Differential regulation of GEF-H1 in pKO-αv and pKO-β1 fibroblasts

HAGA BRANDAO, R.; FÄSSLER, R; COLÓ, GP

Lucca

Conferencia; Fibronectin, Integrins and Related Molecules. Gordon Research Conference; 2019

Gordon Research Conference

Integrins are cell adhesionmolecules expressed on all cells. Upon integrin activation they recruit numerousadaptor and signaling proteins, which in turn trigger signaling outputs thathave profound consequences for cell behavior. To betterunderstand the role of individual integrins, we generated pan-integrin knockoutmouse fibroblasts (pKO) that lack the expression of all 24 integrins. Uponre-expression of individual integrin subunits, we used them to generate either α5β1(pKO-β1) and/or αv-class (pKO-αv) integrin expressing cells. Interestingly,α5β1 expressing pKO-β1 cells formed abundant lamellipodia, almost exclusivelynascent adhesions and low RhoA activation with intermediate force generation, whereasαv-class integrin expressing pKO-αv cells assembled thick stress fiberformation, large focal adhesions and showed highRhoA activation associated with low contractibility when grown on fibronectin.Cells expressing both integrin classes (pKO-αv,β1 cells) presented anintermediate phenotype with high force generation, intermediate RhoA activation,small and large adhesion sites similar to wild type fibroblasts. To further analyze thephenotypes of our genetically engineered cell lines, we compared theirproteomes by mass spectrometry and found GEF-H1 (Lfc in mouse), a RhoA-GEF shownto cross-talk between microtubules and the actin cytoskeleton, to be elevatedin pKO-αv cells. Microtubule-bound GEF-H1 is inactive, whereas once released intothe cytoplasm it becomes active and activates RhoA, which leads to increasedstress fiber formation. Our data suggest that high GEF-H1 levels in thecytoplasm of pKO-αv cells is responsible for their increased stress fiber content.Since GEF-H1 was shown to be released to the cytoplasm either by microtubuledepolymerization or by its phosphorylation/dephosphorylation, we performed a phospho-enrichmentmass spectrometry analysis, which revealed that GEF-H1 is differentiallyphosphorylated in pKO-αv and pKO-β1 grown on fibronectin. Our results suggestthat the fibronectin-induced activation of αvβ3, but not α5β1, leads to increasedactivation of GEF-H1 that contributes to the pKO-av phenotype.