ABSTRACT
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.
Subject(s)
Cytoskeleton/genetics , GTPase-Activating Proteins/genetics , Integrins/genetics , Mechanotransduction, Cellular/genetics , Rho Guanine Nucleotide Exchange Factors/genetics , rac1 GTP-Binding Protein/genetics , Animals , COS Cells , Cell Adhesion , Cell Line , Cell Movement , Chlorocebus aethiops , Computational Biology , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Dogs , Fibroblasts/metabolism , Fibroblasts/ultrastructure , GTPase-Activating Proteins/classification , GTPase-Activating Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Integrins/metabolism , Madin Darby Canine Kidney Cells , Mice , Pan troglodytes , Protein Domains , Rats , Rho Guanine Nucleotide Exchange Factors/classification , Rho Guanine Nucleotide Exchange Factors/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation.
