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J Biol Chem, Vol. 275, Issue 14, 10604-10610, April 7, 2000

Cooperative Signaling between ₆₄ Integrin and ErbB-2 Receptor Is Required to Promote Phosphatidylinositol 3-Kinase-dependent Invasion^*

Daniela Gambaletta, Alessandra Marchetti, Laura Benedetti, Arthur M. Mercurio^§, Ada Sacchi, and Rita Falcioni^¶

From the  Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Via delle Messi d'Oro, 156-00158 Rome, Italy and the ^§ Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously demonstrated that ₄ integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large ₄ cytoplasmic domain that are involved in the interaction of ₆₄ with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of ₄ that lacked either all or portions of the ₄ cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of ₄ was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of ₆₄ to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the ₄ cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of ₆₄ integrin to increase invasion. Interestingly, the extracellular domain of ₄ is not necessary for ₆₄ to stimulate invasion. The association of ₆₄ with ErbB-2 is dependent upon the ₄ cytoplasmic domain and can occur in the absence of ₆₄ heterodimerization. Finally, we observed strong activation of PI3K with ₄ wild type and with those ₄ deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between ₆₄ and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the ₄ cytoplasmic domain (amino acids 854-1183) in this event.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrins are the major family of cell surface receptors that mediate attachment to the extracellular matrix. The interaction between integrins and their ligands is involved in the regulation of many cellular functions, including embryonic development, cell proliferation, as well as tumor growth and metastasis. Integrins are composed of  and  transmembrane subunits that heterodimerize to form different receptors. A single  subunit (e.g. _v or ₆) can associate with different  subunits, (₁, ₃, ₅, or ₁, ₄, respectively), promoting different ligand binding specificity (1-3). The ₆₄ integrin is a receptor for various isoforms of the basement membrane component laminin (4-6), and its expression is restricted to epithelia, endothelia and peripheral nerves (7-9). In many epithelia, ₆₄ is found in hemidesmosomes where it plays an essential role in their organization (10, 11). This integrin can also interact with F-actin and promote the migration of invasive carcinoma cells (12, 13).

The intracellular portion of the ₄ subunit is much larger (1,000 amino acids) than that of all the other known  subunits, and it does not exhibit apparent sequence homology with them (14-16). Increasing evidence indicates that the ability of ₆₄ to regulate cell proliferation, motility, and invasion is dependent upon signal transduction events that are mediated by the ₄ cytoplasmic domain (17-19). Of particular relevance to cancer, ₆₄ has been implicated in carcinoma invasion (19-21) through its ability to activate PI3K (19). Moreover, the ability of ₆₄ to promote invasion is dependent upon the ₄ cytoplasmic domain. The importance of ₆₄ in malignancy is also indicated by the finding that its expression correlates with the progression of squamous, ovarian, thyroid, gastric, and colorectal carcinomas (22-27).

Function and signaling properties of ₆₄ in carcinoma cells are influenced by its association with growth factor receptors. Specifically, we demonstrated that ₆₄ co-immunoprecipitates with ErbB-2 in human mammary and ovarian carcinoma cell lines and that ligation of this integrin increases ErbB-2 phosphorylation (28). Moreover, we also observed that overexpression of ErbB-2 and ₆₄ in NIH3T3 cells increased their invasive capacity (28). In the present study, we sought to identify the portion of ₄ involved in the interaction with ErbB-2 and possibly involved in the development of a more aggressive phenotype. With this aim, we generated different NIH3T3 transfectants that concomitantly overexpress ErbB-2 oncogene and wild type or deletion mutants of ₄ integrin. In vitro invasion assays demonstrated that the portion of ₄ protein involved in the invasive capacity resides in 329 cytoplasmic residues between the amino acids 824 and 1183. Biochemical analysis indicated that ErbB-2/₄ interaction is abolished uniquely when the entire cytoplasmic domain of ₄ is deleted. Thus, we were able to exclude that the interaction of ErbB-2 and ₄ is responsible for increased malignancy of NIH3T3/ErbB-2/₄ cells. The study of the mechanisms by which ₄ cooperates with ErbB-2 to promote increased malignancy showed that the ₆₄ integrin activates the PI3K pathway when both ErbB-2 and ₄ are overexpressed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and cDNA Constructs-- NIH3T3 parental cell line and NIH3T3 cells stably transfected with the human ErbB-2 cDNA (29) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine (Life Technologies, Inc.). NIH3T3 cells and ErbB-2-transduced NIH3T3 cells were transfected, by electroporation, with the pRC/CMV expression vector carrying the wild type human ₄ integrin subunit and truncated ₄ cDNAs (10, 17). Filippo Giancotti (Memorial Sloan-Kettering Cancer Center, New York) kindly provided the cDNAs encoding the full-length and truncated ₄ molecules. Selection of neomycin-positive clones was carried out using 500 µg/ml of G418 (Life Technologies, Inc.).

Antibodies-- The rat monoclonal antibody (mAb)¹ 439-9B and the mouse mAb 450-11A to the human ₄ and the rat mAb 135-13C to ₆ integrin subunits, respectively, were purified as described previously (21, 30). Tom Carey (University of Michigan, Ann Arbor, MI) kindly donated the anti-human ₄ integrin mAb A9 (31). The anti-mouse ₄ integrin mAb 346-11A was prepared and purified from ascitic fluid and used as negative control (32). Purified anti-mouse IgG was from Cappel (Durham, NC). The mouse mAbs to the human ErbB-2 protein used in Western blotting experiments were from Transduction Laboratories (Lexington, KT) or from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated anti-mouse or anti-rabbit IgG were from Bio-Rad. The anti-phosphotyrosine (anti-Tyr(P)) mouse mAb 4G10 was from Upstate Biotechnology, Inc. (Upstate Biotechnology Inc., Lake Placid, NY).

Affinity Chromatography-- Anti-human and anti-mouse ₄ integrin mAbs 439-9B and 346-11A were purified by high performance liquid chromatography and cross-linked to activated immune affinity supports Affi-Gel 10/15 (Bio-Rad). In brief, 4 mg of purified mAb was combined with 1 ml of Affi-Gel in 0.1 M buffer carbonate, pH 8.5, at 4 °C for 4 h. The beads were then washed with Tris-HCl 0.1 M, pH 8, and suspended in PBS containing 0.03% sodium azide (NaN₃). NIH3T3 transfectants were lysed in 20 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, aprotinin (5 µg/ml), leupeptin (10 µg/ml), and pepstatin A (4 µg/ml). Repeated experiments were also performed in the presence of 10 mM CHAPS (Pierce), instead of 1% Nonidet P-40, to disrupt nonspecific protein-protein interactions. Lysates were clarified by centrifugation, and the immune complexes were purified by affinity chromatography using mAbs cross-linked to Affi-Gel (bead-conjugated antibodies).

Immunocomplexes and Total Cell Lysates-- Immunoprecipitations from cells labeled with lactoperoxidase and carrier-free ¹²⁵I showed that the antibodies recognize the appropriate molecules (data not shown). In brief, as described previously (21), 2 × 10⁶ cells were labeled with 1 mCi of ¹²⁵I in the presence of 10 µl of lactoperoxidase (2 mg/ml in 50% of glycerol) (Calbiochem, La Jolla, CA) and 5 µl of a 1:1000 dilution of H₂O₂ (30%). After labeling, cells were washed with PBS and solubilized in lysis buffer containing 5 mg/ml BSA, 1% Nonidet P-40, 1 mM NaN₃, 1 mM phenylmethylsulfonyl fluoride (Sigma), 5 µg/ml leupeptin, 10 µg/ml aprotinin (Sigma), and 10 mM EDTA. The lysates were clarified by centrifugation (30,000 × g) for 3 h at 4 °C and solubilized proteins (1 × 10⁷ cpm) were immunoprecipitated. The immunoprecipitates were analyzed by SDS-PAGE, and autoradiography was performed with X-Omat RP film (Kodak). Direct immunoprecipitations were performed using primary antibodies collected with 50 µl of Protein G-agarose beads (Pierce) suspended in lysis buffer (50% v/v). Total cell lysates were added to the bead-conjugated antibodies, and protein complexes were washed at 4 °C in lysis buffer, boiled, and analyzed by SDS-PAGE.

Kinase Assay-- To assay PI3K activity, after serum starvation for 24 h the cells were washed and lysed in 10 mM Hepes, pH 7.5, 0.15 M NaCl, 10% glycerol in the presence of protease and phosphatase inhibitors. Nuclei were removed by centrifugation at 12,000 × g for 15 min at 4 °C. After lysis, aliquots of cell extracts containing equivalent amounts of protein were incubated overnight at 4 °C with anti-Tyr(P) mAb 4G10 (Upstate Biotechnology, Inc.) and protein G (Pierce). The beads were washed twice with lysis buffer (10 mM Hepes, pH 7.5, 0.15 M NaCl, 10% glycerol, and 1% Nonidet P-40); twice with 0.5 M LiCl; twice with 10 mM Hepes, pH 7.5, 0.15 M NaCl, and 0.2% Nonidet P-40; and once with 10 mM Hepes, pH 7.5, 0.15 M NaCl. After removal of the last wash, the beads were suspended in 30 µl of 30 mM Hepes, pH 7.5, and 30 µl of kinase buffer containing 10 µg (20 µl) of L--phosphatidylinositol from bovin liver (Sigma) sonicated in 10 mM Hepes and 1 mM EDTA, 80 µM ATP (Roche Molecular Biochemicals), 20 µCi of [-³²P]ATP (6000 Ci/mmol), 10 mM MgCl₂, and 400 µM adenosine (Sigma) and incubated for 25 min at room temperature. The reaction was stopped by the addition of 100 µl of 1 M HCl, and 200 µl of 1:1 mixture of chloroform and methanol were added. The organic phase was then washed twice with 300 µl of 1:1 mixture of methanol and HCl. The lipid extracts (20 µl of each reaction) were then resolved by thin layer chromatography plates (TLC Silica gel) (Merck) in chloroform, methanol, and ammonium hydroxide. Dried TLC sheets were developed by autoradiography.

Western Blot Analysis-- Total cell lysates or immune complexes obtained by affinity chromatography or indirect immunoprecipitation were analyzed by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad) or Immobilon-P transfer membrane (Millipore, Bedford, MA). The blots were probed with the following antibodies: 2 µg/ml of anti-ErbB-2 mAb (Clone 3B5) or 2 µg/ml of purified mouse anti-human c-Myc mAb (Clone 9E10). Filters were washed and developed with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Autoradiographies were performed with Hyperfilm ECL (Amersham Pharmacia Biotech).

Flow Cytometry-- The expression levels of ErbB-2 and ₆₄ integrin receptors were detected by flow cytometric analysis of stained cells. Cells harvested using citrate saline buffer (0.134 M KCl, 0.015 M sodium citrate) were washed twice with cold PBS containing 0.002% EDTA and 10 mM NaN₃ (washing buffer). Samples of 1 × 10⁶ cells were incubated for 1 h at 4 °C with saturating concentrations of primary antibodies diluted in PBS containing 0.5% BSA. Control cells were incubated with unrelated antibodies. Cells were then washed three times with washing buffer (PBS containing 0.5% BSA) and incubated for 1 h at 4 °C with 50 µl of fluorescein isothiocyanate-conjugated secondary antibodies (F(ab')₂ (Cappel, West Chester, PA)) diluted 1:20 in PBS/BSA. After three washes, the cells were suspended in 1 ml of washing buffer. Cell suspensions were analyzed by a flow cytometer (Epics XL analyzer, Coulter Corporation, Miami, FL) after addition of 5 µl of propidium iodide (1 mg/ml stock solution) to exclude nonviable cells. At least 1 × 10⁴ cells/sample were analyzed.

Chemoinvasion Assays-- Chemoinvasion assays were carried out in a Boyden chamber as described (33). In brief, 8-mm polycarbonate filters (Nucleopore, Concorezzo, Italy) were coated with Matrigel kindly provided by Dr. A. Albini (Genova, Italy). Optimal Matrigel concentration (12.5 µg/filter from a 250 µg/ml dilution of Matrigel in distilled, cold water) was accurately determined in preliminary experiments. The cells harvested by trypsin-EDTA treatment were washed with serum free Dulbecco's modified Eagle's medium supplemented with 0.1% BSA, and 5 × 10⁵ cells were layered on the top well of a Boyden chamber. The chambers were incubated at 37 °C, 5% CO₂ for 8 h in the presence of Balb/3T3 conditioned medium (added as chemo-attractant) or in the presence of 0.1% BSA (added as negative control). In some assays, the cells were preincubated for 30 min before addition to the Matrigel-coated wells with wortmannin (Biomol, Plymouth Metting, PA). Cells migrated on the lower surface of the filters were fixed in ethanol and stained with Toluidine blue. Five independent fields were counted at 160× with a Zeiss microscope. Each assay was carried out in quadruplicate and repeated at least three times. The ability of the cells to adhere to the filters was verified by staining the upper side of the filters.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Cytoplasmic Domain of 4 Protein Influences the Metastatic Propensity of NIH3T3/ErbB-2-transformed Cells-- We previously demonstrated that the expression of the ₄ integrin subunit in NIH3T3 cells, transformed by the ErbB-2 oncogene, stimulates their in vitro invasion (28). To identify specific domains in the ₄ subunit that confer this invasive potential, we expressed deletion mutants of ₄ in NIH3T3/ErbB-2 cells (Fig. 1). These deletion mutants lacked either all (L) or portions (B, C, and D) of the ₄ cytoplasmic domain. In addition, we used an ecto-domain mutant (F) in which most of the extracellular domain of ₄ was replaced with a c-Myc tag. In agreement with data previously reported (10), all of these mutants were highly expressed on the cell surface of the transfected cells (data not shown). Clones of these transfectants expressing comparable surface expression of the ₄ subunit were chosen for functional and biochemical analyses. The expression levels of ₄ protein corresponding to F deletion mutant were assessed by Western blot analysis using a mouse anti-human c-Myc antibody (Fig. 2). Immunoprecipitation of surface-labeled proteins confirmed the expression of wild type and truncated ₄ proteins on the cell surface of selected clones (data not shown).

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Schematic representation of wild type and mutated 4 cDNAs. , transmembrane domain; amino acids 1-710, extracellular domain; amino acids 734-1752, cytoplasmic domain; 70-660, extracellular domain deletion. Deletions in the cytoplasmic domain are indicated by the amino acids where the molecules were truncated.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Western blot analysis using a mouse anti-human c-Myc antibody of the clones obtained after selection of NIH3T3/ErbB-2 cells transfected with deleted 4 F cDNA. The molecular size of 4 F transfectants (kilodaltons) is indicated by an arrow. W.B., Western blot.

To identify specific domains within the ₄ subunit that are necessary for the ability of ₆₄ to promote invasion, the NIH3T3/ErbB-2 cells that expressed the ₄ deletion mutants were assayed for their ability to invade Matrigel in a standard chemoinvasion assay (Table I). At least two clones from each of the NIH3T3/ErbB-2/₄ transfectant were used for this purpose. In agreement with our previous finding (28), expression of the wild type ₄ subunit increased invasion of NIH3T3/ErbB-2 cells by 2.5-fold. Interestingly, expression of the ₄ C, D, and F mutants resulted in a similar increase in invasion (Table I). However, expression of the ₄ L and B mutants did not enhance the invasiveness of NIH3T3/ErbB-2 cells. Two important conclusions can be drawn from these data. First, the extracellular domain of ₄ does not appear to be necessary for ₆₄ to stimulate invasion based on the results obtained with the ₄ F mutant. Second, a portion of the ₄ cytoplasmic domain between amino acids 854 (the site of the ₄ B deletion) and 1183 (the site of the ₄ C deletion) is critical for the ability of the ₆₄ integrin to stimulate invasion. However, the ₄ mutated proteins we used contain very large deletions, leaving open the possibility for the domains of the ₄ protein, upstream of the 854 amino acid, to stimulate invasion but which at the same time require cooperation with the other downstream domains. We also cannot exclude that such deletions, causing conformational changes of the molecules, could prevent their interaction with intermediate signaling molecules responsible for stimulating invasion.

View this table: [in this window] [in a new window]   Table I Chemoinvasive ability of the NIH3T3 transfectants NIH3T3 transfectants were assayed for their ability to invade Matrigel in the absence or presence of the PI3K inhibitor wortmannin (100 nM. Fibroblast-conditioned serum-free medium was added in the lower compartment of the Boyden Chamber and used as chemoattractant. The data shown are from two individual clones of each transfectant and are the mean values ± S.D. of three experiments done in quadruplicate. Statistical significance (P*) was evaluated according to T-test comparing the median values of NIH3T3/ErbB-2 cells/field versus themselves or NIH3T3/ErbB-2/4 transfectants cells/field in absence or presence of wortmannin. ND, not determined.

Interaction of ₄ with ErbB-2 Is Abrogated by the Lack of ₄ Cytoplasmic Domain-- Given our previous demonstration of a physical association between ₆₄ and ErbB-2, we attempt to determine whether this association is necessary for the ability of ₆₄ and ErbB-2 to promote invasion. Specifically, we examined the ability of ErbB-2 to co-immunoprecipitate with ₆₄ heterodimers that contained specific deletions in the ₄ subunit. To this purpose, these ₆₄ heterodimers were purified by affinity chromatography using either 439-9B mAb, which recognizes an epitope present in the extracellular domain of ₄ or 450-11A mAb, which recognizes an epitope in the ₄ cytoplasmic domain. As negative control, we used 346-11A mAb, which is specific for mouse ₄. The presence of ErbB-2 in the 4 integrin immune complexes was determined by immunoblotting. Using this approach, we detected the 185-kDa ErbB-2 protein in the ₄ immune complexes obtained from extracts of NIH3T3/ErbB-2 cells that expressed the intact ₆₄ heterodimer (Fig. 3, lane 1), as expected (28). In addition, an ErbB-2/₄ complex was detected in extracts obtained from clones 19 and 21 of cells expressing the extracellular domain deletion of ₄ (₄ F) (Fig. 3). In these immune complexes, purified by an anti-₄ mAb specific for the cytoplasmic domain of the molecule, the anti-ErbB-2 mAb was able to detect a 185-kDa protein (Fig. 3, lanes 2 and 3). Moreover, the co-immunoprecipitation of ErbB-2 and ₄ F truncated protein was detected after reprobing the same blot with an anti-c-Myc mAb (Fig. 3, lanes 5 and 6). The same anti-c-Myc antibody did not recognize the ₄ wild type protein (lane 4). Similar experiments performed using extracts from NIH3T3/ErbB-2 cells expressing truncated proteins ₄ C (clones 9 and 17) and D (clones 3 and 25) revealed that all these ₄ truncated proteins co-immunoprecipitate with the ErbB-2 protein (Fig. 4, lanes 1, 3, 5, and 7, respectively). The mAb specific for the mouse ₄ integrin (negative control) did not co-immunoprecipitate ErbB-2 from the same extracts (Fig. 4, lanes 2, 4, 6, and 8). Fig. 5 shows immune complexes from extracts of NIH3T3/ErbB-2 cells transfected with truncated proteins ₄ B (clones 11 and 14) and L (clones 5 and 4). The truncated ₄ B protein still co-precipitated with ErbB-2 (Fig. 5, lanes 3 and 4), whereas ₄ L protein, which was deleted from the entire cytoplasmic domain, did not co-precipitate with ErbB-2 (Fig. 5, lanes 6 and 7). As controls, the ErbB-2 protein was detected in immune complexes derived from lysates of NIH3T3/ErbB-2 cells expressing wild type ₄ protein (Fig. 5, lane 2) but not from lysates of NIH3T3/ErbB-2 cells (Fig. 5, lane 1 and 5). These data show that only the deletion of the entire ₄ cytoplasmic domain abrogates co-immunoprecipitation of ErbB-2 and ₄. Moreover, the finding that the ₄ F protein, which is unable to form ₆₄ heterodimers (10) but is still able to retain the ability to co-precipitate with ErbB-2 indicates that the ₆ subunit is not involved in the ErbB-2/₄ interaction.

View larger version (60K): [in this window] [in a new window]   Fig. 3.   Association of 4 mutant F with ErbB-2. Total cell lysates from NIH3T3/ErbB-2/4 wild type (lanes 1 and 4) and NIH3T3/ErbB-2/4 mutant F cells (clones 19 and 21) (lanes 2, 3, 5, and 6) were incubated with bead-conjugated anti-human 4 mAb 439-9B. Immune complexes were analyzed by SDS-PAGE and probed as shown in the left panel with a mouse anti-human ErbB-2 mAb followed by chemiluminescence detection with an anti-mouse IgG peroxidase-conjugated antibody. The right panel shows the immunoblot on the same filter with a mouse anti-human c-Myc antibody. Molecular sizes (kilodaltons) are indicated. W.B., Western blot.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Association of the 4 mutant C and D molecules with ErbB-2. Total cell lysates from NIH3T3/ErbB-2/4 mutant C and D cells were incubated with bead-conjugated anti-human 4 antibody 439-9B (lanes 1, 3, 5, and 7) or, as negative control, anti-mouse 4 antibody 346-11A (lanes 2, 4, 6, and 8). Immune complexes were analyzed by SDS-PAGE and probed with a mouse anti-human ErbB-2 mAb followed by chemiluminescence detection with anti-mouse IgG peroxidase-conjugated antibody. Molecular sizes (kilodaltons) are indicated. W.B., Western blot.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Association of the 4 mutant B and L molecules with ErbB-2. Total cell lysates from NIH3T3/ErbB-2 (lanes 1 and 5), from NIH3T3/ErbB-2/4 wild type (lane 2), from NIH3T3/ErbB-2/4 mutant B (clone 11 and 14) (lanes 3 and 4), and from NIH3T3/ErbB-2/4 mutant L (clones 5 and 4) (lanes 6 and 7) cells were incubated with bead-conjugated anti-human 4 439-9B antibody. Immune complexes were analyzed by SDS-PAGE and probed with a mouse anti-human ErbB-2 mAb followed by chemiluminescence detection with an anti-mouse IgG peroxidase-conjugated antibody. Molecular sizes (kilodaltons) are indicated. W. B., Western blot.

Cooperativity between ₆₄ and ErbB-2 Is Required to Activate PI3K-- The activation of PI3K by the ₆₄ integrin has been shown to promote the invasion of carcinoma cells (19). Based on our finding that the overexpression of ₆₄ increases the invasiveness of NIH3T3 cells transformed by ErbB-2, we examined the influence of these two surface receptors on PI3K activation. As a prelude to these experiments, we established the involvement of PI3K in the invasion of the NIH3T3/ErbB-2/₄ transfectants using wortmannin. In agreement with previous findings (19, 34), we found that wortmannin inhibited the invasion of NIH3T3 cells expressing either the intact ₆₄ heterodimer or the various ₄ deletion mutants that are still able to increase invasiveness (Table I). Interestingly, we also found that wortmannin did not modify the invasive capacity of NIH3T3/ErbB-2 cells (Table I), confirming the observation that PI3K pathway is involved in ₄-dependent invasive capacity. The fact that wortmannin completely abolishes the invasiveness of NIH3T3/ErbB-2/₄ cells rather than reducing it to the level of parental cell line might be explained by the assumption that the activation of PI3K in NIH3T3/ErbB-2 cells after ₄ expression supersedes other signaling pathways responsible for the invasion of these cells. Based on this hypothesis, it is reasonable to think that wortmannin-mediated inhibition of PI3K activation should abolish the invasive capacity.

To assay PI3K activity, extracts obtained from NIH3T3/ErbB-2 transfectants were immunoprecipitated with an anti-Tyr(P) mAb to capture the activated population of PI3K, and the immunoprecipitates were assayed for their ability to phosphorylate L--phosphatidylinositol. Constitutive PI3K activity was undetectable in NIH3T3 parental cells and also in NIH3T3 cells overexpressing either the ₄ or the ErbB-2 proteins alone (Fig. 6). However, PI3K activation was evident when ₄ protein was co-expressed with ErbB-2 (Fig. 6). These results indicate that both ₆₄ and ErbB-2 expression are required for constitutive PI3K activation in NIH3T3 cells. Furthermore, these data indicate that in the NIH3T3/ErbB-2 cellular context a correlation exists between the ₄-dependent invasive capacity and the ₄-induced PI3K activity. Moreover, the availability of the ₄ deletion mutants enabled us to identify specific domains within the ₄ subunit that are required for PI3K activation and to compare these domains with those that are implicated in invasion based on our results. Cell extracts of the NIH3T3/ErbB-2 transfectants that expressed the ₄ C, D, and F truncated proteins exhibited levels of PI3K activation comparable with that of wild type ₄ (Fig. 7). In contrast, NIH3T3/ErbB-2 transfectants expressing the ₄ L and B proteins that did not enhance the invasiveness of NIH3T3/ErbB-2 cells (Table I) showed minimal activation of PI3K (Fig. 7). To establish a possible link between the formation of the receptor-integrin complex and PI3K activity, we checked the presence of PI3K in the complex. However, using different experimental conditions, we were never able to detect PI3K in ErbB-2/₄ complex (data not shown).

View larger version (7K): [in this window] [in a new window]   Fig. 6.   Analysis of PI3K activity on NIH3T3, NIH3T3/4, NIH3T3/ErbB-2, and NIH3T3/ErbB-2/4 clones. A, aliquots of cell extracts derived from the parental cell line NIH3T3 (lane 1) and NIH3T3 clones (lanes 2-4) containing equivalent amounts of protein were incubated with the anti-phosphotyrosine antibody 4G10 and protein G-Sepharose overnight at 4 °C. After washing, the beads were resuspended in kinase buffer for 20 min at room temperature in presence of L--phosphatidylinositol. The phosphorylated lipids were resolved by thin layer chromatography. B, the amount of radiolabeled lipids was determined by densitometry. The data shown are the mean values ± S.D. from three separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Analysis of PI3K activity on NIH3T3/ErbB-2, NIH3T3/ErbB-2/4 wild type, and/or NIH3T3/ErbB-2 deleted 4 molecules C, D, L, B, and F. A, aliquots of cell extracts containing equivalent amounts of protein were incubated with the anti-Tyr(P) mAb and protein G-Sepharose overnight at 4 °C. After washing, the beads were resuspended in kinase buffer for 20 min at room temperature in the presence of L--phosphatidylinositol. The phosphorylated lipids were resolved by thin layer chromatography. B, the amount of radiolabeled lipids was determined by densitometry. The data shown are the mean values ± S.D. from three separate experiments.

We then questioned whether antibody-mediated ligation of ₆₄ could augment PI3K activation. To this purpose, we used a mAb specific for the ₆ integrin subunit because it allowed us to compare activation of PI3K by the ₆₁ and ₆₄ integrins. However, NIH3T3 cells express ₆₁ but not ₆₄. We observed that antibody-mediated ligation of the ₆ subunit (₆₁) in the NIH3T3/ErbB-2 cells did not stimulate PI3K activation (Fig. 8). However, ligation of the ₆ subunit in the NIH3T3/ErbB-2/₄ transfectants (primarily ₆₄) stimulated PI3K activity (Fig. 8). Finally, we asked whether wortmannin could affect PI3K activity on NIH3T3/ErbB-2/₄ cells. In agreement with previous findings (35), we found that treatment with wortmannin is able to reduce PI3K activity by 90% in these cells (Fig. 9), supporting the effect of the complete inhibition of invasiveness that was obtained after treatment of NIH3T3/ErbB-2/₄ with this inhibitor (Table I). These data further indicate that PI3K activation in NIH3T3/ErbB-2 cells is strictly dependent upon ₄ expression and that in these cells ligand activation of the ₆₁ receptors cannot substitute ₄-dependent signals to generate PI3K activity.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Analysis of PI3K activity on NIH3T3/ErbB-2 and NIH3T3/ErbB-2/4 wild type cells. A, NIH3T3/ErbB-2 and NIH3T3/ErbB-2/4 cells were maintained in suspension (lanes 1 and 3) or plated on dishes coated with the anti-6 specific antibody 135-13C (lanes 2 and 4) and allowed to adhere. Aliquots of cell extracts that contained equivalent amounts of protein were incubated with the anti-Tyr(P) mAb and protein G-Sepharose overnight at 4 °C. After washing, the beads were resuspended in kinase buffer for 20 min at room temperature in the presence of L--phosphatidylinositol. The phosphorylated lipids were resolved by thin layer chromatography. B, densitrometric analysis show that the integrin clustering induces a 2-fold increase of PI3K activation in NIH3T3/ErbB-2/4 cells (36.2 ± 6 versus 80.4 ± 7). The standard deviation of two separate experiments is indicated.

View larger version (9K): [in this window] [in a new window]   Fig. 9.   Analysis of PI3K activity on NIH3T3/ErbB-2/4 cells. A, NIH3T3/ErbB-2/4 cells were maintained in adhesion (lane 1) or treated with wortmannin. Aliquots of cell extracts that contained equivalent amounts of protein were incubated with the anti-Tyr(P) mAb and protein G-Sepharose overnight at 4 °C. After washing, the beads were resuspended in kinase buffer for 20 min at room temperature in presence of L--phosphatidylinositol. The phosphorylated lipids were resolved by thin layer chromatography. B, the percentage of radiolabeled lipids inhibition was determined by densitometry.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recent studies have implicated a key role of the ₆₄ integrin in carcinoma invasion and progression by a mechanism that involves its ability to activate PI3K (19). In addition, we have recently provided evidence that ₆₄ is able to associate with ErbB-2 (28), a growth factor receptor associated with aggressive breast carcinomas (36). In the current study, we defined the mechanism by which ₆₄ and ErbB-2 cooperate to promote invasion using NIH3T3 cells as a model system. We found that the expression of both ₆₄ and ErbB-2 in these cells is required to enhance their PI3K-dependent invasion through Matrigel. More importantly, expression of both ₆₄ and ErbB-2 is also required for the activation of PI3K, an observation that reinforces the importance of PI3K in invasion and has significant implications on how this lipid kinase is activated. The expression of ₄ deletion mutants in NIH3T3/ErbB-2 cells enabled us to identify a specific region within the ₄ cytoplasmic domain (329 amino acids) that is essential for the ability of ₆₄ to stimulate invasion. An important finding, in this context, is that neither the extracellular domain of the ₄ subunit nor ₆₄ heterodimerization are needed for ₆₄ enhancement of invasion. Altogether, our results indicate that the ₄ cytoplasmic domain cooperates with ErbB-2 to activate PI3K and stimulate invasion.

Our finding that the extracellular domain of the ₄ subunit is not required for the stimulation of invasion and activation of PI3K in NIH3T3 cells is interesting in view of recent reports showing the ability of ₆₄ to promote invasion and chemotaxis, which can occur independently of ₆₄ ligation (19, 13). From these observations one can infer that the large ₄ cytoplasmic domain can either initiate signaling events autonomously or influence the function of other receptors such as ErbB-2. This latter possibility is supported by our observation that the c-Myc/₄ cytoplasmic domain chimera is able to associate with ErbB-2 in the absence of the ₄ extracellular domain. Although, the mechanisms by which the ₄ cytoplasmic domain is able to enhance invasion and interact with ErbB-2 are not presently known, the recent findings that the ₄ cytoplasmic is capable of self-association (37) suggest that clustering of this domain could initiate signaling events in the absence of extracellular domain ligation. The independence of the ₄ cytoplasmic domain is also supported by the finding that this domain is sufficient for direct localization of ₄ into adhesive sites (6).

Although the PI3K activation by ₆₄ has been shown to be relevant for invasion, the mechanism by which this or other integrins activate this lipid kinase is not known. In fact, the ₄ cytoplasmic domain lacks the YMXM consensus motif for binding the regulatory p85 subunit of PI3K via SH2 domains. This motif is present in several growth factor receptors that activate PI3K (35, 38). It is clear from our data that both ₆₄ and ErbB-2 are required to induce activation of PI3K. Our finding that the expression of ErbB-2 alone is not sufficient to activate PI3K is in agreement with the report that ErbB-2, by itself, does not recruit PI3K but activates it only after heregulin stimulation and ErbB-2/ErbB-3 dimerization (39-41). Given the fact that heregulin was not present in our experiments, the conclusion can be drawn that the association of ₆₄ with ErbB-2 mimics the ErbB-2/ErbB-3 dimerization that is required for PI3K activation. The fact that ErbB-2 also lacks the consensus motif for p85 binding (42) suggests that the mechanism by which ₆₄ and ErbB-2 cooperate to activate PI3K involves their synergistic activation of signaling intermediates. The identification of the involvement of such signaling intermediates should increase our understanding of PI3K activation and invasion markedly.

Interestingly, expression of both ₆₄ and ErbB-2 in NIH3T3 resulted in the constitutive activation of PI3K in the absence of either ₆₄ ligation or heregulin stimulation. This result reinforces our hypothesis that ₆₄, the ₄ cytoplasmic domain in particular, is able to initiate signaling events in the absence of receptor ligation and clustering. This hypothesis is supported by the recent finding that activation of a cAMP-dependent phosphodiesterase by ₆₄ is independent of ₆₄ ligation (13). Moreover, an increase in the constitutive activation of PI3K upon expression of ₆₄ in a breast carcinoma cell line has been observed (19). In agreement with our findings, constitutive activation of PI3K could be enhanced by antibody-mediated clustering of ₆₄ or by attachment to laminin. It is also apparent from our findings that a threshold of PI3K activation exists for the stimulation of invasion. Specifically, we observed significantly less PI3K activity upon expression of the ₄ L and B mutants in comparison to the wild type and ₄ C, D, and F mutants (Fig. 7), and this activity correlated with the ability of these mutants to stimulate invasion.

An important issue that had not been addressed prior to our study is the identification of a specific region of the large ₄ cytoplasmic domain that is essential for its ability to stimulate invasion and activate PI3K. Indeed, the ₄ cytoplasmic domain has been implicated in many cellular functions including not only invasion and PI3K activation but also hemidesmosome assembly (19, 43), dynamic interactions with F-actin (12), as well as cell proliferation (17) and apoptosis (44, 45). In addition, evidence has been presented for the binding of specific molecules to the ₄ cytoplasmic domain including the adaptor protein Shc and the cytoskeletal-associated protein plectin or HD-1 (46, 47). To date, reasonably good evidence exists for specific sites within the ₄ cytoplasmic domain involved in hemidesmosome assembly and plectin binding (36). However, the ₄ domain involved in invasiveness, which we identified in our study, does not strictly correspond to the ₄ domain able to interact with plectin in the stabilization of the hemidesmosomes.

In conclusion, we have identified a short portion of the ₄ protein sufficient to generate invasive capacity and found that ErbB-2 oncogene and ₄ protein cooperate to generate unexpected levels of PI3K activity, which is at least partially dependent upon signals generated from the ₄ cytoplasmic domain. These findings are relevant not only for the comprehension of malignant phenotype in transformed cells but also for the potential development of specific drugs.

	ACKNOWLEDGEMENTS

We are grateful to Filippo Giancotti for providing us with ₄ deleted cDNAs, Adriana Albini for the Matrigel, and Tom Carey for the anti-₄ A9 antibody. We are also particularly grateful to Robin Bachelder, Kathleen O'Connor, Isaac Rabinovitz, Leslie Shaw, and Silvia Soddu for helpful discussions.

	FOOTNOTES

^* This work was supported by Associazione Italiana Ricerca sul Cancro.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.

^¶ To whom correspondence should be addressed. Tel.: 39-06-4985-2563; Fax: 39-06-4180-526; E-mail: falcioni@crs.ifo.it.

	ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PI3K, phosphatidylinositol 3-kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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