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 α6a integrin

Under normal culture conditions, epithelial cells of the FG line, derived from a pancreatic tumor, characteristically grow in mounds and fail to flatten efficiently onto their substrate. In such cells. . keratin intermediate filaments (IFs) are concentrated in the perinuclear region. Furthermore, the IF associated protein, IFAP300, primarily localizes along these keratin bundles. Additionally, α6β4 integrin heterodimers localize in streaks or spots towards the edges of cells while α3β1 integrin is predominantly at cell-cell surfaces. Neither show any obvious interaction with IF. Remarkably, upon plating FG cells into medium containing soluble rat laminin-5, FG cells rapidly adhere and spread onto their substrate. Moreover, FG cells \u27capture\u27 rat laminin-5 and place it basally in circles or arcs at areas of cell-substrate interaction. Double label immunofluorescence microscopy reveals colocalization of IFAP300 as well as α6β4 and α3β1 integrin with the polarized laminin-5. Concomitantly, α6 integrin undergoes dephosphorylation on serine residue 1041. Laminin-5-induced rapid adhesion can be blocked by antibodies against the α3 integrin subunit. In contrast, while α6 integrin antibodies do not block laminin-5-induced rapid adhesion, they prevent FG cells from assuming an epithelial-like morphology. Keratin IF bundles associate with IFAP300-α6β4/α3β1 integrin complexes along the cell-substratum-attached surface of FG cells coincubated in laminin-5- containing medium. Coprecipitation results suggest that in these complexes, IFAP300 may associate with the α6β4 integrin heterodimer. 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 association with α6β4 integrin. IFAP300 then mediates the interaction of iFs with the cell surface via the α6β4 integrin heterodimer