Low substratum rigidity of collagen gel promotes ERK phosphorylation via lipid raft to augment cell migration.

Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid raft. MbetaCD (Methyl-beta-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation, and cell migration. Overexpression of FAK C-terminal fragment (FRNK) in MDCK cells triggered ERK phosphorylation. Meanwhile, low substratum rigidity induced degradation of FAK into a 35 kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MbetaCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft.