the
adhesive contributions of the
6
4 integrin to normal epithelial function (19, 81). In the absence of the
4 subunit, and
more specifically the
4 cytoplasmic domain, a lethal blistering of the epithelium, which is known as epidermolysis bullosa, occurs (19, 81). In carcinoma cells, the
6
4 integrin also
functions as a laminin receptor and the
4 subunit interacts with the
actin cytoskeleton to promote the formation of actin-rich structures that are important for cell motility (54, 56). However, in addition to its mechanical involvement in mediating adhesive
interactions, the
6
4 integrin activates intracellular signaling
pathways that are essential for the ability of this receptor to promote
tumor progression (51, 52, 64). For example, our analysis
of the mechanism involved in the
6
4-dependent promotion of
invasion revealed that phosphoinositide 3-OH kinase (PI3K) activation
by the receptor is essential for this function (64). The
ability of the
6
4 integrin to promote PI3K activation is greater
than that observed for other
1 integrins, which supports the
increased potential of this integrin to promote carcinoma invasion.
Activation of PI3K by the
6
4 integrin is also required for the
ability of this integrin to promote carcinoma cell survival through the activation of the Akt kinase (2, 3, 74).
PI3K is a lipid kinase that phosphorylates the D3 position of
inositol lipids to form the products phosphatidylinositol (PtdIns)-3-P, PtdIns-3,4-P2, and
PtdIns-3,4,5-P3 (76). These D3
phosphoinositides are expressed at very low levels in unstimulated
cells, but their levels are increased in response to many different
stimuli, supporting their role as second messengers. A major function
of the D3 phosphoinositides is to bind and recruit signaling molecules
to the plasma membrane, where they can interact with other regulatory
and effector molecules (76). The involvement of PI3K in
carcinoma cell biology has been proposed from both direct and indirect
evidence. As mentioned above, PI3K activity promotes carcinoma invasion
and survival, and it has also been implicated in promoting
anchorage-independent growth (20, 34, 35, 64). An avian
sarcoma virus that encodes the catalytic subunit of PI3K transforms
chicken embryo fibroblasts, suggesting that PI3K can also play a role
in the early stages of tumor initiation (11). Finally,
PTEN, a lipid phosphatase that regulates the levels of the PI3K
lipid products, is frequently mutated or deleted in tumors (9,
18). The identification of the PTEN gene as a tumor
suppressor gene demonstrates the importance of tightly regulating the
activity of PI3K. In light of these findings, the relevance of
determining how the
6
4 integrin activates the PI3K signaling
pathway is evident.
The
6
4 integrin is distinct from other integrin receptors because
the
4 subunit contains a 1,000-amino-acid cytoplasmic domain
(27, 70, 72). This large intracellular domain is important
for many of the known
6
4-dependent functions. For example, the
4 cytoplasmic domain is essential for hemidesmosome formation in
normal epithelial cells and it is required for promoting carcinoma cell
invasion (44, 64). In the absence of the
4 cytoplasmic
domain, the
6
4 receptor is not capable of activating PI3K or
other signaling pathways that have been shown to be activated by this
integrin, including the mitogen-activated protein kinase (MAPK) pathway
(64). Although a number of proteins that interact with the
4 cytoplasmic domain in hemidesmosomes have been identified, very
little is known about the structural requirements for signaling by this
integrin or the specific interactions that occur with downstream
effectors to initiate signals (48, 57). Recently, the
binding site in the
4 cytoplasmic domain for the adapter protein
Shc, which recruits Grb2 and Sos to promote Ras activation, has been
identified (16, 43). However, the binding motif for the
p85 regulatory subunit of PI3K, YMXM, is not present in the
4
cytoplasmic domain, which suggests that alternative mechanisms are
required to recruit this lipid kinase (10).
In the present study, we have examined the mechanism by which the
6
4 integrin activates the PI3K signaling pathway. In light of the
fact that the
4 subunit cytoplasmic domain does not contain a
binding site to directly interact with PI3K, we sought to identify the
intermediate proteins that are responsible for recruiting PI3K to the
plasma membrane in response to
6
4 ligation. We identified two
members of the insulin receptor substrate (IRS) family, IRS-1 and
IRS-2, which are specific mediators of
6
4-dependent activation of
PI3K (84). In addition, we investigated the structural
requirements of the
4 subunit cytoplasmic domain for PI3K
activation. Through this analysis we identified a specific
tyrosine residue in the
4 subunit, Y1494, that is required for
6
4-dependent activation of PI3K and, importantly, for the ability
of this integrin receptor to promote carcinoma invasion.
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MATERIALS AND METHODS |
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Cells and antibodies.
MDA-MB-435 cells were grown in
Dulbecco's modified Eagle's medium (DMEM; Biowhittaker) supplemented
with 10% fetal calf serum (Sigma), 1% penicillin-streptomycin
(Gibco), and 1% GlutaMax (Gibco). T47D cells were grown in DMEM
supplemented with 10% fetal calf serum, 1% penicillin-streptomycin,
1% GlutaMax, and 5 µg of insulin (Gibco)/ml. The rat monoclonal
antibody that recognizes the
6-integrin subunit (135-13C) was a gift
from Rita Falcioni, and the mouse monoclonal antibody that recognizes
the
4-integrin subunit (UM-A9) was purchased from Ancell. The
IRS-1-specific polyclonal antibody was purchased from Santa Cruz
Biotechnology. The IRS-2-specific polyclonal antibody and the 4G10
phosphotyrosine-specific monoclonal antibody were purchased from
Upstate Biotechnology Inc. The RC-20 biotinylated-phosphotyrosine-specific monoclonal antibody was purchased
from Transduction Labs. The p85-specific polyclonal antiserum was a
gift from Alex Toker.
Integrin clustering. Cells were removed from their dishes with trypsin and washed twice with RPMI medium containing 25 mM HEPES (RH) and 0.1% heat inactivated bovine serum albumin (BSA; RH-BSA). After being washed, the cells were resuspended in the same buffer at a concentration of 2 × 106 cells/ml and incubated for 30 min with integrin-specific antibodies or in buffer alone. The cells were washed once, resuspended in the same buffer, and added to plates that had been coated overnight with anti-mouse immunoglobulin G (IgG). After a 30-min incubation at 37°C, the cells were washed twice with cold phosphate-buffered saline (PBS) and solubilized at 4°C for 10 min in a 20 mM Tris buffer, pH 7.4, containing 0.14 M NaCl, 1% NP-40, 10% glycerol, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, and 5 µg of aprotinin, pepstatin, and leupeptin/ml. Nuclei were removed by centrifugation at 12,000 × g for 10 min. For laminin attachment assays, cells were added to plates (100 mm in diameter) that had been coated overnight with 150 µg of laminin-1 and incubated for 45 min at 37°C.
Immunoprecipitation and immunoblotting. Aliquots of cell extracts containing equivalent amounts of protein were incubated for 3 h at 4°C with antibodies and protein A- or protein G-Sepharose (Pharmacia) with constant agitation. The beads were washed three times in the extraction buffer. Laemmli sample buffer was added to the samples, which were then incubated at 100°C for 4 min. Immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose filters. The filters were blocked for 1 h using a 50 mM Tris buffer, pH 7.5, containing 0.15 M NaCl and 0.05% Tween 20 (TBST) and 5% (wt/vol) Carnation dry milk. The filters were incubated for 1 h in the same buffer containing primary antibodies. After three 10-min washes in TBST, the filters were incubated for 1 h in blocking buffer containing horseradish peroxidase (HRP)-conjugated secondary antibodies. After three 10-min washes in TBST, proteins were detected by enhanced chemiluminescence (Pierce). For RC-20 phosphotyrosine immunoblots, the filters were blocked for 1 h using a 10 mM Tris buffer, pH 7.5, containing 0.5 M NaCl and 0.1% Tween 20 (RC-20 buffer) and 2% (wt/vol) Carnation dry milk. The filters were washed briefly in RC-20 buffer and then incubated overnight at 4°C in RC-20 buffer containing 3% (wt/vol) BSA and a 1:500 dilution of the RC-20 antibody. After being washed, the filters were incubated for 1 h in blocking buffer containing HRP-conjugated streptavidin, and the proteins were detected by enhanced chemiluminescence.
PI3K kinase assay.
To assay PI3K activity, aliquots of cell
extracts that contained equivalent amounts of protein were incubated
for 3 h at 4°C with antibodies and protein A-Sepharose
(Pharmacia). The Sepharose beads were washed twice with solubilization
buffer and twice with a 10 mM HEPES buffer, pH 7, containing 0.1 mM
EGTA (kinase buffer). After removal of the last wash, the beads were
resuspended in kinase buffer containing 10 µg of sonicated crude
brain lipids (Sigma), 100 µM ATP, 25 mM MgCl2,
and 10 µCi of [
-32P]ATP and incubated for
10 min at room temperature. The reaction was stopped by the addition of
60 µl of 2 N HCl and 160 µl of a 1:1 mixture of chloroform and
methanol. Lipids were resolved by using thin-layer chromatography
plates coated with potassium oxalate.
Site-directed mutagenesis.
The cloning of the human
wild-type
4 cDNA (
4D) and its transfection into the
MDA-MB-435 cell line have been described previously (13,
64). Tyrosine residues 1257 and 1494 in the
4 subunit were
mutated to phenylalanine residues using the Quickchange site-directed mutagenesis kit (Stratagene). Briefly, overlapping primers containing the desired mutations were used to amplify the
4 cDNA and vector by
PCR and the resulting point mutations were confirmed by dideoxy sequencing. The vectors containing the mutant
4 cDNAs were
transfected into the MDA-MB-435 cell line using Lipofectamine (Gibco)
according to the manufacturer's instructions. Neomycin-resistant cells
were isolated by selective growth in medium containing G418 (0.8 mg/ml; Gibco). The stable transfectants were pooled, and subclones of cells
that expressed the mutant
4 subunits on the cell surface were
isolated by fluorescence-activated cell sorting (FACS). The human
4
integrin-specific monoclonal antibody, UM-A9 (Ancell), was used
for this sorting and for subsequent analysis of the transfectants.
Analysis of integrin surface expression.
The relative
surface expression of the
4-integrin subunit on the transfected
MDA-MB-435 subclones was assessed by flow cytometry. For this purpose,
aliquots of cells (5 × 105) were incubated
for 45 min at room temperature with RH-BSA and either the
4-specific
antibody or nonspecific mouse IgG (Sigma). The cells were washed two
times with RH-BSA and then incubated with goat anti-mouse IgG coupled
to Cy2 (Jackson Immunoresearch) for 45 min at room temperature. After
being washed two times with RH-BSA, the cells were resuspended in PBS
and analyzed by flow cytometry.
Invasion assay. Matrigel invasion assays were performed as described previously (63, 64) using 6.5-mm-diameter Transwell chambers (8-µm pore size; Costar). Matrigel purified from the Englebreth-Holm-Swarm tumor was diluted in cold distilled water, added to the Transwells (5 µg/well), and dried in a sterile hood. The Matrigel was then reconstituted with medium for 1 h at 37°C before the addition of cells. Cells (0.5 × 105) were resuspended in serum-free DMEM containing 0.1% BSA, and cells were added to each well. Conditioned NIH 3T3 medium was added to the bottom wells of the chambers. After 4 h, the cells that had not invaded were removed from the upper faces of the filters using cotton swabs and the cells that had invaded to the lower surfaces of the filters were fixed in methanol and then stained with a 0.2% solution of crystal violet in 2% ethanol. Invasion was quantitated by visual counting. The mean of five individual fields in the center of the filter, where invasion was the highest, was obtained for each well.
Adhesion assays. Adhesion assays were performed as described previously (63). Briefly, multiwell tissue culture plates (11.3 mm in diameter) were coated overnight at 4°C with 0.2 ml of PBS containing either murine laminin-1 (20 µg/ml) or rat collagen I (20 µg/ml). The wells were then washed with PBS and blocked with RH-BSA. Cells (105) were resuspended in RH-BSA and added to the protein-coated wells. After a 60-min incubation at 37°C, the wells were washed three times with RH at 37°C, fixed for 15 min with methanol, and stained with a 0.2% solution of crystal violet in 2% ethanol. The crystal violet stain was solubilized with a 1% solution of SDS, and adhesion was quantitated by measuring the absorbance at 595 nm.
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RESULTS |
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Identification of IRS-1 and IRS-2 as intermediates in the
activation of PI3K by the
6
4 integrin.
In previous work, we
demonstrated that ligation of the
6
4 integrin promotes
significantly more PI3K activity than ligation of the
6
1 integrin
or other
1 integrins (64). PI3K is activated by
recruitment of the p85 regulatory subunit to phosphotyrosine-containing binding motifs (pYMXM) (10). Neither the
6- nor the
4-subunit cytoplasmic domain contains the p85 consensus binding
motif, which suggests that additional intermediate proteins are most
likely involved in the
6
4-dependent activation of PI3K. To
identify these intermediates, we analyzed the profile of
phosphoproteins that associate with PI3K after
6
4 ligation. To do
so, cell extracts from mock- (MDA-MB-435/mock) and
4-transfected
(MDA-MB-435/
4) MDA-MB-435 cells that had been clustered with
6-specific antibody 135-13C were immunoprecipitated with a
p85-specific antiserum and the associated proteins were detected by
immunoblotting with phosphotyrosine-specific antibody RC-20. As shown
in Fig. 1A, ligation of both
6
1 and
6
4 resulted in the interaction of PI3K with a 130-kDa
phosphoprotein. However, an additional 180-kDa phosphoprotein
coimmunoprecipitated with PI3K in both of the MDA-MB-435/
4 subclones
after ligation with
6-specific antibodies. To confirm that this
180-kDa protein was specific for
6
4-dependent activation of PI3K,
the MDA-MB-435/
4 transfectants were clustered with
4-specific antibodies, which will not ligate
6
1, and the p85-associated proteins were analyzed. When
6
4 was clustered in the absence of
6
1 ligation, only the 180-kDa protein was observed to
coimmunoprecipitate with PI3K (Fig. 1B). The association of PI3K with
the 180-kDa phosphoprotein only after ligation of the
6
4 integrin
suggests that there is a unique mechanism for PI3K activation by the
6
4 receptor that is not utilized by the
6
1 receptor.
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6
4-dependent PI3K
activation, we sought to identify the 180-kDa protein that was phosphorylated on tyrosine and associated with PI3K in response to
6
4 ligation. Given the molecular mass of this phosphoprotein, we
first investigated if this protein was a growth factor receptor. This
possibility was supported by the fact that the
6
4 integrin can
associate with ErbB2 (21). However, the p85-associated
180-kDa protein was not expressed on the cell surface, as indicated by a test for surface biotinylation (data not shown).
Next, we investigated the IRS adapter proteins. The IRS family members,
which include IRS-1, IRS-2, IRS-3, and IRS-4, are 170- to 180-kDa
proteins (except IRS-3, which is 60 kDa) that function as intermediate
docking proteins downstream of the insulin and insulin-like growth
factor 1 (IGF-1) receptors, as well as a number of cytokine
receptors (84). In addition, the
5
1 integrin can
promote IRS-1 phosphorylation in adipocytes (24).
Importantly, the IRS proteins contain several PI3K binding sites, and
these adapters are known to be involved in the activation of PI3K
downstream of several of the receptors mentioned above (30, 33,
45, 69, 78, 86). To determine if the 180-kDa protein that
coimmunoprecipitated with PI3K was an IRS family member, we first
analyzed the expression of each IRS homolog in MDA-MB-435 cells. As
shown in Fig. 2A, MDA-MB-435 cells
express very low levels of IRS-1 but express high levels of IRS-2.
IRS-3 and IRS-4 were not detected in these cells (data not shown). To
investigate the potential involvement of IRS-2 in the
6
4-dependent activation of PI3K, cell extracts from
MDA-MB-435/mock and MDA-MB-435/
4 cells that had been clustered with
6-specific antibodies were assayed for IRS-2 phosphorylation. As
shown in Fig. 2B, IRS-2 was phosphorylated on tyrosine in response to
clustering with
6-specific antibodies in the MDA-MB-435/
4 transfectants but not in the MDA-MB-435/mock transfectants. In addition, ligation with a
4-specific antibody also increased the
tyrosine phosphorylation of IRS-2. Most importantly, the p85 subunit of
PI3K associated with IRS-2 after ligation of the
6
4 receptor
(Fig. 2B, bottom). As a positive control for IRS-2 phosphorylation, the
MDA-MB-435/
4 cells were treated with IGF-1, which increased the
phosphorylation of IRS-2 and its association with PI3K (Fig. 2B). The
MDA-MB-435/mock and -
4 transfectants express equivalent levels of
IRS-2, and therefore the lack of IRS-2 phosphorylation after ligation
of
6
1 in the MDA-MB-435/mock transfectants is not due to a
relative difference in protein expression levels (Fig. 2A).
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6
4
ligation, in vitro kinase assays were performed on IRS-2 immune
complexes. MDA-MB-435/mock and MDA-MB-435/
4 transfectants were
clustered with
6- and
4-specific antibodies, and the cell extracts were immunoprecipitated with IRS-2 antibodies. The IRS-2 immunoprecipitates were assayed for their ability to phosphorylate crude brain phosphoinositides. As shown in Fig. 2C, ligation of the
6
4 integrin with both
6- and
4-specific antibodies resulted in a marked increase in PI3K activity associated with IRS-2, as demonstrated by the appearance of the
PtdIns-3,4,5-P3 lipid product. In
contrast, ligation of
6
1 in the MDA-MB-435/mock transfectants resulted in minimal IRS-2-associated PI3K activity.
IRS-1 and IRS-2 have considerable structural homology and both homologs
contain multiple binding sites for PI3K (84). To determine
if IRS-1 can also be involved in the activation of PI3K by the
6
4
integrin, we used T47D breast carcinoma cells, which express
high levels of IRS-1 and low levels of IRS-2 (Fig. 2A). As shown in
Fig. 2D (top), ligation of the
6
4 receptor using either
6- or
4-specific antibodies increased the association of PI3K with a
170-kDa phosphoprotein. An IRS-1-specific antibody recognized this
phosphoprotein (bottom). Based on these results we conclude that both
IRS-1 and IRS-2 can function as intermediate signaling proteins in the
activation of PI3K by the
6
4 integrin. The 130-kDa phosphoprotein
that associates with PI3K in response to
6
1 ligation has not been
conclusively identified.
Adhesion to laminin-1 promotes
6
4-dependent IRS
phosphorylation.
To confirm that the
6
4-dependent IRS
signaling pathway that we identified by antibody clustering occurs in
response to ligation of
6
4 by a natural extracellular matrix
ligand, we assessed the phosphorylation of IRS-2 after adhesion of the
MDA-MB-435/mock and MDA-MB-435/
4 transfectants to a laminin-1
substratum. As shown in Fig. 3, adhesion
of the MDA-MD-435/mock transfectants to laminin-1 did not result in an
increase in the tyrosine phosphorylation of IRS-2. Given that
6
1
is the major laminin receptor on the surfaces of these cells, these
results confirm the data obtained using antibodies to cluster the
6
1 integrin (Fig. 2B). In contrast, adhesion of the
MDA-MB-435/
4 transfectants to laminin-1 promoted the tyrosine
phosphorylation of IRS-2 and the recruitment of PI3K to this adapter
protein. Therefore, ligation of the
6
4 integrin by either
laminin-1 or receptor-specific antibodies can activate the IRS-PI3K
signaling pathway.
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Analysis of tyrosine phosphorylation of the
4 subunit.
Having identified IRS-1 and IRS-2 as signaling intermediates in the
pathway utilized by the
6
4 integrin to activate PI3K, we next
investigated the mechanism by which this integrin receptor activates
this pathway. It had been previously demonstrated that the
4 subunit
is phosphorylated on tyrosine after ligation of the
6
4 receptor
(43). We confirmed this finding in our transfected MDA-MB-435/
4 cells. As shown in Fig.
4A, a
time-dependent increase in the tyrosine phosphorylation of the
4
subunit was observed after ligation of the
6
4 receptor with
6-specific antibodies. Addition of sodium orthovanadate markedly
increased the level of tyrosine phosphorylation, indicating that
tyrosine phosphorylation of the
4 subunit is regulated by tyrosine
phosphatases in these cells (Fig. 4A) (43). To evaluate
the role of
4-tyrosine phosphorylation in the activation of PI3K by
the
6
4 integrin, we analyzed the
4 cytoplasmic domain for
tyrosine residues that were located within known consensus binding or
phosphorylation motifs. We identified two tyrosines that were of
potential interest, Y1257 and Y1494 (Fig. 3B). Both of these tyrosines
are located within immune T-cell inhibitory motifs (ITIM) that have the
consensus sequence of L/VXXpYXL/V and that were initially
identified in immune cell inhibitory coreceptors (79).
ITIM sites are involved in regulating B- and T-cell receptor signaling,
and they have been characterized as binding motifs for the SH2 domains
of the SH2-containing tyrosine phosphatase 1 (SHP-1) and -2 protein
tyrosine phosphatases and also for the SH2-containing inositol
polyphosphate 5-phosphatase 1 (SHIP-1) and -2 lipid phosphatases
(79).
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6
4-dependent activation of PI3K, Y1257 and Y1494 in the
4
subunit were individually mutated to phenylalanine residues. In
addition, we also mutated both Y1257 and Y1494 to generate a
double-ITIM-mutant
4 subunit (Y1257F/Y1494F). The mutant
4 subunits were stably expressed in the MDA-MB-435 cells, which lack
endogenous
4 expression, and subclones expressing the
4 mutant
proteins on the cell surface were isolated by FACS. As shown in the
flow cytometry profiles for two individual subclones of each
transfectant in Fig. 4C, all of the mutant
4 subunits were expressed
on the cell surface. To confirm that the mutant
4 subunits
associated with endogenous
6 subunits, cell extracts of the
4
mutant-expressing subclones were immunoprecipitated with an
6-specific antibody and then immunoblotted with an antiserum that
recognizes the C terminus of the
4 subunit. As shown in Fig. 4D, all
of the mutant
4 subunits formed heterodimers with the endogenous
6 subunits. Moreover, all of the mutants were recognized by the
C-terminal antiserum, which indicates that they are all expressed as
full-length proteins.
To determine if the
4 cytoplasmic domain is phosphorylated on either
Y1257 or Y1494 in response to
6
4 ligation, we assayed the
tyrosine phosphorylation of the mutant
4 subunits after clustering the receptors with
4-specific antibodies. Mutation of either Y1257
or Y1494 resulted in a significant decrease in the level of
4-tyrosine phosphorylation (2 to 7% of the wild-type level) after
clustering with
4-specific antibodies (Fig.
5). Addition of sodium orthovanadate to
the cells during the clustering markedly increased the tyrosine
phosphorylation of the mutant
4 subunits. However, when the results
were normalized for total
4 protein, a significant decrease
in the tyrosine phosphorylation of the mutant proteins compared to the
level of phosphorylation of the wild-type
4 subunit was still
observed.
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Tyrosine 1494 in the
4 subunit is required for
6
4-dependent activation of PI3K.
To evaluate the impact of
mutating Y1257 and Y1494 in the
4-subunit cytoplasmic domain on the
ability of the
6
4 receptor to activate downstream signaling
pathways, we initially examined the ability of these
6
4 mutant
receptors to promote increases in total cellular tyrosine
phosphorylation after clustering with
4-specific antibodies.
MDA-MB-435 cells that expressed the wild-type
4 and each of the
mutant
4 subunits were clustered with
4-specific antibodies, and
the cell extracts were incubated with phosphotyrosine-specific antibody
4G10. As shown in Fig. 6A (top), ligation
of wild-type
6
4 resulted in a marked increase in total cellular
tyrosine phosphorylation levels. A similar increase in tyrosine
phosphorylation was observed in two individual subclones that expressed
the Y1257F
4 subunit, indicating that Y1257 is not essential for
6
4-dependent promotion of tyrosine phosphorylation (Fig. 6A).
Although one of the Y1257F subclones had a lower level of tyrosine
phosphorylation than was observed for the wild-type
4 subclone, this
level of phosphorylation correlated with the levels of surface
expression of the
4 subunit in these cells (Fig. 4C). In contrast,
none of the MDA-MB-435 subclones that expressed the Y1494F or the
double-mutant Y1257F/Y1494F
4 subunits showed increases in
cellular tyrosine phosphorylation levels in response to
6
4
clustering. These results suggest that Y1494 is essential for
6
4-dependent promotion of tyrosine phosphorylation.
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4 cytoplasmic domain is
required for PI3K activation by the
6
4 receptor, we assessed the
association of PI3K with the phosphotyrosine immune complexes after
ligation of
6
4 in the subclones that expressed these mutant
4
subunits. As shown in Fig. 6A (bottom), p85 association with the
phosphotyrosine immune complexes increased after the clustering of the
wild-type
6
4 and the Y1257F mutant
6
4 receptors. In
contrast, an increase in p85 association with phosphotyrosine immune
complexes in response to the clustering of the Y1494F and Y1257F/Y1494F
mutant
6
4 receptors was not observed. The level of p85 subunit
association with the phosphotyrosine immune complexes correlated well
with the level of PI3K activity observed in in vitro kinase assays
(Fig. 6B).
Our data suggest that IRS-2 is an important intermediate in the
activation of PI3K by the
6
4 integrin. Therefore, we examined the
ability of the mutant
6
4 receptors to promote IRS-2 tyrosine phosphorylation. As shown in Fig. 7A
(top), mutation of Y1494 inhibited the ability of the
6
4 receptor
to promote IRS-2 phosphorylation. Tyrosine phosphorylation of IRS-2 was
also inhibited by the double Y1257F Y1494F
4 mutations.
Moreover, the recruitment of p85 to IRS-2 in response to
6
4
ligation was prevented in the Y1494F-expressing and Y1257F
Y1494F-expressing subclones (Fig. 6A, bottom). Although lower levels of
IRS-2 expression were observed in the Y1494F-expressing and Y1257F- and
Y1494F-expressing subclones, IRS-2 phosphorylation in response to
6
4 ligation was not detected even after prolonged exposure of the
immunoblot. To confirm that the lack of IRS-2 phosphorylation in the
subclones expressing the mutant
4 subunits was specific to
6
4-dependent signaling, the transfectants were treated with
IGF-1, which promotes IRS-2 phosphorylation through IGF-1R. As shown in
Fig. 7B, IRS-2 phosphorylation and PI3K recruitment in the wild-type
and
4 mutant-expressing subclones after IGF-1 stimulation were
equivalent. Taken together, our results indicate that Y1494 in the
4
subunit plays a pivotal role in the ability of the
6
4 integrin to
activate PI3K.
|
Tyrosine 1494 in the
4 subunit is required for
6
4-dependent invasion.
Expression of the
4 subunit
increases the invasive potential of MDA-MB-435 cells, and we have
hypothesized that this ability to promote invasion involves activation
of PI3K by the
6
4 receptor (64). If this prediction
is correct, expression of the Y1494F and Y1257F/Y1494F
4 mutant
subunits in MDA-MB-435 cells should not increase their invasive
potential. To address this question, subclones expressing the wild-type
and mutant
4 subunits were assayed for their ability to invade
Matrigel using a modified Boyden chamber assay. The MDA-MB-435
subclones expressing the Y1257F mutant subunit invaded to the same
extent as the MDA-MB-435 subclone that expressed the wild-type
4
subunit (Fig. 8A). In contrast, the
MDA-MB-435 subclones that expressed the Y1494F and Y1257F/Y1494F mutant
4 subunits did not invade. In fact, invasion was reduced below the
level observed for the MDA-MB-435/mock transfectants, which suggests
that the Y1494F and Y1257F/Y1494F mutant
4 subunits act in a
dominant-negative manner for invasion.
|
4 transfectants could result from
a deficiency in adhesion. To examine if the observed decrease in
invasion of the Y1494F and Y1257F/Y1494F transfectants was related to a
decrease in cell adhesion, the ability of these cells to adhere to a
laminin-1 or collagen I substrate was assessed. As shown in Fig. 8B,
the Y1494F and Y1257F/Y1494F transfectants demonstrated a 1.5- to
2-fold higher level of adhesion to both substrates than the mock,
wild-type
4, and Y1257 transfectants. The increased adhesion was not
due to higher levels of receptor expression in the Y1494F and
Y1257F/Y1494F transfectants (data not shown). Interestingly, the
haptotactic migration of the Y1494F and Y1257F/Y1494F transfectants was
significantly diminished on both laminin-1 and collagen I substrata
(data not shown). The increased adhesive strength of the mutant
4
transfectants may inhibit their motility, which would impede the
ability of these cells to invade (37). These results
suggest that the Y1494 of the
4 subunit is important for regulating
dynamic adhesion and that, in the absence of the IRS-2-PI3K signaling
pathway activated through this tyrosine residue, other signals from the
6
4 receptor may promote stable adhesion.
| |
DISCUSSION |
|---|
|
|
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Our results establish that the
6
4 integrin activates PI3K
through the signaling adapters IRS-1 and IRS-2. This is a unique mechanism for
6
4 because the IRS proteins are not involved in PI3K activation downstream of the
6
1 receptor. We have also identified a specific tyrosine residue in the
4 cytoplasmic domain, Y1494, which is essential for the activation of PI3K and for the ability of the
6
4 integrin to promote carcinoma invasion. Taken together, our findings highlight a novel mechanism for
6
4-dependent signaling and for the ability of this integrin to
promote carcinoma invasion.
Invasion is thought to be the essential first step for tumor cells in
the metastatic cascade (14). The correlation between metastasis and patient mortality provides a strong impetus to elucidate
the mechanisms involved in this transition. One approach to
understanding how carcinoma cells acquire a motile, invasive phenotype
is to dissect the cellular alterations that occur to drive this complex
process. In this regard, previous work, including our own, has
established that the
6
4 integrin can promote carcinoma invasion
(12, 21, 64, 67). We determined that activation of PI3K
was not only essential but also sufficient to increase the invasive
potential of carcinoma cells, which emphasized the importance of
investigating further the mechanism of
6
4 activation of this
signaling pathway (64). We have now added to our
understanding of this "invasion pathway" by establishing that IRS
proteins IRS-1 and IRS-2 are upstream mediators in the activation of
PI3K by the
6
4 integrin. This is the first report of an
involvement of the IRS family in tumor invasion, and it establishes a
new area of research for these proteins, which have been studied
primarily for their role in metabolic regulation (84).
The role of the IRS proteins in
6
4-dependent promotion of tumor
progression adds to other studies that have demonstrated an involvement
of the IRS family in cancer. IRS-1 and IRS-2 are essential downstream
effectors of IGF-1R, which is frequently overexpressed in tumors and
which is a prognostic indicator of tumor recurrence and reduced patient
survival (39, 77). Although only a limited number of
studies have been performed to address directly the contribution of IRS
function to cancer, the data to date support an important role for
these proteins in tumor biology. For example, in breast cancer, an
essential role for IRS-1 in IGF-1-dependent breast carcinoma cell
survival and an involvement in IGF-1-dependent breast carcinoma cell
growth have been observed (50). IRS-1 expression is
regulated by estrogen, and the levels of IRS-1 are decreased in
response to antiestrogens such as tamoxifen and ICI 182,780 (25, 60). This regulation of IRS-1 expression has been
hypothesized to be a mechanism by which these antiestrogens inhibit
breast carcinoma growth. Finally, high levels of IRS-1 expression in
primary human breast cancers predict a greater incidence of recurrence
and a decreased patient survival rate (58). Increased
IRS-1 expression levels have also been observed in early stages of
hepatocellular carcinoma, and a dominant-negative IRS-1 protein can
reverse the malignant phenotype of transformed hepatocellular carcinoma
cell lines (49, 73). Finally, IRS-1 and IRS-2 are both
overexpressed in pancreatic cancer (36). It is intriguing
to speculate that the correlations of IRS expression with tumor
progression are related at least in part to the functions of the IRS
proteins downstream of the
6
4 integrin.
The identification of IRS-1 and IRS-2 as signaling intermediates for
the
6
4 integrin is significant because these proteins have the
potential to regulate multiple signaling pathways downstream of this
integrin receptor. IRS-1 and IRS-2 are cytoplasmic adapter proteins
that do not contain intrinsic kinase activity but rather function by
recruiting proteins to surface receptors, where they organize signaling
complexes (84). These proteins belong to the IRS family,
which includes IRS-1, IRS-2, IRS-3, and IRS-4. IRS-1 and IRS-2 are
expressed ubiquitously, whereas the IRS-3 and IRS-4 homologs are more
restricted in their localization (84). All of the IRS
family members have homology and contain multiple binding motifs that
are essential for their interaction with downstream effectors, which
can include PI3K, Grb-2, SHP-2, Nck, Fyn, phospholipase C-
, and Crk
(4, 40, 46, 47, 66, 68, 69). With their ability to recruit
such a variety of signaling molecules, the IRS proteins, not
surprisingly, have been implicated in numerous cellular functions
including mitogenesis, cell survival, gene transcription, and glucose
transport (84). With regard to the involvement of the IRS
proteins in
6
4-dependent signaling, we have identified PI3K as
one downstream effector that is recruited to these adapter proteins in
response to receptor ligation. The IRS-dependent activation of PI3K is
important for the ability of
6
4 to promote invasion, an essential
function of metastatic cells. Moreover, given the potential of the IRS
proteins to interact with many other signaling effectors, other
6
4-dependent signals may also be regulated through these adapter
proteins. For example, although we have observed MAPK activation in
response to
6
4 ligation in the MDA-MB-435/
4 transfectants, we
have not observed Shc phosphorylation, a proposed mechanism for MAPK
activation (16, 43). An alternative mechanism for MAPK
activation downstream of the
6
4 integrin could be IRS recruitment
of Grb2. In support of this, we have not observed MAPK
activation in the Y1494 mutant transfectants (data not shown).
Our demonstration that the
6
4 integrin is capable of stimulating
the phosphorylation of both IRS-1 and IRS-2 raises the question of
whether these homologs serve identical or distinct functions downstream
of this integrin receptor. Although IRS-1 and IRS-2 share overall
structural features and have some common effector binding sites, they
also have unique phosphorylation sites (84). Furthermore,
there are a number of reports that suggest that these homologs have
different functions. For example, overexpression of IRS-1, but not
IRS-2, in IRS-1 null fibroblasts restores IGF-1 stimulated cell cycle
progression to the level observed in normal fibroblasts
(8). In addition, IGF-1 stimulation of fetal brown
adipocytes results in the association of Grb-2 with IRS-1 but not with
IRS-2 (80). Differences in intracellular localization have
also been demonstrated for IRS-1 and IRS-2, and this may explain some
of the functional distinctions between these two homologs
(28). The most striking evidence for functional differences in IRS-1 and IRS-2 comes from the phenotype of the IRS-1
and IRS-2 knockout mice. IRS-1 null mice are stunted in their growth,
but they do not develop diabetes (1). In contrast, IRS-2
null mice develop insulin resistance in the liver and skeletal muscle
and progressively lose their ability to regulate glucose homeostasis
(85). The question of distinct IRS homolog function in
carcinoma cells is relevant because differences in the expression and
activity of IRS-1 and IRS-2 in tumor cells have been reported. For
example, IRS-1 and IRS-2 are predominantly expressed in estrogen receptor positive (ER+) and
ER
breast carcinoma cells, respectively
(29). If IRS-1 and IRS-2 activate distinct downstream
pathways, the function of the
6
4 receptor would depend on which
IRS homolog was activated in response to receptor ligation. Therefore,
although the
6
4 integrin can promote the phosphorylation of both
IRS-1 and IRS-2, it is intriguing to speculate that
6
4-dependent
signaling in carcinoma cells could be influenced by factors that
differentially regulate the expression of the IRS proteins.
One issue that arises from our identification of IRS-1 and IRS-2 as
downstream effectors of the
6
4 receptor is how the
phosphorylation of these adapters is regulated by this integrin
receptor. The IRS proteins were first discovered as signaling
intermediates of the insulin receptor (IR), and they bind to a
consensus sequence in the IR, where they are phosphorylated directly by
the intrinsic receptor kinase domain (84). Although the
IRS consensus binding site is also present in the IGF-1R and
interleukin-4 (IL-4) receptor, not all receptors that promote IRS
phosphorylation contain this binding motif, including the
6
4
integrin (15, 26, 32). In addition, none of these
receptors possess intrinsic kinase domains. Therefore, alternative
mechanisms for the recruitment and phosphorylation of the IRS proteins
are necessary. Several models for
6
4-dependent activation of the
IRS proteins could be proposed based on the mechanisms utilized by
other members of this group of receptors that lack the IRS binding
motif. One potential model involves the JAK family of tyrosine kinases,
which includes JAK1, JAK2, JAK3, and Tyk2 (42). Several
receptors, including those for alpha interferon, prolactin, growth
hormone, and cytokines IL-2, -4, -7, and -15, associate with members of the JAK family, and these kinases recruit and phosphorylate the IRS
proteins upon receptor stimulation (30, 53, 86). A second model involves the phosphorylation of the IRS proteins by members of
the src-kinase family (17). However, it is not clear how the IRS proteins are recruited to receptor complexes for this src-dependent phosphorylation to occur. Focal adhesion kinase (FAK) has
also been shown to associate with and promote phosphorylation of IRS-1
(38). However, the FAK-dependent phosphorylation may be
indirect due to recruitment of src to the protein complexes. Finally,
the possibility that the
6
4 receptor interacts with the IRS
proteins through an association with other surface receptors is also
valid. For example, the
v
3 integrin can recruit IRS-1 indirectly to receptor complexes through an interaction with the insulin receptor (61, 83). The elucidation of the
mechanism involved in the
6
4-dependent phosphorylation of the IRS
proteins will provide additional targets for the disruption of tumor invasion.
We have identified a single amino acid in the
4 cytoplasmic domain,
Y1494, which is involved in promoting the
6
4-IRS-PI3K invasion
pathway. Mutation of this tyrosine residue inhibited not only
6
4-dependent activation of PI3K but also the ability of this
receptor to promote carcinoma invasion. The fact that mutation of Y1494
had such a dramatic impact on
6
4-dependent signaling emphasizes
the likelihood that this residue is an essential binding site for
downstream effectors of the
6
4 receptor. We specifically selected
Y1257 and Y1494 for mutation based on the location of these tyrosines
within ITIM consensus binding motifs (79). The presence of
both of these tyrosines within ITIM motifs might suggest that these
residues have similar functions. However, it is clear that mutation of
Y1257 in the
4 subunit does not disrupt the signaling functions of
the
6
4 integrin, as was observed when Y1494 was mutated, even
though both of these tyrosines appear to be phosphorylated in response
to
6
4 ligation. These findings indicate that the two ITIM motifs
in the
4 subunit do not function equally and that the specific
sequences surrounding Y1257 and Y1494 must be important for regulating
6
4-dependent signals. ITIM motifs are known to be potential
binding sites for the SH2 domains of tyrosine phosphatases SHP-1 and
SHP-2 and lipid phosphatases SHIP-1 and SHIP-2 (79). SHP-1
and -2 phosphatases regulate tyrosine kinase signaling pathways,
whereas the SHIP-1 and -2 phosphatases regulate the PI3K signaling
pathway (reviewed in references 59 and 65). Disruption of
either pathway could produce the phenotype we observed with the mutant
receptors. For example, the phosphorylation of the
4 subunit is
tightly regulated by tyrosine phosphatases, and this supports the
potential involvement of SHP-2 (Fig. 5). The fact that IRS-2 is not
phosphorylated in response to ligation of the Y1494 mutant receptor
indicates that this tyrosine could be involved in the recruitment of
IRS-2 to the receptor. However, it is also possible that Y1494 is
essential for recruiting the kinase that regulates the phosphorylation
of IRS-2 or other essential binding sites in the
4 subunit.
In summary, we have identified a novel mechanism for the activation of
PI3K by the
6
4 integrin that involves IRS-1 and IRS-2 and
requires Y1494 in the
4 cytoplasmic domain. Activation of this
6
4-IRS-PI3K signaling pathway promotes carcinoma invasion.
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ACKNOWLEDGMENTS |
|---|
I thank Yumiko Honzako for excellent technical assistance.
This work was supported by NIH grant CA81325 and U.S. Army grant DAMD17-97-1-7313.
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FOOTNOTES |
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* Mailing address: Department of Pathology, Beth Israel Deaconess Medical Center-RN227, 330 Brookline Ave., Boston, MA 02215. Phone: (617) 667-1430. Fax: (617) 667-3616. E-mail: lshaw{at}caregroup.harvard.edu.