Fgd5 is a Rac1-specific Rho GEF that is selectively inhibited by aurintricarboxylic acid

Abstract

Rho proteins are signalling molecules that control cellular dynamics, movement and morphological changes. They are activated by Rho guanine-nucleotide exchange factors (Rho GEFs) that transduce upstream signals into Rho-mediated activation of downstream processes. Fgd5 is a Rho GEF involved in angiogenesis and its target Rho protein for this process has been linked to Cdc42 activation. Here, we examined the function of purified Fgd5, specifically, which Rho proteins it activates and pinpoint the structural domains required for enzymatic activity. Using a GEF enzyme assay, we found that purified Fgd5 showed preferential activation of Rac1 and direct binding of Rac1 in pull-down and co-immunoprecipitation assays. Structural comparisons showed that the Fgd5 DH domain is highly similar to the Rac1 GEF, TrioN, supporting a role for Fgd5 as a Rac1 GEF. Compounds that bind to purified Fgd5 DH-PH protein were identified by screening a small molecule library via surface plasmon resonance. The effects of eleven ligands were further examined for their ability to inhibit the Fgd5 GEF enzymatic activity and Rac1 interaction. From these studies, we found that the compound aurintricarboxylic acid, and to a lesser extent mitoxantrone dihydrochloride, inhibited both Fgd5 GEF activation of Rac1 and their interaction. Aurintricarboxylic acid had no effect on the activity or binding of the Rac1 GEF, TrioN, thus demonstrating the feasibility of selectively disrupting Rho GEF activators.

Targeting Rac and Cdc42 GEFs in Metastatic Cancer

“…From the human FYVE domain containing proteins, those within the Fgd subfamily act as Rac/Cdc42 GEFs due to their DH domain and two PH domains (Eitzen et al, 2019). Recently, biochemical analysis of Fgd5, a member of the Fgd family of proteins, showed specificity for Rac1 activation (Park et al, 2019), even though FGD1 has been described as a GEF for Cdc42 (Egorov et al, 2009). Fgd5 has a DH domain similar to Trio with preferential activation of Rac1, and has been related to poorer prognosis in breast cancer patients (Valla et al, 2017).…”

“…Fgd5 has a DH domain similar to Trio with preferential activation of Rac1, and has been related to poorer prognosis in breast cancer patients (Valla et al, 2017). Park et al (2019) identified the small molecule aurintricarboxylic acid through a surface plasmon resonance screening as a compound that binds to Fgd5, thus inactivating Rac1 with an IC 50 of 157 nM. Further studies are needed to validate these results in vivo and to delineate the utility of targeting Fgd5 to block cancer progression.…”

FGD5 regulates endothelial cell PI3 kinase‐β to promote neo‐angiogenesis

“…Initial work identified FGD5‐dependent Cdc42‐GTP increased in VEGF‐stimulated EC 23 . A more recent study shows FGD5 preferentially elicits GTP exchange on Rac1 in vitro 31 . Our data indicate that FGD5 RhoGEF activity is required to elicit CXCL12‐stimulated PI3 kinase‐β activity in EC, since a FGD5 Dbl domain‐deletant both acted as a dominant‐negative and failed to rescue native endothelial FGD5 loss.…”

“…FGD5 RhoGEF activity has been attributed to Cdc42, but recent work using the truncated RhoGEF Dbl-PH domains from FGD5, identified that FGD5 acts as an exchange factor for Rac1 in preference to Cdc42 in vitro. 31 First, EC were pre-treated with the Rac1 inhibitor, EHT-1864 (10 µM), or carrier, then stimulated with CXCL12. We found the Rac1 inhibitor markedly blunted CXCL12-stimulated Akt phosphorylation versus the carrier control treatment (Figure 4A).…”

Upregulation of FGD6 Predicts Poor Prognosis in Gastric Cancer

“…There are seven members of the FGD family, namely, FGD1, FGD2, FGD3, FGD4, FGD5, FGD6, and FRG (9). For example, several researches have revealed that FGD family proteins regulated culler functions by specifically activating Cdc42 (10)(11)(12).…”