Search tips
Search criteria 


Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1996 February 2; 132(4): 727–740.
PMCID: PMC2199869

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein


Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells.

Full Text

The Full Text of this article is available as a PDF (3.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams JC, Watt FM. Regulation of development and differentiation by the extracellular matrix. Development. 1993 Apr;117(4):1183–1198. [PubMed]
  • Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed]
  • Burridge K, Petch LA, Romer LH. Signals from focal adhesions. Curr Biol. 1992 Oct;2(10):537–539. [PubMed]
  • Carter WG, Wayner EA. Characterization of the class III collagen receptor, a phosphorylated, transmembrane glycoprotein expressed in nucleated human cells. J Biol Chem. 1988 Mar 25;263(9):4193–4201. [PubMed]
  • Carter WG, Kaur P, Gil SG, Gahr PJ, Wayner EA. Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J Cell Biol. 1990 Dec;111(6 Pt 2):3141–3154. [PMC free article] [PubMed]
  • Carter WG, Wayner EA, Bouchard TS, Kaur P. The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol. 1990 Apr;110(4):1387–1404. [PMC free article] [PubMed]
  • Carter WG, Ryan MC, Gahr PJ. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes. Cell. 1991 May 17;65(4):599–610. [PubMed]
  • Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken. Science. 1995 Apr 14;268(5208):233–239. [PubMed]
  • Damsky CH, Werb Z. Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Curr Opin Cell Biol. 1992 Oct;4(5):772–781. [PubMed]
  • Domloge-Hultsch N, Gammon WR, Briggaman RA, Gil SG, Carter WG, Yancey KB. Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease. J Clin Invest. 1992 Oct;90(4):1628–1633. [PMC free article] [PubMed]
  • Falanga V, Grinnell F, Gilchrest B, Maddox YT, Moshell A. Workshop on the pathogenesis of chronic wounds. J Invest Dermatol. 1994 Jan;102(1):125–127. [PubMed]
  • Filvaroff E, Stern DF, Dotto GP. Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation. Mol Cell Biol. 1990 Mar;10(3):1164–1173. [PMC free article] [PubMed]
  • Filvaroff E, Calautti E, McCormick F, Dotto GP. Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation. Mol Cell Biol. 1992 Dec;12(12):5319–5328. [PMC free article] [PubMed]
  • Franke WW, Goldschmidt MD, Zimbelmann R, Mueller HM, Schiller DL, Cowin P. Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4027–4031. [PubMed]
  • Fuchs E. Epidermal differentiation: the bare essentials. J Cell Biol. 1990 Dec;111(6 Pt 2):2807–2814. [PMC free article] [PubMed]
  • Gil SG, Brown TA, Ryan MC, Carter WG. Junctional epidermolysis bullosis: defects in expression of epiligrin/nicein/kalinin and integrin beta 4 that inhibit hemidesmosome formation. J Invest Dermatol. 1994 Nov;103(5 Suppl):31S–38S. [PubMed]
  • Gipson IK, Spurr-Michaud S, Tisdale A, Elwell J, Stepp MA. Redistribution of the hemidesmosome components alpha 6 beta 4 integrin and bullous pemphigoid antigens during epithelial wound healing. Exp Cell Res. 1993 Jul;207(1):86–98. [PubMed]
  • Guadagno TM, Ohtsubo M, Roberts JM, Assoian RK. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science. 1993 Dec 3;262(5139):1572–1575. [PubMed]
  • Guan JL, Trevithick JE, Hynes RO. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul. 1991 Nov;2(11):951–964. [PMC free article] [PubMed]
  • Hay ED. Extracellular matrix alters epithelial differentiation. Curr Opin Cell Biol. 1993 Dec;5(6):1029–1035. [PubMed]
  • Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. [PubMed]
  • Ingber DE, Prusty D, Frangioni JV, Cragoe EJ, Jr, Lechene C, Schwartz MA. Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J Cell Biol. 1990 May;110(5):1803–1811. [PMC free article] [PubMed]
  • Jones JC, Green KJ. Intermediate filament-plasma membrane interactions. Curr Opin Cell Biol. 1991 Feb;3(1):127–132. [PubMed]
  • Jones JC, Kurpakus MA, Cooper HM, Quaranta V. A function for the integrin alpha 6 beta 4 in the hemidesmosome. Cell Regul. 1991 Jun;2(6):427–438. [PMC free article] [PubMed]
  • Juliano RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993 Feb;120(3):577–585. [PMC free article] [PubMed]
  • Kaur P, Carter WG. Integrin expression and differentiation in transformed human epidermal cells is regulated by fibroblasts. J Cell Sci. 1992 Nov;103(Pt 3):755–763. [PubMed]
  • Kaur P, McDougall JK, Cone R. Immortalization of primary human epithelial cells by cloned cervical carcinoma DNA containing human papillomavirus type 16 E6/E7 open reading frames. J Gen Virol. 1989 May;70(Pt 5):1261–1266. [PubMed]
  • Kornberg LJ, Earp HS, Turner CE, Prockop C, Juliano RL. Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8392–8396. [PubMed]
  • Kornberg L, Earp HS, Parsons JT, Schaller M, Juliano RL. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem. 1992 Nov 25;267(33):23439–23442. [PubMed]
  • Kurpakus MA, Quaranta V, Jones JC. Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing. J Cell Biol. 1991 Dec;115(6):1737–1750. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lin CQ, Bissell MJ. Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J. 1993 Jun;7(9):737–743. [PubMed]
  • Lin TH, Yurochko A, Kornberg L, Morris J, Walker JJ, Haskill S, Juliano RL. The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes. J Cell Biol. 1994 Sep;126(6):1585–1593. [PMC free article] [PubMed]
  • Mainiero F, Pepe A, Wary KK, Spinardi L, Mohammadi M, Schlessinger J, Giancotti FG. Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J. 1995 Sep 15;14(18):4470–4481. [PubMed]
  • Miyamoto S, Akiyama SK, Yamada KM. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science. 1995 Feb 10;267(5199):883–885. [PubMed]
  • Niessen CM, Cremona O, Daams H, Ferraresi S, Sonnenberg A, Marchisio PC. Expression of the integrin alpha 6 beta 4 in peripheral nerves: localization in Schwann and perineural cells and different variants of the beta 4 subunit. J Cell Sci. 1994 Feb;107(Pt 2):543–552. [PubMed]
  • Rankin S, Rozengurt E. Platelet-derived growth factor modulation of focal adhesion kinase (p125FAK) and paxillin tyrosine phosphorylation in Swiss 3T3 cells. Bell-shaped dose response and cross-talk with bombesin. J Biol Chem. 1994 Jan 7;269(1):704–710. [PubMed]
  • Riddelle KS, Hopkinson SB, Jones JC. Hemidesmosomes in the epithelial cell line 804G: their fate during wound closure, mitosis and drug induced reorganization of the cytoskeleton. J Cell Sci. 1992 Oct;103(Pt 2):475–490. [PubMed]
  • Rousselle P, Lunstrum GP, Keene DR, Burgeson RE. Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments. J Cell Biol. 1991 Aug;114(3):567–576. [PMC free article] [PubMed]
  • Ryan MC, Tizard R, VanDevanter DR, Carter WG. Cloning of the LamA3 gene encoding the alpha 3 chain of the adhesive ligand epiligrin. Expression in wound repair. J Biol Chem. 1994 Sep 9;269(36):22779–22787. [PubMed]
  • Schaller MD, Parsons JT. Focal adhesion kinase: an integrin-linked protein tyrosine kinase. Trends Cell Biol. 1993 Aug;3(8):258–262. [PubMed]
  • Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. [PubMed]
  • Sonnenberg A, Calafat J, Janssen H, Daams H, van der Raaij-Helmer LM, Falcioni R, Kennel SJ, Aplin JD, Baker J, Loizidou M, et al. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol. 1991 May;113(4):907–917. [PMC free article] [PubMed]
  • Sonnenberg A, de Melker AA, Martinez de Velasco AM, Janssen H, Calafat J, Niessen CM. Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line and mechanisms involved in adherence of these cells to laminin and kalinin. J Cell Sci. 1993 Dec;106(Pt 4):1083–1102. [PubMed]
  • Sporn SA, Eierman DF, Johnson CE, Morris J, Martin G, Ladner M, Haskill S. Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J Immunol. 1990 Jun 1;144(11):4434–4441. [PubMed]
  • Stepp MA, Spurr-Michaud S, Tisdale A, Elwell J, Gipson IK. Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8970–8974. [PubMed]
  • Streuli CH, Bailey N, Bissell MJ. Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity. J Cell Biol. 1991 Dec;115(5):1383–1395. [PMC free article] [PubMed]
  • Verrando P, Hsi BL, Yeh CJ, Pisani A, Serieys N, Ortonne JP. Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes. Exp Cell Res. 1987 May;170(1):116–128. [PubMed]
  • Verrando P, Blanchet-Bardon C, Pisani A, Thomas L, Cambazard F, Eady RA, Schofield O, Ortonne JP. Monoclonal antibody GB3 defines a widespread defect of several basement membranes and a keratinocyte dysfunction in patients with lethal junctional epidermolysis bullosa. Lab Invest. 1991 Jan;64(1):85–92. [PubMed]
  • Wayner EA, Carter WG. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. [PMC free article] [PubMed]
  • Wayner EA, Carter WG, Piotrowicz RS, Kunicki TJ. The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa. J Cell Biol. 1988 Nov;107(5):1881–1891. [PMC free article] [PubMed]
  • Wayner EA, Gil SG, Murphy GF, Wilke MS, Carter WG. Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes. J Cell Biol. 1993 Jun;121(5):1141–1152. [PMC free article] [PubMed]
  • Werb Z, Tremble PM, Behrendtsen O, Crowley E, Damsky CH. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol. 1989 Aug;109(2):877–889. [PMC free article] [PubMed]
  • Wu H, Parsons JT. Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J Cell Biol. 1993 Mar;120(6):1417–1426. [PMC free article] [PubMed]
  • Zachary I, Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992 Dec 11;71(6):891–894. [PubMed]
  • Zhao Y, Sudol M, Hanafusa H, Krueger J. Increased tyrosine kinase activity of c-Src during calcium-induced keratinocyte differentiation. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8298–8302. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press