Tyrosine Phosphorylation of the β4 Integrin Cytoplasmic Domain Mediates Shc Signaling to Extracellular Signal-regulated Kinase and Antagonizes Formation of Hemidesmosomes

Ligation of the α6β4 integrin induces tyrosine phosphorylation of the β4 cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of β4 that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr1440, or secondarily Tyr1422, interacts with the SH2 domain of Shc, whereas Tyr1526, or secondarily Tyr1642, interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by α6β4. In addition, mutation of β4 Tyr1526, which binds to the PTB domain of Shc, but not of Tyr1422 and Tyr1440, which interact with its SH2 domain, abolishes the activation of ERK by α6β4. Phenylalanine substitution of the β4 tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of α6β4 in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of β4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type β4. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of β4containing phenylalanine substitutions at Tyr1422 and Tyr1440, at Tyr1526 and Tyr1642, or at all four tyrosine phosphorylation sites. These results suggest that β4 Tyr1526 interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes. Ligation of the α6β4 integrin induces tyrosine phosphorylation of the β4 cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of β4 that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr1440, or secondarily Tyr1422, interacts with the SH2 domain of Shc, whereas Tyr1526, or secondarily Tyr1642, interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by α6β4. In addition, mutation of β4 Tyr1526, which binds to the PTB domain of Shc, but not of Tyr1422 and Tyr1440, which interact with its SH2 domain, abolishes the activation of ERK by α6β4. Phenylalanine substitution of the β4 tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of α6β4 in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of β4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type β4. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of β4containing phenylalanine substitutions at Tyr1422 and Tyr1440, at Tyr1526 and Tyr1642, or at all four tyrosine phosphorylation sites. These results suggest that β4 Tyr1526 interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes. Src homology 2 phosphotyrosine binding epidermal growth factor extracellular signal-regulated kinase monoclonal antibody glutathione S-transferase horseradish peroxidase fluorescein isothiocyanate Dulbecco's modified Eagle's medium fetal bovine serum human umbilical vein endothelial cell phosphate-buffered saline bovine serum albumin polyacrylamide gel electrophoresis Tris-buffered saline fibronectin tyrosine-based activation motif Basement membranes regulate the survival, proliferation, and differentiation of cells through ligation of integrin receptors (1Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2812) Google Scholar, 2Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3809) Google Scholar, 3Howe A. Aplin A.E. Alahari S.K. Juliano R.L. Curr. Opin. Cell Biol. 1998; 10: 220-231Crossref PubMed Scopus (584) Google Scholar, 4Schwartz M.A. J. Cell Biol. 1997; 139: 575-578Crossref PubMed Scopus (304) Google Scholar). The α6β4 integrin is a major receptor for the basement membrane component laminin-5 and is expressed in a variety of epithelial cells, in Schwann cells, in certain endothelial cells, and in CD4− CD8− T cells (5Giancotti F.G. J. Cell Sci. 1996; 109: 1165-1172Crossref PubMed Google Scholar, 6Borradori L. Sonnenberg A. Curr. Opin. Cell Biol. 1996; 8: 647-656Crossref PubMed Scopus (198) Google Scholar). The cytoplasmic domain of β4, which is unusually long and dissimilar in amino acid sequence from the corresponding portions of other integrin β subunits, enables α6β4to recruit the adaptor protein Shc as well as to promote the assembly of hemidesmosomes (5Giancotti F.G. J. Cell Sci. 1996; 109: 1165-1172Crossref PubMed Google Scholar, 6Borradori L. Sonnenberg A. Curr. Opin. Cell Biol. 1996; 8: 647-656Crossref PubMed Scopus (198) Google Scholar). Shc is an SH2/PTB1 domain adaptor protein that couples a variety of receptor and nonreceptor tyrosine kinases, cytokine receptors, immune receptors, and adhesion receptors to Ras signaling (7Bonfin L. Migliacci E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 8Xu Y. Guo D.F. Davidson M. Inagami T. Carpenter G. J. Biol. Chem. 1997; 272: 13463-13466Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In most cases, Shc binds to upstream tyrosine phosphorylated molecules through its SH2 domain, PTB domain, or both. It is then phosphorylated on tyrosine and recruits the Grb2/SOS complex, which can subsequently activate Ras. By this mechanism, Shc participates in mediating the proliferative functions of many receptors. In addition, it regulates cell migration (9Pelicci G. Giordano S. Zhen Z. Salcini A.E. Lanfrancone L. Bardelli A. Panayotou G. Waterfield M.D. Ponzetto C. Pelicci P.G. 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Cell Biol. 1998; PubMed Scopus Google Scholar). have that ligation of α6β4 phosphorylation of the β4 cytoplasmic domain through activation of an Src M. M. and G. in M. L. and G. in Src are to activate Shc S. T. Sci. S. A. 1996; PubMed Scopus Google it is that α6β4 Shc through the Src kinase of binding of Shc to the cytoplasmic domain of β4. We have Shc signaling binding of Shc to the domain. Our results indicate that the SH2 and PTB domain of Shc to in the cytoplasmic domain of β4. The interaction by the PTB domain is for Shc signaling to ERK in whereas that by the SH2 domain is In addition, that phosphorylation of the Shc binding sites in β4 formation of hemidesmosomes. The antibody and the the extracellular domain of β4 C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar, F.G. M.A. S. E. Ruoslahti E. J. Cell Biol. PubMed Scopus Google Scholar). The to on a from of with The and protein from The recombinant antibody from The from the and horseradish peroxidase from The monoclonal antibody to from The to from The to and the from and for and protein from from protein and from human and wild type and versions of β4 the of the The of the of β4, and the versions C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar, L. F.G. Biol. PubMed Scopus Google and A. L. M. J. F.G. J. 1995; PubMed Scopus Google the by the the with the from wild type β4. The by the from wild type β4 with the the The β4 by β4 with and followed by The by β4 with and followed by which an The β4 by β4 with and followed by in an β4 and by of the and or and sites of β4 A. and tyrosine to phenylalanine of β4 then by of or in 804G cells, β4 the of wild type and Shc in the PTB domain or SH2 domain or and by and J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Shc A. C. F.G. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). 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PubMed Google Scholar). by for at with an of and at serum cells or for at with orthovanadate and with and in with phosphatase and and and of for with of of by and to with for at and with Shc SH2 or PTB domain in and A. with the membranes with antibody in and then with protein in with and with the membranes with and with for to phosphorylation of β4, the membranes for at in and with and then by with to the for of β4, membranes with and by with in followed by protein cells serum and as β4 with the from 2 of and to and Far Western as in the or of of phosphorylated or by the of β4 β4 β4 β4 and β4 the binding for the SH2 domain of Shc at in the growth factor receptor and the binding for the PTB domain of Shc in the T J. Biol. 1995; PubMed Scopus Google Scholar, S. J. T. L. J. Biol. Chem. Full Text PDF PubMed Google used as tyrosine phosphorylation of Shc, the cells serum with 2 with at in and in on for with of with of in of of and at for one with the cells and in of with phosphatase and Shc with a by and to The membranes by with of followed by tyrosine phosphorylation of β4, with versions of β4 as to for serum and with orthovanadate and in β4 with of and of protein and by with with for 2 at with and then with at cells with 2 with and in in for at then for at and in and phosphatase and of by and by with an to 804G cells on for in and then in in the of the of The cells with at for and with followed by 2 with a assays with have that the SH2 and PTB domain of Shc can interact with the tyrosine phosphorylated β4 in C. A. M. F.G. 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It is that the in and is for with a tyrosine phosphatase able to the of the β4 tyrosine phosphorylation sites in to the tyrosine kinase that The the fibronectin type and not phosphorylated on can with Src it is that the major tyrosine phosphorylation sites in β4 of the type suggest that the SH2 domain of Shc binds to phosphorylated tyrosines the of β4. The tyrosine phosphorylation sites that to the for binding to the SH2 domain of Shc Z. J. T. M. T. S. L. Biol. PubMed Scopus Google Scholar). We tyrosines and to phenylalanine or in The versions of β4 for to the SH2 domain of Shc. 2 that the SH2 domain of Shc does not to and binds to a to β4 By the SH2 domain of Shc interacts with β4 These results suggest that the SH2 domain of Shc binds to phosphorylated and secondarily to phosphorylated Tyr1422 in the of β4. The PTB domain of Shc binds to phosphorylated tyrosines in the of S. T. Sci. S. A. 1996; PubMed Scopus Google Scholar, J. Biol. 1995; PubMed Scopus Google Scholar). The cytoplasmic domain of β4 one in the the domain and the type and the other of the one in the and the other in the type in 2 the PTB domain of Shc to phosphorylated β4 as as to phosphorylated wild type β4, but it not interact with phosphorylated M. This that the major binding for the PTB domain of Shc in β4 of the and to the motif in the or that in the type versions of β4 phenylalanine substitutions of the tyrosines of sites and by Far Western with the Shc PTB domain 2 Phenylalanine substitution of Tyr1526 or binding of the PTB domain to β4, with substitution of Tyr1526 in a of sites in the binding of PTB domain to β4. These results suggest that the PTB domain of Shc binds to Tyr1526 and secondarily to in β4. competition assays to the of the binding sites for the SH2 or PTB domain of Shc in β4 for used which α6β4. or with to and with the SH2 or PTB domain of Shc in the or of tyrosine phosphorylated the Tyr1422, Tyr1440, Tyr1526, or in β4 used tyrosine phosphorylated the binding sites for the SH2 and PTB domain of Shc in the growth factor receptor and T J. Biol. 1995; PubMed Scopus Google Scholar, S. J. T. L. J. Biol. Chem. Full Text PDF PubMed Google of used in competition growth factor receptor in a in the β4 and β4 the binding of the SH2 domain of Shc to wild type β4 to a that Tyr1422 and are binding sites for the SH2 domain of Shc. It is that the SH2 domain of Shc binds to 2 this tyrosine is phosphorylated Tyr1422 in A. L. M. J. F.G. J. 1995; PubMed Scopus Google Scholar, C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar). the binding for the SH2 domain of Shc in β4. β4 a able to the binding of the SH2 domain of Shc to β4. that the phosphorylated β4 but not the binding of the PTB domain of Shc to wild type β4. as well as phosphorylated with of analysis 2 results indicate that Tyr1526 is the binding for the PTB domain of Shc in β4. Shc signaling by α6β4 required interaction of the SH2 PTB domain of Shc with the β4 cytoplasmic domain, used versions of Shc inactivating in the SH2 domain PTB domain or The SH2 domain mutation the motif and the interaction with whereas the PTB domain mutation interaction with the at and with the at in the motif Z. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). in of the binding of a protein the corresponding domain of Shc to tyrosine phosphorylated β4 in We the of α6β4 to activate in versions of Shc inactivating in or the SH2 and PTB cells with wild type or versions of Shc. cells are and used to α6β4 signaling C. A. M. F.G. J. 1997; PubMed Scopus Google as well as receptor activation of the Shc J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). cells in with the followed by an The recombinant Shc with an antibody and by with in wild type and SH2 Shc phosphorylated on tyrosine in to ligation of α6β4. By the PTB and versions of Shc not phosphorylated on tyrosine upon α6β4 four versions of Shc phosphorylated upon of the cells with as J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These results indicate that a PTB domain is for Shc signaling and that Shc to to β4 through this domain to phosphorylated on tyrosine by the In cells, α6β4 signaling to ERK through Shc C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar). We the binding of Shc to required for activation of this to not α6β4 and can with versions of β4. These cells a for α6β4 signaling cells α6β4 is expressed in certain endothelial cells in (5Giancotti F.G. J. Cell Sci. 1996; 109: 1165-1172Crossref PubMed Google Scholar, 6Borradori L. Sonnenberg A. Curr. Opin. Cell Biol. 1996; 8: 647-656Crossref PubMed Scopus (198) Google Scholar). In addition, ERK is in cell cells as the a of the activation of that α6β4 ERK in a manner in cells with or in with of a of Shc phenylalanine at binding sites. serum the cells and with the in Shc activation of ERK in a that α6β4 signaling to ERK through Shc in We the of phosphorylation of the β4 tyrosines in binding to the SH2 and PTB domain of Shc. phosphopeptide as a major tyrosine phosphorylation in β4 and that β4 is phosphorylated at tyrosines in A. L. M. J. F.G. J. 1995; PubMed Scopus Google C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar). with of phenylalanine substitutions at one or all four tyrosines Tyr1440, Tyr1526, and of binding to Shc and with The phosphorylation of β4 by with Phenylalanine substitution of or Tyr1526 in a in phosphorylation of β4, whereas phenylalanine substitutions at Tyr1422 or a of all four tyrosines to phenylalanine in an in tyrosine the that the not interact with with all phosphorylated tyrosines in β4, results suggest that and Tyr1526 are the major phosphorylation sites in β4. the interaction of Shc with β4 required for signaling to the with in with versions of β4 to to the SH2 domain or the PTB domain of Shc. then and by with in ligation of β4 activation of ligation of wild type β4, whereas ligation of β4 not activation of with a antibody the extracellular domain of β4 and cell analysis not that the versions of β4 expressed at These results that α6β4 signaling to ERK interaction of the PTB domain of Shc with β4. This is with the that α6β4 signaling to ERK through Shc and the activation of Shc by α6β4 a PTB domain the of the PTB binding sites in β4 for activation of in ligation of β4 not activation of whereas ligation of β4 activation of wild type β4. These results indicate that the binding for the PTB domain of Shc, is required for activation of The cytoplasmic domain of β4, and in the the type and the is required for formation of hemidesmosomes C. M. F.G. 1998; PubMed Scopus Google Scholar, L. F.G. Biol. PubMed Scopus Google Scholar). Our that Tyr1422 and a tyrosine-based activation motif or for incorporation of recombinant β4 the by 804G cells in A. L. M. J. F.G. J. 1995; PubMed Scopus Google Scholar). however, that to in the β4 cytoplasmic domain are not required for the formation of by cells L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google Scholar, L. Sonnenberg A. J. Cell Sci. 1998; Google Scholar). this have a of an analysis of the sequence of the used in that the β4 the or in A. L. M. J. F.G. J. 1995; PubMed Scopus Google from a of β4 that an of the amino this sequence is in the membrane of the cytoplasmic domain of β4, which to for incorporation in adhesion L. F.G. Biol. PubMed Scopus Google Scholar, L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google not it sequence The and of the amino to this the in with the mutation incorporation of β4 hemidesmosomes in 804G cells, in with The 804G cells are a cell that α6β4 and in of that the of cells are in a J. Cell Sci. Google Scholar). We 804G cells with human versions of the or of or of cells expressing recombinant of and with the that human but not β4. in the and to hemidesmosomes as as wild type β4 whereas the β4 a to to hemidesmosomes. mutation of the β4 of β4 to hemidesmosomes in the of the of β4. These results indicate that the of Tyr1422 and is not required for incorporation of β4 in as by L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google Scholar). the the mutation and the that Tyr1422 and a in assembly of hemidesmosomes. It is that a of this of the assembly of hemidesmosomes in or in are that adhesion of epithelial cells to the basement membranes and are cell migration J. Cell Sci. Google Scholar, L. Sonnenberg A. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). phenylalanine substitution of the tyrosines that to Shc does not incorporation of the of β4 in it is that the phosphorylation of sites participates in the assembly of hemidesmosomes L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google and We have that with induces tyrosine phosphorylation of β4 and of hemidesmosomes in not a A. M. Y. F.G. J. Cell Biol. 1996; PubMed Scopus Google Scholar). tyrosine phosphorylation of β4 of used 804G cell expressing wild type β4 or the β4 or cells of with or orthovanadate for to tyrosine phosphorylation of β4. orthovanadate is in the of it as a inhibitor of tyrosine whereas in the of it is to the inhibitor S. J. J. G. C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). We used orthovanadate for to cell with orthovanadate tyrosine phosphorylation of wild type β4 but not of the in 804G cells not in orthovanadate of hemidesmosomes in 804G cells expressing wild type β4 but not in expressing of the versions of or In the of most 804G cells expressing wild type β4 and whereas cells expressing the versions of β4 from of hemidesmosomes to expressing the β4 the most whereas expressing the β4 to a degree and expressing the β4 the phosphorylation of tyrosines can to a of hemidesmosomes and a for with for in a and of hemidesmosomes in all four not that the of hemidesmosomes by in 804G cells an mechanism, as A. J. Cell Biol. 1999; PubMed Scopus Google Scholar). Ligation of the α6β4 integrin induces tyrosine phosphorylation of the cytoplasmic domain of β4, recruitment of Shc, and activation of mitogen-activated protein kinase A. L. M. J. F.G. J. 1995; PubMed Scopus Google Scholar, C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar). and that signaling of and epithelial cells C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar, C. M. F.G. 1998; PubMed Scopus Google Scholar). the of Shc in it is to the by which Shc binds to β4 and this binding is required for activation of signaling The results of this that the PTB domain of Shc interacts in a phosphorylation-dependent manner with Tyr1526 in β4, whereas the SH2 domain binds to phenylalanine substitution of Tyr1526 in β4 and of the PTB domain in Shc phosphorylation of Shc and signaling to By mutation of the and binding for the Shc SH2 domain in β4 or of the SH2 domain a on Shc signaling to These suggest that the binding of the PTB domain of Shc to Tyr1526 in β4 is for subsequent phosphorylation of Shc by the kinase and activation of It however, and that the interaction by the SH2 domain of Shc to to the of the of Shc with β4 in The β4 binding for the PTB domain of Shc, Tyr1526, is in the type all type a tyrosine at this type are not in extracellular and and cell receptors growth and but in cytoplasmic and the β4 cytoplasmic Sci. S. A. PubMed Scopus Google Scholar). this is the to a phosphorylation in a type of have the in β4, it is to the of β4 Tyr1526 and amino The to tyrosine that are for interaction with the PTB domain of Shc, the and at the and are to in a the and of the on its in a β this sequence motif is to to in the This its phosphorylation and subsequent interaction with the PTB domain of Shc. In addition, is that the of the β4 binds to a the type and the in L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google Scholar). this interaction in it the type to the membrane and the interaction of the Src kinase with Tyr1526 in β4 and the recruitment of Shc. This Shc to Grb2/SOS in to its Ras. The SH2 domain of Shc interacts in a phosphorylation-dependent manner with and secondarily with Tyr1422 in β4. In with the that sites are to one and well to the for binding to the SH2 domain of Shc results that sites to a the interaction of the SH2 domain of Shc with β4. It is that is in in and in it is phosphorylated A. L. M. J. F.G. J. 1995; PubMed Scopus Google and C. A. M. F.G. J. 1997; PubMed Scopus Google Scholar). on the that phenylalanine substitutions at Tyr1422 and incorporation of β4 in the of 804G cells, the that phosphorylation of which a required for assembly of hemidesmosomes A. L. M. J. F.G. J. 1995; PubMed Scopus Google Scholar). This to with the results of by L. S. Sonnenberg A. J. Cell Biol. 1997; PubMed Scopus Google Scholar, L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google indicate that the phosphorylation of as well as that of Tyr1526 and Tyr1642, disrupts hemidesmosomes or The is that tyrosines of β4 interact with a as L. S. Sonnenberg A. J. Cell Biol. 1997; PubMed Scopus Google Scholar, L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google that to hemidesmosomes. of tyrosines with the of β4 with this component and hemidesmosomes. This is it phenylalanine substitution an phosphorylation of and In a that phenylalanine substitution of tyrosines of with hemidesmosomes L. S. Sonnenberg A. J. Cell Biol. 1997; PubMed Scopus Google Scholar, L. Sonnenberg A. J. Cell Biol. 1998; PubMed Scopus Google and have that it incorporation of a of β4 amino in the of 804G cells. required to this Our results indicate that tyrosine phosphorylation of Shc signaling but formation of that It that of the laminin-5 assembly of hemidesmosomes and cell migration G. J. Science. 1997; PubMed Scopus Google Scholar, J. Cell Biol. 1998; PubMed Scopus Google Scholar, G. J. Cell Biol. PubMed Scopus Google Scholar). It to this α6β4 signaling to Shc. of laminin-5 α6β4 ligation to tyrosine phosphorylation and Shc signaling or The that the of the β4 cytoplasmic domain required for Shc signaling is from that required for formation on the of α6β4 analysis of expressing a of β4 to through Shc but able to promote assembly of hemidesmosomes a of the of α6β4 We and for and of for We are to for in to map the major tyrosine phosphorylation sites of β4 by

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