The signaling pathways mediated by Rho family GTPases have been implicated in many aspects of cell biology. The specificity of the pathways is achieved in part by the selective interaction between Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. Here, we report a first-generation small-molecule inhibitor of Rac GTPase targeting Rac activation by GEF. The chemical compound NSC23766 was identified by a structure-based virtual screening of compounds that fit into a surface groove of Rac1 known to be critical for GEF specification. In vitro it could effectively inhibit Rac1 binding and activation by the Rac-specific GEF Trio or Tiam1 in a dose-dependent manner without interfering with the closely related Cdc42 or RhoA binding or activation by their respective GEFs or with Rac1 interaction with BcrGAP or effector PAK1. In cells, it potently blocked serum or platelet-derived growth factor-induced Rac1 activation and lamellipodia formation without affecting the activity of endogenous Cdc42 or RhoA. Moreover, this compound reduced Trio or Tiam1 but not Vav, Lbc, Intersectin, or a constitutively active Rac1 mutant-stimulated cell growth and suppressed Trio, Tiam1, or Ras-induced cell transformation. When applied to human prostate cancer PC-3 cells, it was able to inhibit the proliferation, anchorage-independent growth and invasion phenotypes that require the endogenous Rac1 activity. Thus, NSC23766 constitutes a Rac-specific small-molecule inhibitor that could be useful to study the role of Rac in various cellular functions and to reverse tumor cell phenotypes associated with Rac deregulation.
Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.