From the Laboratoire d'Etude de la Differenciation
et de l'Adhérence Cellulaires, Unité Mixte de
Recherche UJF/CNRS 5538, Institut Albert Bonniot, Faculte de
Médecine de Grenoble, La Tronche F38706 cedex, France, the
Department of Genetics, Biology, and Biochemistry, University of
Torino, Torino 10126, Italy, and the § Department of
Molecular Medicine, Max Planck Institute for Biochemistry, Am
Klopferspitz 18A, Martinsried D-82152, Germany
Received for publication, November 4, 2002
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ABSTRACT |
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Regulation of integrin affinity and clustering
plays a key role in the control of cell adhesion and migration. The
protein ICAP-1 Interactions of cells with the extracellular matrix are essential
for survival, differentiation, and proliferation of cells (1). They are
mainly mediated by type I On the other hand, it has been reported that intracellular calcium
plays a key role in cell adhesion (8). Calcium-dependent cycles between high and low affinity states of integrins seem to be
crucial for cell migration (9-12). More recently, we found that the
affinity state of the Integrin cytoplasmic domain-associated protein-1 In this report we show that ICAP-1 Antibodies--
The anti- Cells and Cell Culture--
The murine NIH3T3, the hamster CHO,
and the human HeLa cell lines were grown in Protein Purifications--
ICAP-1
The polypeptide corresponding to the Transfection in Mammalian Cells and Selection of Stable
Clones--
Full-length human ICAP-1 Immunofluorescence Microscopy--
Immunofluorescence was
carried out using standard procedures. Stained cells were analyzed with
an inverted fluorescence microscope (Olympus Provis AX70) equipped with
a Plan Apo ×63 oil immersion, numerical aperture 1.40 objective lens.
For all double-staining experiments, the appropriate controls were
performed to ensure that no undesired cross-reactivity occurred between
the primary and secondary antibodies.
Purification of Ventral Plasma Membranes--
The purification
of HeLa, GD25- Solid-phase Assays--
The interaction between ICAP-1 Microinjection into NIH3T3 Cells--
NIH3T3 cells were seeded
onto fibronectin-coated glass coverslips overnight at 37 °C. All
injections were carried out with the aid of a micromanipulator 5171 connected to an Eppendorf microinjector unit (Transjector 5246). The
cells were microinjected with PBS containing a final concentration of 1 mg/ml of the freshly purified recombinant ICAP-1 ICAP-1
To have direct access to focal adhesion proteins, ventral plasma
membranes were obtained from HeLa cells grown overnight on fibronectin.
Double immunostaining was performed with an anti-vinculin antibody and
anti-ICAP-1 Interaction of ICAP-1
Next, we tested whether ICAP-1
Finally, we expressed ICAP-1 ICAP-1
Finally, disruption of focal adhesions by ICAP-1 Disruption of Focal Adhesions by ICAP-1
ICAP-1 Talin and ICAP-1 We examined the cellular localization of the endogenous ICAP-1 Even though ICAP-1 Several reports have shown that talin is crucial for the formation of
focal adhesions (27, 28, 40). A simple explanation for the negative
effect of ICAP-1 The distribution of ICAP-1 Recently, a 20-kDa protein named TAP-20 (with marked homology with
(integrin cytoplasmic domain-associated protein-1
)
binds to the cytoplasmic domain of the
1A
integrin and controls cell spreading on fibronectin. Here, we
demonstrate that, despite its ability to interact with
1A integrin, ICAP-1
is not recruited in focal
adhesions, whereas it is colocalized with the integrin at the ruffling
edges of the cells. ICAP-1
induced a rapid disruption of focal
adhesions, which may result from the ability of ICAP-1
to inhibit
the association of
1A integrin with talin, which is crucial for the assembly of these structures. ICAP-1
-mediated dispersion of
1A integrins is not observed with
1D integrins that do not bind ICAP. This strongly
suggests that ICAP-1
action depends on a direct interaction between
ICAP-1
and the cytoplasmic domain of the
1 chains.
Altogether, these results suggest that ICAP-1
plays a key role in
cell adhesion by acting as a negative regulator of
1
integrin avidity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimer transmembrane receptors
named integrins (2). Integrin-mediated cell adhesion is a highly
controlled process that can be modulated very rapidly by two
mechanisms: the modulation of the receptor affinity by a conformational
change and the modulation of receptor avidity by lateral diffusion and
clustering into highly ordered structures named focal adhesions. As
shown for the platelet integrin
IIb
3, the
effects of integrin clustering and affinity modulation are additive and
seem to play complementary roles (3). The conformational change that
modulates the affinity of some integrins is mediated by monomeric G
proteins of the Ras family. R-Ras seems to prevent H-Ras-dependent decrease in integrin affinity (4-6).
However, proteins involved in this signaling pathway are still largely unknown (6, 7).
5
1 integrin in
CHO1 cells may be switched by
the balance between two antagonistic enzymatic activities: calcineurin
and calcium/calmodulin-dependent protein kinase of type II
(CaMKII) (13, 14). A CaMKII-dependent inside-out signaling
was also described as the molecular basis of the cross-talk between
v
3 and
5
1
(15). Although this regulatory pathway remains to be unraveled,
calcineurin has been shown to control
v
3
and
5
1 integrin affinity in neutrophils and CHO cells, respectively (16, 17). Finally a complex between
1 integrin and CaMKII was observed in breast cancer
MCF-7 cells (18). Although the regulation of integrin function may
involve phosphorylation events on the threonine doublet TT788-789 of
the
1A chain (19) or on the threonine triplet
TTT758-760 of the
2 chain (20), these phosphorylation
sites do not seem to be directly linked to the
CaMKII-dependent control of integrin affinity. Therefore,
it is likely that this latter signaling pathway occurs via an
intermediate regulatory protein. This hypothesis was further supported
by the fact that ectopically expressed
cytoplasmic domains have a
dominant negative effect on integrin function, suggesting that some
control proteins are titrated by the overexpression of
cytoplasmic
tails (21, 22).
(ICAP-1
) was
identified in a yeast two-hybrid screen as a protein specifically associated with the cytoplasmic domain of
1A integrins
(23). This protein has two isoforms named
and
of 200 and 150 amino acids, respectively. ICAP-1 is expressed throughout development and also in adult tissues (24). ICAP-1
but not ICAP-1
interacts with the cytoplasmic tail of the
1A chain in a manner
that depends on the conserved NPXY integrin motif
(25). ICAP-1
contains a number of putative phosphorylation sites,
including a phosphorylation motif for the CaMKII around threonine 38. We could show that a point mutation T38D (that mimics the
phosphorylated form) or T38A (which cannot be phosphorylated) in
ICAP-1
and expression of the corresponding recombinant proteins
reduced or increased cell spreading on fibronectin, respectively. These
data suggest that phosphorylation of ICAP-1
on threonine 38 by
CaMKII modulates
5
1 integrin function
(13). A further involvement of ICAP-1
in the regulation of
1 integrin function was suggested by experiments indicating that its overexpression increases cell motility on a
1-dependent substrate such as fibronectin
(26).
, despite its ability to interact
directly and specifically with the
1 integrin
cytoplasmic domain in vitro, was never observed in focal
adhesions. In addition, ICAP-1
could inhibit the interaction between
talin and the
1 cytoplasmic tail in vitro.
Because talin recruitment is a prerequisite for focal adhesion assembly
(27, 28), we have analyzed the effect of ICAP-1
on the organization
of these structures and showed that this protein was able to
disorganize focal adhesions in a manner dependent on its direct
interaction with the
1 cytoplasmic tail. These results
strongly suggest that ICAP-1
is a key regulator of cell adhesion
mediated through
1 integrin and focal adhesion dynamic
by weakening talin binding to the
1 integrin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 tail serum (anti
cyto-
1) was raised against a synthetic peptide
corresponding to the cytoplasmic domain of the
1 chain
covalently coupled to keyhole limpet hemocyanin. Anti-talin monoclonal
antibody (8d4) was purchased from Sigma (St. Louis, MO). The monoclonal
antibody 9EG7 directed against the
1 subunit was kindly
supplied by Dr. D. Vestweber (Muenster, Germany). The monoclonal
antibody 7E2 directed against the hamster
1 subunit was
a generous gift of Dr. R. Juliano (Chapel Hill, NC). Polyclonal antibody directed against the human ICAP-1
protein was previously described (13). Cyanin3-, Alexa-, or rhodamine-conjugated goat anti-mouse or anti-rabbit from Molecular Probes (Eugene, OR) or Immunotech (Marseille, France) were used as secondary antibodies.
-minimal essential
medium supplemented with 10% fetal calf serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin. The murine GD25,
GD25-
1A, and GD25-
1D were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
GD25 cells do not express the
1 integrin chain due to a
null mutation in both alleles (29). GD25 cells transfected with either
the murine
1A, or the human
1A and
1D full-length cDNA are called
GD25-
1A and GD-
1D, respectively, and have
been described earlier (30, 31). All transfected cells were grown in
complete medium supplemented with the appropriate antibiotics for the
selection of the transfected cells.
and ICAP-1
fragments
fused to a polyhistidine tag at the N-terminal position were purified
from the BL21(DE3) Escherichia coli strain containing the
vector pET19b-ICAP-1
. Briefly, human ICAP-1
cDNA cloned in
pBluescript was used as a template in a PCR reaction using primers with
an XhoI site in the 5' position. In the sense primer the
XhoI site is in-frame with the first methionine of
ICAP-1
. Then the XhoI-digested PCR product was cloned
into the XhoI site of pET-19b vector (Novagen). Fragments
were obtained by insertion of stop codons at different positions using
the QuikChange mutagenesis kit (Stratagene). All constructs used in
this study have been sequenced by the Eurogentec direct sequencing
department (Belgium). Purification was carried out using the
nickel-charged resin nickel-nitrilotriacetic acid from Qiagen.
Inclusion bodies were solubilized in urea. Protein refolding was
performed directly on the column by progressive removal of the
chaotropic agent. The purity of the protein was checked by SDS-PAGE and
Coomassie Blue staining and was greater than 90-95%. All experiments
were carried out with freshly purified proteins. Before each
experiment, the capacity of each batch of the purified protein to
interact with the
1 cytoplasmic domain was estimated in
a solid-phase assay.
1
integrin cytoplasmic domain was produced from the BLR(DE3)pLysS
E. coli strain containing the vector pET19b-cyto
1. This
construct allows the production of the fragment 752-798 of the
1 integrin cytoplasmic domain. This peptide was
recognized by a polyclonal antibody raised against a synthetic
1 cytoplasmic peptide coupled to keyhole limpet
hemocyanin. Talin and
-actinin were purified as previously
described (32), and fibronectin was purified according to a previous
study (33).
was excised from the
pBS-ICAP-1
vector as an EcoRI/XbaI fragment
and inserted into the pcDNA3.1(+) vector (Invitrogen, The
Netherlands). Stable GD25-
1A cell lines expressing
ICAP-1
were obtained by electroporation of 4 × 106
cells in 400 µl of PBS at 280 V with 15 µg of
pcDNA3.1(+)-ICAP-1
vector. Transfected cells were selected in
complete medium with Zeocin (Invitrogen, The Netherlands) at a final
concentration of 300 µg/ml. The expression of ICAP-1
was monitored
by indirect immunofluorescence and Western blot analysis using the
ICAP-1
polyclonal antibodies.
1, or NIH3T3 ventral plasma membranes was
performed as previously described by Cattelino et al. (34).
The cells were grown overnight on fibronectin-coated coverslips in
complete medium. After two washes in PBS, the cells were incubated with
cold water for 2 min and then flushed with a 1000-µl tip. Cell
disruption was confirmed by microscopy. Ventral plasma membranes were
either immediately fixed with paraformaldehyde or were preincubated for
30 min at 4 °C with ICAP-1
or ICAP-1
fragments at the
concentration of 5 µM in a VPM buffer containing 125 mM potassium acetate, 2.5 mM MgCl2,
12 mM glucose, and 25 mM HEPES, pH 7.5, prior
to fixation.
and
the cyto-
1 peptide or the whole
5
1 integrin was carried out using a
solid-phase assay. Briefly, a 96-well tray (MaxiSorp, Nunc) was coated
with the whole ICAP-1
protein or ICAP-1
fragments for 16 h
at 4 °C and blocked with a 3% BSA/PBS solution for 1 h at room
temperature. A Triton X-100 CHO cell lysate made in PBS supplemented
with 1% Triton X-100 (w:v) or the cyto-
1 peptide were
incubated for 1 h at 37 °C. After three washes in PBS
containing 3% BSA and 0.01% Tween-20, detection of the
5
1 integrin from the CHO cell lysate was
performed using the 7E2 monoclonal antibody, whereas the detection of
the cyto-
1 peptide was achieved with a polyclonal
antibody directed against a synthetic peptide corresponding to the
1 tail.
protein, or the
N-terminal (1-100) or C-terminal (101-200) fragments, in the presence
of 100 µM tetramethylrhodamine-dextran amine
(Mr 3000, Molecular Probes, Interchim,
France) to view the injected cells. Three hours (whole ICAP-1
protein) or 30 min (ICAP-1
fragments) after microinjection, the
cells were fixed with 3% paraformaldehyde and 2% sucrose in PBS for
10 min at 37 °C and then immunostained for vinculin localization.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Does Not Localize in Focal Adhesions--
The protein
ICAP-1
was isolated as a
1A-interacting protein in a
yeast two-hybrid screen (23) and was shown to modulate CHO cell
adhesion (13) and to promote cell motility (26). In epithelial cells or
in cell lines derived from epithelial cells such as HeLa, ICAP-1
could be detected in a cell lysate by Western blot using a polyclonal
antibody raised against the full-length recombinant protein (Fig.
1A). The endogenous human
ICAP-1
protein in HeLa cells migrates in SDS-PAGE like the
ectopically expressed protein in rodent fibroblast-like GD25 cells
(Fig. 1B). GD25, CHO, and NIH3T3 cells showed no detectable
ICAP-1
expression as monitored by Western blot analysis. To
determine the physiological relevance of the interaction between
ICAP-1
and the
1 integrin, we carried out
immunomicroscopy experiments of ICAP-1
in different cell lines. In
HeLa cells, ICAP-1
showed a diffuse expression pattern and often
some nuclear localization (Fig. 1C, panel a). Surprisingly, no accumulation of ICAP-1
was observed in focal adhesions visualized by vinculin staining (Fig. 1C,
panels a-c). Similarly, we reported previously that in the
Hs68 cell line, ICAP-1
and
1 colocalize in ruffles
but not in focal adhesions (35). A direct competition of endogenous
ICAP-1
with the purified recombinant protein revealed a dramatic
decrease in ICAP-1
immunostaining and confirmed the specificity of
the immunolabeling (Fig. 1C, panels d-f).
Despite the diffuse ICAP-1
localization, these cells were able to
form well-organized focal adhesions connected to stress fibers as
judged by double labeling using a monoclonal antibody directed against
vinculin and phalloidin-rhodamine-stained stress fibers (not
shown).
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Fig. 1.
Antibodies characterization and
cellular localization of the protein
ICAP-1 . A, the proteins
of a HeLa cell lysate in radioimmune precipitation assay buffer were
resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride
membrane. The protein ICAP-1
was detected with polyclonal
antibodies. B, Western blots of ICAP-1
protein in NIH3T3
cells, HeLa cells, CHO cells, GD-25 cells, and GD-25 cells transfected
with ICAP-1
cDNA. C, HeLa cells were cultured
overnight on fibronectin, fixed, permeabilized, and processed for
double immunofluorescence labeling. In a, HeLa cells are
stained using polyclonal antibodies directed against ICAP-1
. In
d, HeLa cells are stained with the same polyclonal
antibodies directed against ICAP-1
, which has been incubated with
the recombinant ICAP-1
protein to compete with the
ICAP-1
-specific labeling. In b and e, HeLa
cells are stained using a monoclonal antibody directed against
vinculin. In c and f is shown the merged images
of a with b and d with e,
respectively. D, ventral plasma membranes (VPM)
from HeLa cells were isolated, and double labeling of ICAP-1
(a) and vinculin (b) was carried out with
specific primary antibodies. These results are representative of three
independent experiments. Bar, 10 µm.
polyclonal antibodies. In these membrane preparations,
focal adhesions could be viewed by vinculin staining (Fig.
1D, panel b) or by talin or
1
staining (not shown), whereas anti-ICAP-1
antibodies showed a faint
background staining that was barely detectable (Fig. 1D,
panel a). Altogether these results suggest that ICAP-1
is
not present in focal adhesions.
with the
5
1
Integrin--
The absence of ICAP-1
in focal adhesions prompted us
to study the interaction of ICAP-1
with
1 integrins
in more detail. ICAP-1
and the
1 cytoplasmic domains
were expressed in bacteria as polyhistidine fusion proteins. Fig.
2A shows that the purified ICAP-1
protein interacted specifically with the purified
1 cytoplasmic domain in a solid-phase assay, which is
consistent with previous reports (23, 26). As a control, we used a
1 cytoplasmic domain bearing the point mutation Y to S
in the NPXY membrane distal (cyto3) domain. In full
agreement with a previous report (23), this mutation abolished the
interaction between ICAP-1
and the
1 cytoplasmic tail
(Fig. 2A).
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Fig. 2.
ICAP-1
interacts specifically and directly with
1 integrins. A, the capacity
of ICAP-1
to interact with a peptide corresponding to the
1 integrin cytoplasmic domain was checked in a
solid-phase binding assay. A constant amount of purified recombinant
ICAP-1
(10 µg/well) or BSA (3% w:v) was used to coat a 96-well
tray overnight at 4 °C. After a blocking step, increasing amounts of
the wild type
1-cyto peptide or YS
1-cyto
mutant were added into the wells and detected with a specific
polyclonal antibodies. Each experimental point was obtained from
triplicate experiments, and background values of BSA coating have been
subtracted. These results are representative of three independent
experiments using different preparations of the purified ICAP-1
protein and cyto-
1 peptides. B, increasing
amounts of the recombinant ICAP-1
protein were used to coat plastic
wells of a 96-well tray. Subsequently, a constant amount (300 µg/well) of a CHO cell lysate was added. The
1
integrin receptors bound to ICAP-1
were detected using the
non-blocking monoclonal antibody 7E2 (raised against the hamster
1 chain). The results from three independent experiments
using different preparations of the purified ICAP-1
were averaged,
and standard deviations are shown. C, polyhistidine-tagged
ICAP-1
fragments were used in the
1 binding assay
described above. The wells were coated with 10 µg of the ICAP-1
recombinant fragments and then incubated with 300 µg of CHO cell lysate proteins. The bound
5
1
was immunodetected by the 7E2 anti-hamster
1 monoclonal
antibody. Each histogram represents mean ± S.D. of three
independent experiments.
was able to interact with the whole
5
1 integrin from a CHO cell lysate. This
was crucial, because beta subunits do not exist in isolation in cells,
and therefore, two hybrid experiments with integrins may be prone to
artifacts. Increasing amounts of the recombinant ICAP-1
protein were
used to coat 96-well trays. The protein concentration during coating
was maintained constant by adding BSA. An equal amount of a CHO cell
lysate in Triton X-100 was subsequently incubated in each coated well.
A dose-dependent and -specific binding of the
1 integrin was detected by a specific antibody (Fig.
2B). These data indicate that ICAP-1
expressed in
bacteria is able to interact with the
1A cytoplasmic
domain, and that the cytoplasmic domain of the
subunit did not
impair the interaction with ICAP-1
.
fragments in bacteria and used them in
a solid-phase binding assay to map the
1 binding site. Only the C-terminal moiety (amino acids 100-200) of the protein was
able to bind to the
1 integrin (Fig. 2C). But
neither the fragment corresponding to amino acids 1-150 nor the
fragment corresponding to amino acids 151-200 of ICAP-1
were found
to interact strongly with the
5
1 integrin
from cell lysate (Fig. 2C).
Disorganizes Focal Adhesions ex Vivo--
Despite its
specific and direct association with the
1 integrin,
ICAP-1
was not localized in focal adhesions. One possible explanation for these contradictory results could be that ICAP-1
might act as a negative regulator of the recruitment of focal adhesion
components. To investigate this possibility we microinjected ICAP-1
recombinant protein into the cytoplasm of NIH3T3 cells and monitored
focal adhesion organization by staining for vinculin. Although
microinjection of dextran-coupled rhodamine alone had no significant
effect on the localization of vinculin (Fig.
3, A-C), talin, and
-actinin (not shown), microinjection of the full-length ICAP-1
in
the dextran-coupled rhodamine buffer induced a rapid delocalization of
vinculin (Fig. 3, D-F) or talin and
-actinin (not shown)
observed in 70% of the cells. Microinjection of the C-terminal moiety
of ICAP-1
(amino acids 101-200) that encompasses the
1 binding site had similar effects (Fig. 3,
J-L) in 77% of the injected cells. Because the N-terminal
fragment (amino acids 1-100) does not bind the
1
integrin domain (Fig. 2C), we made use of this recombinant
fragment as a control. Indeed, the microinjection of this part of
ICAP-1
did not interfere with vinculin staining (Fig. 3,
G-I).
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Fig. 3.
Microinjection of purified
ICAP-1 causes focal adhesion disassembly.
NIH3T3 cells were seeded onto fibronectin-coated coverslips and allowed
to spread overnight at 37 °C. Then a PBS solution of
dextran-rhodamine alone (A-C) or supplemented with the
purified recombinant ICAP-1
protein at 1 mg/ml
(D-F), ICAP-1
1-100 fragment
(G-I), or ICAP-1
100-200 fragment (J-L),
was microinjected into the cells. After microinjection, the cells were
fixed, permeabilized as described under "Experimental Procedures"
and immunostained for vinculin. These panels are representative of four
independent experiments using different preparations of purified
recombinant ICAP-1
protein and fragments.
was also
investigated in a cellular context after stable transfection into GD25-
1A cells of a vector containing human ICAP-1
cDNA. This cell line expresses functional
1
integrins at the cell surface (19) that can be monitored by the 9EG7
monoclonal antibody, which recognizes a ligand-induced binding
site epitope correlating with the occupied conformational state of
1 integrins (36, 37). Under our experimental conditions,
immunofluorescence microscopy did not reveal any detectable staining
for endogenous ICAP-1
in GD25-
1A cells (Fig.
4A). On the other hand, these
cells exhibited surface expression of
1A integrins
confined to focal adhesions that could be monitored by the 9EG7
antibody (Fig. 4B). In a non-clonal population of
GD25-
1A cells transfected with a cDNA encoding the
human ICAP-1
, a positive immunofluorescence signal for ICAP-1
was
diffusely present within the cytoplasm (Fig. 4C).
Simultaneously, a diminution of cell spreading and loss of 9EG7
monoclonal antibody staining was observed, suggesting that
1 integrins were no longer occupied and involved in
focal adhesions (Fig. 4D).
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Fig. 4.
ICAP-1 expression
disrupts
1 integrin-containing
focal adhesions. GD25-
1A cells were stably
transfected either with vector alone (A and B) or
with a cDNA coding for the full-length ICAP-1
protein
(C and D). Transfected cells were spread
overnight at 37 °C on fibronectin-coated coverslips. The expression
of ICAP-1
was visualized with polyclonal antibodies (A
and C) and the high affinity conformational state of the
1 integrin with the 9EG7 monoclonal antibody
(B and D). Note that the reduction of 9EG7
staining correlated with the expression of ICAP-1
. Bar,
10 µm.
Requires Direct
Interaction with the
1 Integrin Chain--
The action
of ICAP-1
on focal adhesions might be indirect, for instance due to
the interference with some regulatory pathways. Therefore, the purified
recombinant ICAP-1
was also tested for its ability to disassemble
focal adhesions in vitro in a cytosol-free ventral plasma
membrane preparation (VPM). These preparations are depleted in
nucleotide triphosphate and soluble signaling enzymes. The cell
membranes were incubated for 30 min at 4 °C with a solution of
purified ICAP-1
in acetate buffer and glucose. Although buffer alone
did not interfere with the detection of focal adhesion proteins such as
vinculin (Fig. 5A), the
incubation with ICAP-1
efficiently displaced vinculin from focal
adhesions (Fig. 5B). A similar result was also observed for
talin and
-actinin (not shown). The same result was obtained by the
incubation of the C-terminal part (amino acids 101-200) of ICAP-1
(Fig. 5D). Finally, incubation of these ventral membranes
with the N-terminal purified fragment (amino acids 1-100) had no
effect on focal adhesion organization (Fig. 5C).
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Fig. 5.
Purified ICAP-1
disrupts focal adhesions in vitro. Ventral plasma
membranes from NIH3T3 cells were prepared as described under
"Experimental Procedures." The membranes were incubated at 4 °C
for 30 min in the absence (A) or in the presence
(B) of purified recombinant ICAP-1
(5 µM).
Alternatively, the purified N-terminal moiety of ICAP-1
(amino acids
1-100) shown in C or the C-terminal moiety of ICAP-1
(amino acids 101-200) shown in D were added at a
concentration of 5 µM. The membranes were subsequently
fixed and stained for vinculin. Note the dramatic reduction of vinculin
staining upon the addition of recombinant ICAP-1
or the C-terminal
domain (B and D). Photographs were taken with
identical exposure times. These observations are representative of four
independent experiments using different preparations of purified
recombinant ICAP-1
. Bar, 10 µm.
was suggested to have a GDP dissociating inhibitor
activity for Rac and Cdc42 (38), two monomeric G proteins of the Rho
family involved in the regulation of cytoskeleton organization. This
activity might account for ICAP-1
destabilizing action on focal
adhesions of ventral plasma membranes. To assess whether ICAP-1
action on focal adhesions was due to its direct binding on
1 integrin chains or to some interference with Rho
signaling pathways, we performed similar experiment on VPM from
GD-25
1A and GD-25
1D cells lines. The
1D and
1A isoforms are functionally similar with regard to integrin-mediated signaling (39), but the former
strongly binds talin (31) and does not bind ICAP-1
(38). Upon
addition of the ICAP-1
fragment 100-200, the dispersion of
1A integrins initially clustered into focal adhesions
was observed (Fig. 6, A and
C), whereas
1D-containing focal adhesions remained unaffected (Fig. 6, B and D). This
result strongly suggests that a direct interaction between ICAP-1
and the
1 chain is a prerequisite for focal adhesion
disassembly.
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Fig. 6.
ICAP-1 integrin
binding domain displaces
1A but
not
1D integrins from focal
adhesions. Ventral plasma membranes from GD25-
1A
(A and C) and GD25-
1D
(B and D) cells were prepared as described under
"Experimental Procedures." The membranes were incubated at 4 °C
for 30 min in the absence (A and B) or in the
presence (C and D) of the C-terminal moiety of
ICAP-1
(amino acids 101-200) added at a concentration of 5 µM. The membranes were subsequently fixed and stained for
1 integrins using the monoclonal antibody 4B7R.
Photographs were taken with identical exposure times. These
observations are representative of four independent experiments using
different preparations of purified recombinant ICAP-1
.
Bar, 10 µm.
Compete for Binding on the Cytosolic Domain of
the
1 Integrin Chain--
Because talin interacts
directly with the
1 integrin cytoplasmic domain and is
crucial for focal adhesion assembly, one attractive hypothesis is that
ICAP-1
is involved in the control of talin-integrin interaction.
Therefore, we tested whether ICAP-1
could modulate the binding of
talin to the integrin
1 cytoplasmic domain. In an
in vitro solid-phase assay, ICAP-1
could inhibit talin
binding to the cytoplasmic tail of the
1A chain in a
dose-dependent manner (Fig.
7A). These data suggest that
the displacement of talin from its binding site on
1A
may be sufficient for focal adhesion disruption and, consequently, for
a decrease in the integrin avidity. Moreover, the competition of
ICAP-1
and talin for the binding to
1 was specific,
because it could not be observed either with
-actinin, another
1 interacting protein (Fig. 7B), or with the
1-100 ICAP-1
moiety (Fig. 7C).
View larger version (18K):
[in a new window]
Fig. 7.
ICAP-1 competes with
talin but not with
-actinin binding to
the
1 cytoplasmic domain.
A, increasing amounts of purified recombinant ICAP-1
were
preincubated with 1 µg of the cyto-
1 peptide and then
incubated in a 96-well tray coated with equal amounts (10 µg/well) of
talin purified from human platelets. The binding of the
cyto-
1 peptide to talin was detected by polyclonal
antibodies raised against the cytoplasmic domain of the
1 integrin chain and a biotin-conjugated anti rabbit
secondary antibody. B, an amount of 2 µg of the
recombinant protein ICAP-1
was preincubated with 1 µg of the
cyto-
1 peptide and incubated in 96-well plastic trays
coated with 10 µg of purified talin (from human platelets) or
-actinin (from chicken gizzard). The binding of the
cyto-
1 peptide to talin or
-actinin was detected by
polyclonal antibodies raised against the cytoplasmic domain of the
1 integrin chain and a biotin-conjugated anti-rabbit
secondary antibody. C, a concentration of 1.5 µg of
ICAP-1
fragments 1-100 and 101-200 was preincubated with 1 µg of
the cyto-
1 peptide and incubated in 96-well plastic
trays coated with 10 µl of purified talin. The binding of the
cyto-
1 peptide to talin was detected by polyclonal
antibodies raised against the cytoplasmic domain of the
1 integrin chain and a biotin-conjugated anti rabbit
secondary antibody. Each experiment was performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein. Surprisingly, this protein was never detected in focal
adhesions, but instead, exhibited a diffuse pattern within the cell,
although a significant amount of the protein was associated within the
Triton X-100-insoluble fraction (not shown) and often, a nuclear
staining was observed. Using purified ventral membrane preparation from
HeLa cells, we never observed ICAP-1
colocalized with vinculin or
talin, which were used as markers of focal adhesions.
was not detected in focal adhesions, the purified
recombinant protein interacted strongly with the cytoplasmic domain of
the
1A integrin chain as reported previously (23, 26).
Additionally, this interaction also occurred with the whole integrin
receptors purified from a cell lysate. The strong binding of ICAP-1
to the cytoplasmic domain of the
1 integrin and its complete absence from focal adhesions suggested that this interaction may disrupt focal adhesion structures. To confirm this hypothesis we
microinjected ICAP-1
in NIH3T3 cells, and we indeed observed a rapid
disorganization of focal adhesions. In addition, recombinant ICAP-1
was able to disaggregate focal adhesions when added to purified ventral
plasma membranes from NIH3T3 and GD-25
1A cells. Conversely, the
1D-containing integrins were resistant
to ICAP-1
. This latter experiment strongly suggests that the
disassembly of focal adhesions is due to a direct interaction with the
1A integrin subunit and is independent of a cellular
signaling pathway. Furthermore, the focal adhesion disruption mediated
by ICAP-1
is in good correlation with our previous data, which have
shown that ectopic expression of ICAP-1
-regulated CHO cell spreading (13).
on focal adhesion structure could be its ability to
disrupt the direct association between the integrin and talin. To
investigate this hypothesis we performed an in vitro assay
and found that talin and ICAP-1
compete for binding to the
1A cytoplasmic domain. On the other hand, we found that
the interaction between
-actinin and the
1 integrin
is not inhibited by the presence of ICAP-1
. This shows that
ICAP-1
inhibits the interaction between
1A integrins
and talin in a specific manner and confirms previous reports showing
that the interaction of
-actinin with the
1
cytoplasmic domain is not sufficient to stabilize focal adhesion sites
(40). The lack of effect of ICAP-1
on
1D localization
suggests that, under our experimental conditions, this action is direct
and not dependent on the GDP dissociating inhibitor activity
recently suggested (38). Based on these findings we propose that
ICAP-1
and talin compete for integrin
1A binding and
thereby modulate focal adhesion assembly and/or dynamic. How ICAP-1
interferes with talin binding on the
1 integrin needs
further investigation. The talin binding site is not unambiguously
defined. Recent reports have demonstrated that the talin N-terminal
head binds to the
3,
1A, and
1D cytoplasmic domains (41, 42). Some data indicated
that the binding site of the talin head could be located on the
proximal membrane region of the integrin
chain (41). Conversely,
other reports indicate that a phosphotyrosine binding-like
subdomain of the FERM domain of talin head is the major
binding site that triggers the activation of the
IIb
3 integrin (43). This finding is very
interesting, because it offers some molecular basis of ICAP-1
and
talin competition. Indeed, sequence homology and molecular modeling
favor the view that ICAP-1
is a phosphotyrosine binding
domain protein. It was suggested that the interaction specificity with
the
1A cytosolic tail was due to the interaction of
Val-787 on the integrin and an hydrophobic pocket created by Leu-82 and
Tyr-144 of ICAP-1
(25). This is fairly consistent with the lack of
interaction of ICAP-1
with the
1D isoform that do not
have a valine at this position. This latter residue is very close to
the tyrosine 783 on the human
1A chain. The tyrosine at
this position on the
1 chain or on the homologous
position 747 on the
3 chain seems to be crucial for
integrin conformational switch and talin head binding. Moreover, talin
C-terminal rod domain contains another binding site located within the
residues 1984-2541 (44). Because the talin-active form is an
anti-parallel homodimer (32, 45), the head and tail integrin binding
sites in the adjacent talin molecules would be in close proximity with
each other. Therefore, it is likely that talin and ICAP-1
binding
sites on the integrin
1A tail overlap.
in ruffles and its absence from focal
adhesions suggest that the interaction between ICAP-1
and the
1 integrin cytoplasmic domain is regulated. It is
possible that ICAP-1
is sequestered inside the cell and that the
interaction between a sequestering protein and ICAP-1
may be the
regulated event. Alternatively, the interaction of ICAP-1
with the
cytoplasmic domain of the
1 integrin may be modulated by
post-translational modifications (like phosphorylation). Indeed we have
previously shown that a point mutation into the CaMKII putative
phosphorylation site dramatically affected cell spreading (13).
Moreover, pull-down assays showed that only a small fraction of
ICAP-1
was able to interact with
1A (26). How the
interaction of ICAP-1
and the integrin is regulated is not yet
understood and requires further investigations.
3-endonexin) was shown to interact specifically with the
5 cytosolic domain of the
v
5 integrin (46). Overexpression of this
protein leads to decreased adhesion and focal adhesion formation, and
enhances migration. These properties are quite reminiscent of those of
ICAP-1
, suggesting that a family of negative regulators may control
specific integrin classes in a similar fashion.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Frank Gertler for suggestions and critical reading of the manuscript and Dr. R. Juliano and Dr. D. Vestweber for kindly providing monoclonal antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Fédération Nationale des Ligues Contre le Cancer, the CNRS (Program Biologie Cellulaire: du Normal au Pathologique), and the Association pour la Recherche contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a fellowship from the Ministère de la Recherche and the Association pour la Recherche contre le Cancer and presently supported by a Marie-Curie fellowship.
** To whom correspondence should be addressed. Tel.: 33-476-54-95-70; Fax: 33-476-54-94-25; E-mail: marc.block@ujf-grenoble.fr.
Published, JBC Papers in Press, December 7, 2002, DOI 10.1074/jbc.M211258200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CHO, Chinese hamster
ovary;
CaMKII, calmodulin-dependent protein kinase of type
II;
ICAP-1, integrin cytoplasmic domain-associated protein-1
;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
VPM, ventral
plasma membrane.
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