Disruption of Focal Adhesions by Integrin Cytoplasmic Domain-associated Protein-1alpha *

Daniel BouvardDagger §, Lucile VignoudDagger , Sandra Dupé-ManetDagger , Nadia AbedDagger , Henri-Noël FournierDagger , Carole Vincent-MonegatDagger , Saverio Francesco Retta||, Reinhard Fässler§, and Marc R. BlockDagger **

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-1alpha (integrin cytoplasmic domain-associated protein-1alpha ) binds to the cytoplasmic domain of the beta 1A integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with beta 1A integrin, ICAP-1alpha is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1alpha induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1alpha to inhibit the association of beta 1A integrin with talin, which is crucial for the assembly of these structures. ICAP-1alpha -mediated dispersion of beta 1A integrins is not observed with beta 1D integrins that do not bind ICAP. This strongly suggests that ICAP-1alpha action depends on a direct interaction between ICAP-1alpha and the cytoplasmic domain of the beta 1 chains. Altogether, these results suggest that ICAP-1alpha plays a key role in cell adhesion by acting as a negative regulator of beta 1 integrin avidity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interactions of cells with the extracellular matrix are essential for survival, differentiation, and proliferation of cells (1). They are mainly mediated by type I alpha beta 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 alpha IIbbeta 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).

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 alpha 5beta 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 alpha vbeta 3 and alpha 5beta 1 (15). Although this regulatory pathway remains to be unraveled, calcineurin has been shown to control alpha vbeta 3 and alpha 5beta 1 integrin affinity in neutrophils and CHO cells, respectively (16, 17). Finally a complex between beta 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 beta 1A chain (19) or on the threonine triplet TTT758-760 of the beta 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 beta  cytoplasmic domains have a dominant negative effect on integrin function, suggesting that some control proteins are titrated by the overexpression of beta  cytoplasmic tails (21, 22).

Integrin cytoplasmic domain-associated protein-1alpha (ICAP-1alpha ) was identified in a yeast two-hybrid screen as a protein specifically associated with the cytoplasmic domain of beta 1A integrins (23). This protein has two isoforms named alpha  and beta  of 200 and 150 amino acids, respectively. ICAP-1 is expressed throughout development and also in adult tissues (24). ICAP-1alpha but not ICAP-1beta interacts with the cytoplasmic tail of the beta 1A chain in a manner that depends on the conserved NPXY integrin motif (25). ICAP-1alpha 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-1alpha and expression of the corresponding recombinant proteins reduced or increased cell spreading on fibronectin, respectively. These data suggest that phosphorylation of ICAP-1alpha on threonine 38 by CaMKII modulates alpha 5beta 1 integrin function (13). A further involvement of ICAP-1alpha in the regulation of beta 1 integrin function was suggested by experiments indicating that its overexpression increases cell motility on a beta 1-dependent substrate such as fibronectin (26).

In this report we show that ICAP-1alpha , despite its ability to interact directly and specifically with the beta 1 integrin cytoplasmic domain in vitro, was never observed in focal adhesions. In addition, ICAP-1alpha could inhibit the interaction between talin and the beta 1 cytoplasmic tail in vitro. Because talin recruitment is a prerequisite for focal adhesion assembly (27, 28), we have analyzed the effect of ICAP-1alpha 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 beta 1 cytoplasmic tail. These results strongly suggest that ICAP-1alpha is a key regulator of cell adhesion mediated through beta 1 integrin and focal adhesion dynamic by weakening talin binding to the beta 1 integrin.

    EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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Antibodies-- The anti-beta 1 tail serum (anti cyto-beta 1) was raised against a synthetic peptide corresponding to the cytoplasmic domain of the beta 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 beta 1 subunit was kindly supplied by Dr. D. Vestweber (Muenster, Germany). The monoclonal antibody 7E2 directed against the hamster beta 1 subunit was a generous gift of Dr. R. Juliano (Chapel Hill, NC). Polyclonal antibody directed against the human ICAP-1alpha 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.

Cells and Cell Culture-- The murine NIH3T3, the hamster CHO, and the human HeLa cell lines were grown in alpha -minimal essential medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The murine GD25, GD25-beta 1A, and GD25-beta 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 beta 1 integrin chain due to a null mutation in both alleles (29). GD25 cells transfected with either the murine beta 1A, or the human beta 1A and beta 1D full-length cDNA are called GD25-beta 1A and GD-beta 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.

Protein Purifications-- ICAP-1alpha and ICAP-1alpha 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-1alpha . Briefly, human ICAP-1alpha 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-1alpha . 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 beta 1 cytoplasmic domain was estimated in a solid-phase assay.

The polypeptide corresponding to the beta 1 integrin cytoplasmic domain was produced from the BLR(DE3)pLysS E. coli strain containing the vector pET19b-cytobeta 1. This construct allows the production of the fragment 752-798 of the beta 1 integrin cytoplasmic domain. This peptide was recognized by a polyclonal antibody raised against a synthetic beta 1 cytoplasmic peptide coupled to keyhole limpet hemocyanin. Talin and alpha -actinin were purified as previously described (32), and fibronectin was purified according to a previous study (33).

Transfection in Mammalian Cells and Selection of Stable Clones-- Full-length human ICAP-1alpha was excised from the pBS-ICAP-1alpha vector as an EcoRI/XbaI fragment and inserted into the pcDNA3.1(+) vector (Invitrogen, The Netherlands). Stable GD25-beta 1A cell lines expressing ICAP-1alpha were obtained by electroporation of 4 × 106 cells in 400 µl of PBS at 280 V with 15 µg of pcDNA3.1(+)-ICAP-1alpha 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-1alpha was monitored by indirect immunofluorescence and Western blot analysis using the ICAP-1alpha polyclonal antibodies.

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-beta 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-1alpha or ICAP-1alpha 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.

Solid-phase Assays-- The interaction between ICAP-1alpha and the cyto-beta 1 peptide or the whole alpha 5beta 1 integrin was carried out using a solid-phase assay. Briefly, a 96-well tray (MaxiSorp, Nunc) was coated with the whole ICAP-1alpha protein or ICAP-1alpha 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-beta 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 alpha 5beta 1 integrin from the CHO cell lysate was performed using the 7E2 monoclonal antibody, whereas the detection of the cyto-beta 1 peptide was achieved with a polyclonal antibody directed against a synthetic peptide corresponding to the beta 1 tail.

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-1alpha 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-1alpha protein) or 30 min (ICAP-1alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ICAP-1alpha Does Not Localize in Focal Adhesions-- The protein ICAP-1alpha was isolated as a beta 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-1alpha 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-1alpha 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-1alpha expression as monitored by Western blot analysis. To determine the physiological relevance of the interaction between ICAP-1alpha and the beta 1 integrin, we carried out immunomicroscopy experiments of ICAP-1alpha in different cell lines. In HeLa cells, ICAP-1alpha showed a diffuse expression pattern and often some nuclear localization (Fig. 1C, panel a). Surprisingly, no accumulation of ICAP-1alpha 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-1alpha and beta 1 colocalize in ruffles but not in focal adhesions (35). A direct competition of endogenous ICAP-1alpha with the purified recombinant protein revealed a dramatic decrease in ICAP-1alpha immunostaining and confirmed the specificity of the immunolabeling (Fig. 1C, panels d-f). Despite the diffuse ICAP-1alpha 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-1alpha . 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-1alpha was detected with polyclonal antibodies. B, Western blots of ICAP-1alpha protein in NIH3T3 cells, HeLa cells, CHO cells, GD-25 cells, and GD-25 cells transfected with ICAP-1alpha 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-1alpha . In d, HeLa cells are stained with the same polyclonal antibodies directed against ICAP-1alpha , which has been incubated with the recombinant ICAP-1alpha protein to compete with the ICAP-1alpha -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-1alpha (a) and vinculin (b) was carried out with specific primary antibodies. These results are representative of three independent experiments. Bar, 10 µm.

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-1alpha polyclonal antibodies. In these membrane preparations, focal adhesions could be viewed by vinculin staining (Fig. 1D, panel b) or by talin or beta 1 staining (not shown), whereas anti-ICAP-1alpha antibodies showed a faint background staining that was barely detectable (Fig. 1D, panel a). Altogether these results suggest that ICAP-1alpha is not present in focal adhesions.

Interaction of ICAP-1alpha with the alpha 5beta 1 Integrin-- The absence of ICAP-1alpha in focal adhesions prompted us to study the interaction of ICAP-1alpha with beta 1 integrins in more detail. ICAP-1alpha and the beta 1 cytoplasmic domains were expressed in bacteria as polyhistidine fusion proteins. Fig. 2A shows that the purified ICAP-1alpha protein interacted specifically with the purified beta 1 cytoplasmic domain in a solid-phase assay, which is consistent with previous reports (23, 26). As a control, we used a beta 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-1alpha and the beta 1 cytoplasmic tail (Fig. 2A).


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Fig. 2.   ICAP-1alpha interacts specifically and directly with beta 1 integrins. A, the capacity of ICAP-1alpha to interact with a peptide corresponding to the beta 1 integrin cytoplasmic domain was checked in a solid-phase binding assay. A constant amount of purified recombinant ICAP-1alpha (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 beta 1-cyto peptide or YS beta 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-1alpha protein and cyto-beta 1 peptides. B, increasing amounts of the recombinant ICAP-1alpha 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 beta 1 integrin receptors bound to ICAP-1alpha were detected using the non-blocking monoclonal antibody 7E2 (raised against the hamster beta 1 chain). The results from three independent experiments using different preparations of the purified ICAP-1alpha were averaged, and standard deviations are shown. C, polyhistidine-tagged ICAP-1alpha fragments were used in the beta 1 binding assay described above. The wells were coated with 10 µg of the ICAP-1alpha recombinant fragments and then incubated with 300 µg of CHO cell lysate proteins. The bound alpha 5beta 1 was immunodetected by the 7E2 anti-hamster beta 1 monoclonal antibody. Each histogram represents mean ± S.D. of three independent experiments.

Next, we tested whether ICAP-1alpha was able to interact with the whole alpha 5beta 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-1alpha 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 beta 1 integrin was detected by a specific antibody (Fig. 2B). These data indicate that ICAP-1alpha expressed in bacteria is able to interact with the beta 1A cytoplasmic domain, and that the cytoplasmic domain of the alpha  subunit did not impair the interaction with ICAP-1alpha .

Finally, we expressed ICAP-1alpha fragments in bacteria and used them in a solid-phase binding assay to map the beta 1 binding site. Only the C-terminal moiety (amino acids 100-200) of the protein was able to bind to the beta 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-1alpha were found to interact strongly with the alpha 5beta 1 integrin from cell lysate (Fig. 2C).

ICAP-1alpha Disorganizes Focal Adhesions ex Vivo-- Despite its specific and direct association with the beta 1 integrin, ICAP-1alpha was not localized in focal adhesions. One possible explanation for these contradictory results could be that ICAP-1alpha might act as a negative regulator of the recruitment of focal adhesion components. To investigate this possibility we microinjected ICAP-1alpha 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 alpha -actinin (not shown), microinjection of the full-length ICAP-1alpha in the dextran-coupled rhodamine buffer induced a rapid delocalization of vinculin (Fig. 3, D-F) or talin and alpha -actinin (not shown) observed in 70% of the cells. Microinjection of the C-terminal moiety of ICAP-1alpha (amino acids 101-200) that encompasses the beta 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 beta 1 integrin domain (Fig. 2C), we made use of this recombinant fragment as a control. Indeed, the microinjection of this part of ICAP-1alpha did not interfere with vinculin staining (Fig. 3, G-I).


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Fig. 3.   Microinjection of purified ICAP-1alpha 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-1alpha protein at 1 mg/ml (D-F), ICAP-1alpha 1-100 fragment (G-I), or ICAP-1alpha 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-1alpha protein and fragments.

Finally, disruption of focal adhesions by ICAP-1alpha was also investigated in a cellular context after stable transfection into GD25-beta 1A cells of a vector containing human ICAP-1alpha cDNA. This cell line expresses functional beta 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 beta 1 integrins (36, 37). Under our experimental conditions, immunofluorescence microscopy did not reveal any detectable staining for endogenous ICAP-1alpha in GD25-beta 1A cells (Fig. 4A). On the other hand, these cells exhibited surface expression of beta 1A integrins confined to focal adhesions that could be monitored by the 9EG7 antibody (Fig. 4B). In a non-clonal population of GD25-beta 1A cells transfected with a cDNA encoding the human ICAP-1alpha , a positive immunofluorescence signal for ICAP-1alpha 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 beta 1 integrins were no longer occupied and involved in focal adhesions (Fig. 4D).


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Fig. 4.   ICAP-1alpha expression disrupts beta 1 integrin-containing focal adhesions. GD25-beta 1A cells were stably transfected either with vector alone (A and B) or with a cDNA coding for the full-length ICAP-1alpha protein (C and D). Transfected cells were spread overnight at 37 °C on fibronectin-coated coverslips. The expression of ICAP-1alpha was visualized with polyclonal antibodies (A and C) and the high affinity conformational state of the beta 1 integrin with the 9EG7 monoclonal antibody (B and D). Note that the reduction of 9EG7 staining correlated with the expression of ICAP-1alpha . Bar, 10 µm.

Disruption of Focal Adhesions by ICAP-1alpha Requires Direct Interaction with the beta 1 Integrin Chain-- The action of ICAP-1alpha on focal adhesions might be indirect, for instance due to the interference with some regulatory pathways. Therefore, the purified recombinant ICAP-1alpha 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-1alpha 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-1alpha efficiently displaced vinculin from focal adhesions (Fig. 5B). A similar result was also observed for talin and alpha -actinin (not shown). The same result was obtained by the incubation of the C-terminal part (amino acids 101-200) of ICAP-1alpha (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-1alpha 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-1alpha (5 µM). Alternatively, the purified N-terminal moiety of ICAP-1alpha (amino acids 1-100) shown in C or the C-terminal moiety of ICAP-1alpha (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-1alpha 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-1alpha . Bar, 10 µm.

ICAP-1alpha 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-1alpha destabilizing action on focal adhesions of ventral plasma membranes. To assess whether ICAP-1alpha action on focal adhesions was due to its direct binding on beta 1 integrin chains or to some interference with Rho signaling pathways, we performed similar experiment on VPM from GD-25beta 1A and GD-25beta 1D cells lines. The beta 1D and beta 1A isoforms are functionally similar with regard to integrin-mediated signaling (39), but the former strongly binds talin (31) and does not bind ICAP-1alpha (38). Upon addition of the ICAP-1alpha fragment 100-200, the dispersion of beta 1A integrins initially clustered into focal adhesions was observed (Fig. 6, A and C), whereas beta 1D-containing focal adhesions remained unaffected (Fig. 6, B and D). This result strongly suggests that a direct interaction between ICAP-1alpha and the beta 1 chain is a prerequisite for focal adhesion disassembly.


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Fig. 6.   ICAP-1alpha integrin binding domain displaces beta 1A but not beta 1D integrins from focal adhesions. Ventral plasma membranes from GD25-beta 1A (A and C) and GD25-beta 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-1alpha (amino acids 101-200) added at a concentration of 5 µM. The membranes were subsequently fixed and stained for beta 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-1alpha . Bar, 10 µm.

Talin and ICAP-1alpha Compete for Binding on the Cytosolic Domain of the beta 1 Integrin Chain-- Because talin interacts directly with the beta 1 integrin cytoplasmic domain and is crucial for focal adhesion assembly, one attractive hypothesis is that ICAP-1alpha is involved in the control of talin-integrin interaction. Therefore, we tested whether ICAP-1alpha could modulate the binding of talin to the integrin beta 1 cytoplasmic domain. In an in vitro solid-phase assay, ICAP-1alpha could inhibit talin binding to the cytoplasmic tail of the beta 1A chain in a dose-dependent manner (Fig. 7A). These data suggest that the displacement of talin from its binding site on beta 1A may be sufficient for focal adhesion disruption and, consequently, for a decrease in the integrin avidity. Moreover, the competition of ICAP-1alpha and talin for the binding to beta 1 was specific, because it could not be observed either with alpha -actinin, another beta 1 interacting protein (Fig. 7B), or with the 1-100 ICAP-1alpha moiety (Fig. 7C).


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Fig. 7.   ICAP-1alpha competes with talin but not with alpha -actinin binding to the beta 1 cytoplasmic domain. A, increasing amounts of purified recombinant ICAP-1alpha were preincubated with 1 µg of the cyto-beta 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-beta 1 peptide to talin was detected by polyclonal antibodies raised against the cytoplasmic domain of the beta 1 integrin chain and a biotin-conjugated anti rabbit secondary antibody. B, an amount of 2 µg of the recombinant protein ICAP-1alpha was preincubated with 1 µg of the cyto-beta 1 peptide and incubated in 96-well plastic trays coated with 10 µg of purified talin (from human platelets) or alpha -actinin (from chicken gizzard). The binding of the cyto-beta 1 peptide to talin or alpha -actinin was detected by polyclonal antibodies raised against the cytoplasmic domain of the beta 1 integrin chain and a biotin-conjugated anti-rabbit secondary antibody. C, a concentration of 1.5 µg of ICAP-1alpha fragments 1-100 and 101-200 was preincubated with 1 µg of the cyto-beta 1 peptide and incubated in 96-well plastic trays coated with 10 µl of purified talin. The binding of the cyto-beta 1 peptide to talin was detected by polyclonal antibodies raised against the cytoplasmic domain of the beta 1 integrin chain and a biotin-conjugated anti rabbit secondary antibody. Each experiment was performed in triplicate.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the cellular localization of the endogenous ICAP-1alpha 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-1alpha colocalized with vinculin or talin, which were used as markers of focal adhesions.

Even though ICAP-1alpha was not detected in focal adhesions, the purified recombinant protein interacted strongly with the cytoplasmic domain of the beta 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-1alpha to the cytoplasmic domain of the beta 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-1alpha in NIH3T3 cells, and we indeed observed a rapid disorganization of focal adhesions. In addition, recombinant ICAP-1alpha was able to disaggregate focal adhesions when added to purified ventral plasma membranes from NIH3T3 and GD-25beta 1A cells. Conversely, the beta 1D-containing integrins were resistant to ICAP-1alpha . This latter experiment strongly suggests that the disassembly of focal adhesions is due to a direct interaction with the beta 1A integrin subunit and is independent of a cellular signaling pathway. Furthermore, the focal adhesion disruption mediated by ICAP-1alpha is in good correlation with our previous data, which have shown that ectopic expression of ICAP-1alpha -regulated CHO cell spreading (13).

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-1alpha 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-1alpha compete for binding to the beta 1A cytoplasmic domain. On the other hand, we found that the interaction between alpha -actinin and the beta 1 integrin is not inhibited by the presence of ICAP-1alpha . This shows that ICAP-1alpha inhibits the interaction between beta 1A integrins and talin in a specific manner and confirms previous reports showing that the interaction of alpha -actinin with the beta 1 cytoplasmic domain is not sufficient to stabilize focal adhesion sites (40). The lack of effect of ICAP-1alpha on beta 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-1alpha and talin compete for integrin beta 1A binding and thereby modulate focal adhesion assembly and/or dynamic. How ICAP-1alpha interferes with talin binding on the beta 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 beta 3, beta 1A, and beta 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 beta  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 alpha IIbbeta 3 integrin (43). This finding is very interesting, because it offers some molecular basis of ICAP-1alpha and talin competition. Indeed, sequence homology and molecular modeling favor the view that ICAP-1alpha is a phosphotyrosine binding domain protein. It was suggested that the interaction specificity with the beta 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-1alpha (25). This is fairly consistent with the lack of interaction of ICAP-1alpha with the beta 1D isoform that do not have a valine at this position. This latter residue is very close to the tyrosine 783 on the human beta 1A chain. The tyrosine at this position on the beta 1 chain or on the homologous position 747 on the beta 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-1alpha binding sites on the integrin beta 1A tail overlap.

The distribution of ICAP-1alpha in ruffles and its absence from focal adhesions suggest that the interaction between ICAP-1alpha and the beta 1 integrin cytoplasmic domain is regulated. It is possible that ICAP-1alpha is sequestered inside the cell and that the interaction between a sequestering protein and ICAP-1alpha may be the regulated event. Alternatively, the interaction of ICAP-1alpha with the cytoplasmic domain of the beta 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-1alpha was able to interact with beta 1A (26). How the interaction of ICAP-1alpha and the integrin is regulated is not yet understood and requires further investigations.

Recently, a 20-kDa protein named TAP-20 (with marked homology with beta 3-endonexin) was shown to interact specifically with the beta 5 cytosolic domain of the alpha vbeta 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-1alpha , 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-1alpha , integrin cytoplasmic domain-associated protein-1alpha ; PBS, phosphate-buffered saline; BSA, bovine serum albumin; VPM, ventral plasma membrane.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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