Anchorage mediated by integrin alpha 6 beta 4 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 alpha 6 beta 4 and alpha 3 beta 1 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 alpha 6 beta 4 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 alpha 6 beta 4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha 6 beta 4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha 3 beta 1 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 alpha 6 beta 4, while motility via alpha 3 beta 1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha 6 beta 4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha 6 beta 4-dependent cell anchorage to laminin 5 at 4 degrees C. In contrast, adhesion at 37 degrees C via alpha 3 beta 1 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, alpha 6 beta 4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton-insoluble cell fraction that lacks alpha 3 beta 1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha 6 beta 4-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.