LAMININ-5 AND MODULATION OF KERATIN CYTOSKELETON ARRANGEMENT IN FG PANCREATIC-CARCINOMA CELLS - INVOLVEMENT OF IFAP300 AND EVIDENCE THAT LAMININ-5 CELL INTERACTIONS CORRELATE WITH A DEPHOSPHORYLATION OF ALPHA-6A INTEGRIN/

Under normal culture conditions, epithelial cells of the FG line, deri ved from a pancreatic tumor, characteristically grow in mounds and fai l to flatten efficiently onto their substrate. In such cells, keratin intermediate filaments (Ifs) are concentrated in the perinuclear regio n. Furthermore, the IF associated protein, IFAP300, primarily localize s along these keratin bundles. Additionally, alpha 6 beta 4 integrin h eterodimers localize in streaks or spots towards the edges of cells wh ile alpha 3 beta 1 integrin is predominantly at cell-cell surfaces. Ne ither show any obvious interaction with IF. Remarkably, upon plating F G cells into medium containing soluble rat laminin-5, FG cells rapidly adhere and spread onto their substrate. Moreover, FG cells ''capture' ' rat laminin-5 and place it basally in circles or arcs at areas of ce ll-substrate interaction. Double label immunofluorescence microscopy r eveals colocalization of 1FAP300 as well as alpha 6 beta 4 and alpha 3 beta 1 integrin with the polarized laminin-5. Concomitantly, alpha 6 integrin undergoes dephosphorylation on serine residue 1041. Laminin-5 -induced rapid adhesion can be blocked by antibodies against the alpha 3 integrin subunit. In contrast, while alpha 6 integrin antibodies do not block laminin-5-induced rapid adhesion, they prevent FG cells fro m assuming an epithelial-like morphology. Keratin IF bundles associate with IFAP300-alpha 6 beta 4/alpha 3 beta 1 integrin complexes along t he cell-substratum-attached surface of FG cells coincubated in laminin -5-containing medium. Coprecipitation results suggest that in these co mplexes, 1FAP300 may associate with the alpha 6 beta 4 integrin hetero dimer. Based on our results and published evidence that IFAP300 binds keratin in vitro [Skalli et al., 1994; J. Cell Biol. 125:159-170], we propose that laminin-5/FG cell interaction results in a novel integrin dephosphorylation event, which subsequently induces IFAP300 associati on with alpha 6 beta 4 integrin. IFAP300 then mediates the interaction of Ifs with the cell surface via the alpha 6 beta 4 integrin heterodi mer. (C) 1997 Wiley-Liss, Inc.