Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho 
guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK). 
FAK is recruited to adhesions and activated by integrin receptors binding to 
matrix proteins, such as fibronectin (FN). Canonical models place Rgnef 
downstream of integrin-FAK signaling in regulating Rho GTPase activity and cell 
movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK 
localization to early peripheral adhesions and promoting FAK activation upon FN 
binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion 
formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to 
peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these 
defects, but requires Rgnef-FAK binding. A mutation in the Rgnef pleckstrin 
homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397 
and paxillin-Y118 phosphorylation without disrupting the Rgnef-FAK interaction. 
A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion 
localization, but not paxillin-Y118 phosphorylation. This suggests that, 
downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a 
mechanism distinct from adhesion formation and FAK activation. These results 
support a scaffolding role for Rgnef in FAK localization and activation at early 
adhesions in a PH-domain-dependent but GEF-activity-independent manner.