(1) Following ECM binding and integrin activation, Rgnef may be recruited to the leading edge of cells by increased plasma membrane phosphatidylinositol lipids (red ovals) through the Rgnef PH domain name (red region within Rgnef)
(1) Following ECM binding and integrin activation, Rgnef may be recruited to the leading edge of cells by increased plasma membrane phosphatidylinositol lipids (red ovals) through the Rgnef PH domain name (red region within Rgnef). adhesions upon re-plating on FN. Rgnef re-expression rescues these defects, but requires RgnefCFAK binding. A mutation in the Rgnef pleckstrin homology (PH) domain name inhibits adhesion formation, FAK localization, and FAK-Y397 and paxillin-Y118 phosphorylation without disrupting the RgnefCFAK conversation. 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 unique 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. (Cai et al., 2008). When activated, FAK binds to a variety of signaling (Src), adaptor [p130Cas (also known as BCAR1), Grb2], and cytoskeletal proteins (paxillin, cortactin), some of which can also bind to phosphatidylinositol lipids (Schaller, 2010; Schlaepfer and Mitra, 2004). Although multiple and overlapping protein binding interactions might stabilize integrin signaling complexes (FAK binds p130Cas, Src binds p130Cas, and FAK binds Src), a subset of FAK protein binding interactions are unique (Schaller, 2010). One of these is with Rgnef (also known as p190RhoGEF or Arhgef28), a ubiquitous Rho guanine-nucleotide-exchange factor (GEF) that contains central Dbl and pleckstrin homology domains (DH and PH, respectively) linked to Rho GTPase activation and lipid binding, respectively (van Horck et al., 2001). FAK binds to Rgnef residues 1292C1301 and this direct interaction is not shared with other GEFs (Zhai et al., 2003). Rgnef activates RhoA and RhoC GTPases (Bravo-Cordero et al., 2011; van Horck et al., 2001), which function as bi-molecular switches alternating between inactive GDP- and active GTP-bound says (Hall, 1998). It is the coordination of GEF and GTPase-activating protein (Space) activity that control cycles of Rho GTPase activation. Cell adhesion to ECM through integrins promotes quick FAKCSrc activation, p190RhoGAP Benorylate (also known as ARHGAP35) tyrosine phosphorylation, and Space activation leading Rabbit Polyclonal to HEY2 to the transient RhoA inhibition during cell distributing (Arthur et al., 2000; Huveneers and Danen, 2009; Ren et al., 2000; Tomar et al., 2009). The formation and maturation of adhesions are required for optimal cell migration and this depends upon RhoA reactivation occurring 1?hour after cell adhesion to ECM (Ren et al., 1999). Mutation of the Rgnef DH domain name (Y1003A) eliminates Rgnef exchange activity (van Horck et al., 2001) and prevents Rgnef-mediated activation of RhoA in cells (Lim et al., 2008b). Rgnef knockout and studies with Rgnef-null mouse embryonic fibroblasts (MEFs) confirm that Rgnef is usually a key regulator of RhoA reactivation and adhesion establishment downstream of integrins (Miller et al., 2012). Studies with MEFs or dominant-negative inhibition of RgnefCFAK binding in colon cancer cells show that this signaling axis is usually important for normal and tumor cell motility (Lim et al., 2008b; Miller et al., 2012; Yu et al., Benorylate 2011). Herein, we show that Rgnef expression facilitates FAK localization to early adhesion sites and FAK Y397 phosphorylation upon MEF binding to fibronectin (FN). Analyses of Rgnef re-expression in Rgnef-null MEFs show that FAK activation is dependent upon RgnefCFAK binding and Rgnef-PH domain name function. However, Rgnef-mediated FAK Y397 phosphorylation and localization is usually impartial of intrinsic Rgnef GEF activity. Our results support a new, non-canonical, GEF-independent and PH-domain-associated scaffolding role for Rgnef in promoting FAK localization and activation at early adhesions. Results Rgnef promotes FAK localization to early adhesions and FAK Y397 phosphorylation Integrin-stimulated protein tyrosine phosphorylation occurs rapidly upon MEF adhesion to FN and continues during the processes of cell distributing. To elucidate biochemical signaling changes occurring upon cell adhesion to FN, lysates of wild-type (Rgnef+/+) and Rgnef-null (Rgnef?/?) MEFs were made from cells held in suspension or suspended and then re-plated on FN-coated plates for 5, 15 and 30?moments (Fig.?1A). FAK activation, as measured by increased FAK Y397 phosphorylation, is usually observed within 5?moments upon MEF adhesion and reaches a maximum level within 30?moments during Rgnef+/+ MEF spreading on FN (Fig.?1A; data pooled from three impartial experiments in Fig.?1B). Interestingly, FAK Y397 phosphorylation is usually significantly attenuated in lysates of Rgnef?/? compared to Rgnef+/+ MEFs at 30?moments on FN (Fig.?1A,B). A similar reduction in FAK Y397 phosphorylation occurs upon Rgnef knockdown in HEY human carcinoma cells when plated on Benorylate FN (Fig.?1C). These results implicate Rgnef in the early signaling events following integrin binding to FN. Notably, MEF adhesion and distributing between 5 and 30?moments on FN is associated with low levels of Rgnef and RhoA GTPase activity (Miller et al., 2012; Ren et al., 2000). Rgnef becomes activated by 60?moments on FN and this corresponds to increased RhoA GTPase activity.