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Cooperative Signaling between α6β4Integrin and ErbB-2 Receptor Is Required to Promote Phosphatidylinositol 3-Kinase-dependent Invasion*

Open AccessPublished:April 07, 2000DOI:https://doi.org/10.1074/jbc.275.14.10604
      We previously demonstrated that β4 integrin subunit overexpression increasesin vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large β4 cytoplasmic domain that are involved in the interaction of α6β4 with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of β4 that lacked either all or portions of the β4 cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of β4 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 α6β4 to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the β4 cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of α6β4 integrin to increase invasion. Interestingly, the extracellular domain of β4 is not necessary for α6β4to stimulate invasion. The association of α6β4 with ErbB-2 is dependent upon the β4 cytoplasmic domain and can occur in the absence of α6β4 heterodimerization. Finally, we observed strong activation of PI3K with β4 wild type and with those β4 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 α6β4 and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the β4 cytoplasmic domain (amino acids 854–1183) in this event.
      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
      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 α and β transmembrane subunits that heterodimerize to form different receptors. A single α subunit (e.g.αv or α6) can associate with different β subunits, (β1, β3, β5, or β1, β4, respectively), promoting different ligand binding specificity (
      • Hemler M.E.
      ,
      • Sonnenberg A.
      ,
      • Sonnenberg A.
      • Modderman P.W.
      • Hogervorst F.
      ). The α6β4integrin is a receptor for various isoforms of the basement membrane component laminin (
      • Lee E.C.
      • Lotz M.M.
      • Steele G.D.
      • Mercurio A.M.
      ,
      • Lotz M.M.
      • Corzelius C.A.
      • Mercurio A.M.
      ,
      • Spinardi L.
      • Einheber S.
      • Cullen T.
      • Milner T.A.
      • Giancotti F.G.
      ), and its expression is restricted to epithelia, endothelia and peripheral nerves (
      • Sonnenberg A.
      • Linders C.J.T.
      • Daams J.H.
      • Kennel S.J.
      ,
      • Kennel S.J.
      • Falcioni R.
      • Wesley J.W.
      ,
      • Quattrini A.
      • Previtali S.
      • Feltri M.L.
      • Canal N.
      • Nemni R.
      • Wrabetz L.
      ). In many epithelia, α6β4 is found in hemidesmosomes where it plays an essential role in their organization (
      • Spinardi L.
      • Ren Y.L.
      • Sanders R.
      • Giancotti F.G.
      ,
      • Schaapveld R.Q.
      • Borradori L.
      • Geerts D.
      • van Leusden M.R.
      • Kuikman I.
      • Nievers M.G.
      • Niessen C.M.
      • Steenbergen R.D.
      • Snijders P.J.
      • Sonnenberg A.
      ). This integrin can also interact with F-actin and promote the migration of invasive carcinoma cells (
      • Rabinovitz I.
      • Mercurio A.M.
      ,
      • O'Connor K.L.
      • Shaw L.M.
      • Mercurio A.M.
      ).
      The intracellular portion of the β4 subunit is much larger (1,000 amino acids) than that of all the other known β subunits, and it does not exhibit apparent sequence homology with them (
      • Suzuki S.
      • Naitoh Y.
      ,
      • Feltri M.L.
      • Arona M.
      • Scherer S.S.
      • Wrabetz L.
      ,
      • Hogervorst F.
      • Kuikman I.
      • von dem Borne A.E.
      • Sonnenberg A.
      ). Increasing evidence indicates that the ability of α6β4 to regulate cell proliferation, motility, and invasion is dependent upon signal transduction events that are mediated by the β4 cytoplasmic domain (
      • Mainiero F.
      • Murgia C.
      • Wary K.K.
      • Curatola A.M.
      • Pepe A.
      • Blumemberg M.
      • Westwick J.K.
      • Der C.J.
      • Giancotti F.G.
      ,
      • Vivinus-Nebot M.
      • Ticchioni M.
      • Mary F.
      • Hofman P.
      • Quaranta V.
      • Rousselle P.
      • Bernard A.
      ,
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ). Of particular relevance to cancer, α6β4 has been implicated in carcinoma invasion (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ,
      • Chao C.
      • Lotz M.M.
      • Clarke A.C.
      • Mercurio A.M.
      ,
      • Falcioni R.
      • Sacchi A.
      • Resau J.
      • Kennel S.J.
      ) through its ability to activate PI3K (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ). Moreover, the ability of α6β4 to promote invasion is dependent upon the β4 cytoplasmic domain. The importance of α6β4 in malignancy is also indicated by the finding that its expression correlates with the progression of squamous, ovarian, thyroid, gastric, and colorectal carcinomas (
      • Kimmel K.A.
      • Carey T.E.
      ,
      • Bottini C.
      • Miotti S.
      • Fiorucci S.
      • Facheris P.
      • Menàrd S.
      • Colnaghi M.I.
      ,
      • Serini G.
      • Trusolino L.
      • Saggiorato E.
      • Cremona O.
      • Derossi M.
      • Angeli A.
      • Orlandi F.
      • Marchisio P.C.
      ,
      • Tani T.
      • Karttunen T.
      • Kiviluoto T.
      • Kivilaakso E.
      • Burgeson R.E.
      • Sipponen P.
      • Virtanen I.
      ,
      • Falcioni R.
      • Turci V.
      • Vitullo P.
      • Navarra G.
      • Ficari F.
      • Cavaliere F.
      • Sacchi A.
      • Mariani-Costantini R.
      ,
      • Rabinovitz I.
      • Mercurio A.M.
      ).
      Function and signaling properties of α6β4in carcinoma cells are influenced by its association with growth factor receptors. Specifically, we demonstrated that α6β4 co-immunoprecipitates with ErbB-2 in human mammary and ovarian carcinoma cell lines and that ligation of this integrin increases ErbB-2 phosphorylation (
      • Falcioni R.
      • Antonini A.
      • Nisticò P.
      • Di Stefano S.
      • Crescenzi M.
      • Natali P.G.
      • Sacchi A.
      ). Moreover, we also observed that overexpression of ErbB-2 and α6β4 in NIH3T3 cells increased their invasive capacity (
      • Falcioni R.
      • Antonini A.
      • Nisticò P.
      • Di Stefano S.
      • Crescenzi M.
      • Natali P.G.
      • Sacchi A.
      ). In the present study, we sought to identify the portion of β4 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 β4 integrin. In vitro invasion assays demonstrated that the portion of β4 protein involved in the invasive capacity resides in 329 cytoplasmic residues between the amino acids 824 and 1183. Biochemical analysis indicated that ErbB-2/β4 interaction is abolished uniquely when the entire cytoplasmic domain of β4 is deleted. Thus, we were able to exclude that the interaction of ErbB-2 and β4 is responsible for increased malignancy of NIH3T3/ErbB-2/β4cells. The study of the mechanisms by which β4 cooperates with ErbB-2 to promote increased malignancy showed that the α6β4 integrin activates the PI3K pathway when both ErbB-2 and β4 are overexpressed.

      DISCUSSION

      Recent studies have implicated a key role of the α6β4 integrin in carcinoma invasion and progression by a mechanism that involves its ability to activate PI3K (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ). In addition, we have recently provided evidence that α6β4 is able to associate with ErbB-2 (
      • Falcioni R.
      • Antonini A.
      • Nisticò P.
      • Di Stefano S.
      • Crescenzi M.
      • Natali P.G.
      • Sacchi A.
      ), a growth factor receptor associated with aggressive breast carcinomas (
      • Hynes N.E.
      • Stern D.F.
      ). In the current study, we defined the mechanism by which α6β4 and ErbB-2 cooperate to promote invasion using NIH3T3 cells as a model system. We found that the expression of both α6β4 and ErbB-2 in these cells is required to enhance their PI3K-dependent invasion through Matrigel. More importantly, expression of both α6β4 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 β4 deletion mutants in NIH3T3/ErbB-2 cells enabled us to identify a specific region within the β4 cytoplasmic domain (329 amino acids) that is essential for the ability of α6β4 to stimulate invasion. An important finding, in this context, is that neither the extracellular domain of the β4 subunit nor α6β4 heterodimerization are needed for α6β4 enhancement of invasion. Altogether, our results indicate that the β4 cytoplasmic domain cooperates with ErbB-2 to activate PI3K and stimulate invasion.
      Our finding that the extracellular domain of the β4subunit 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 α6β4 to promote invasion and chemotaxis, which can occur independently of α6β4 ligation (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ,
      • O'Connor K.L.
      • Shaw L.M.
      • Mercurio A.M.
      ). From these observations one can infer that the large β4 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/β4cytoplasmic domain chimera is able to associate with ErbB-2 in the absence of the β4 extracellular domain. Although, the mechanisms by which the β4 cytoplasmic domain is able to enhance invasion and interact with ErbB-2 are not presently known, the recent findings that the β4 cytoplasmic is capable of self-association (
      • Gunther A.R.
      • de Pereda J.M.
      • Reipert S.
      • Wiche G.
      ) suggest that clustering of this domain could initiate signaling events in the absence of extracellular domain ligation. The independence of the β4 cytoplasmic domain is also supported by the finding that this domain is sufficient for direct localization of β4 into adhesive sites (
      • Spinardi L.
      • Einheber S.
      • Cullen T.
      • Milner T.A.
      • Giancotti F.G.
      ).
      Although the PI3K activation by α6β4 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 β4 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 (
      • Carraway III, K.L.
      • Soltoff S.P.
      • Diamonti A.J.
      • Cantlet L.C.
      ,
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ). It is clear from our data that both α6β4 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 (
      • Tan M.
      • Grijalva R.
      • Yu D.
      ,
      • Alimandi M.
      • Romano A.
      • Curia M.C.
      • Muraro R.
      • Fedi P.
      • Aaronson S.A.
      • Di Fiore P.P.
      • Kraus M.H.
      ,
      • Ram T.G.
      • Ethier S.P.
      ). Given the fact that heregulin was not present in our experiments, the conclusion can be drawn that the association of α6β4 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 (
      • Bargmann C.I.
      • Hung M.C.
      • Weinberg R.A.
      ) suggests that the mechanism by which α6β4 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 α6β4 and ErbB-2 in NIH3T3 resulted in the constitutive activation of PI3K in the absence of either α6β4 ligation or heregulin stimulation. This result reinforces our hypothesis that α6β4, the β4 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 α6β4 is independent of α6β4 ligation (
      • O'Connor K.L.
      • Shaw L.M.
      • Mercurio A.M.
      ). Moreover, an increase in the constitutive activation of PI3K upon expression of α6β4 in a breast carcinoma cell line has been observed (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ). In agreement with our findings, constitutive activation of PI3K could be enhanced by antibody-mediated clustering of α6β4 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 β4 L and B mutants in comparison to the wild type and β4 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 β4cytoplasmic domain that is essential for its ability to stimulate invasion and activate PI3K. Indeed, the β4 cytoplasmic domain has been implicated in many cellular functions including not only invasion and PI3K activation but also hemidesmosome assembly (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.F.
      • Toker A.
      • Mercurio A.M.
      ,
      • Schaapveld R.Q.J.
      • Borradori L.
      • Geerts D.
      • van Leusden M.R.
      • Kuikman I.
      • Nierves M.G.
      • Niessen C.M.
      • Steenbergen R.D.M.
      • Snijders P.J.F.
      • Sonnenberg A.
      ), dynamic interactions with F-actin (
      • Rabinovitz I.
      • Mercurio A.M.
      ), as well as cell proliferation (
      • Mainiero F.
      • Murgia C.
      • Wary K.K.
      • Curatola A.M.
      • Pepe A.
      • Blumemberg M.
      • Westwick J.K.
      • Der C.J.
      • Giancotti F.G.
      ) and apoptosis (
      • Clarke A.S.
      • Lotz M.M.
      • Chao C.
      • Mercurio A.M.
      ,
      • Bachelder R.E.
      • Marchetti A.
      • Falcioni R.
      • Soddu S.
      • Mercurio A.M.
      ). In addition, evidence has been presented for the binding of specific molecules to the β4 cytoplasmic domain including the adaptor protein Shc and the cytoskeletal-associated protein plectin or HD-1 (
      • Mainiero F.
      • Pepe A.
      • Yeon M.
      • Ren Y.
      • Giancotti F.G.
      ,
      • Niessen C.M.
      • Hulsman E.H.M.
      • Oomen L.C.J.
      • Kuikman I.
      • Sonnenberg A
      ). To date, reasonably good evidence exists for specific sites within the β4 cytoplasmic domain involved in hemidesmosome assembly and plectin binding (
      • Hynes N.E.
      • Stern D.F.
      ). However, the β4 domain involved in invasiveness, which we identified in our study, does not strictly correspond to the β4 domain able to interact with plectin in the stabilization of the hemidesmosomes.
      In conclusion, we have identified a short portion of the β4 protein sufficient to generate invasive capacity and found that ErbB-2 oncogene and β4 protein cooperate to generate unexpected levels of PI3K activity, which is at least partially dependent upon signals generated from the β4cytoplasmic 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.

      Acknowledgments

      We are grateful to Filippo Giancotti for providing us with β4 deleted cDNAs, Adriana Albini for the Matrigel, and Tom Carey for the anti-β4 A9 antibody. We are also particularly grateful to Robin Bachelder, Kathleen O'Connor, Isaac Rabinovitz, Leslie Shaw, and Silvia Soddu for helpful discussions.

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