Ubiquitin-mediated regulation of RhoGTPase signalling: IAPs and HACE1 enter the fray.

The EMBO Journal, 14–28 (2012); published online November252011 [PMC free article] [PubMed] Activation of members of the Rho-like family of guanosine triphosphatases GTPases (RhoGTPases) controls diverse physiological processes and is frequently found in cancer, contributing to tumour malignancy, cancer cell migration, invasion and metastasis. While the regulation of nucleotide binding to RhoGTPases is well understood, little is currently known regarding the molecular mechanisms through which RhoGTPase signalling is regulated by ubiquitylation. Two reports in this issue of The EMBO Journal and Developmental Cell now identify inhibitor of apoptosis (IAP) proteins and HACE1 as E3 ubiquitin (Ub)-protein ligases for Rac1 regulating Rac1 levels and activity. Most members of the RhoGTPase protein family function as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound state (Vega and Ridley, 2008). Binding of GTP to RhoGTPases, such as Rac1, triggers a conformational change that allows binding and activation of downstream effector proteins, through which Rac1 modulates actin assembly, actomyosin contractility, and microtubule formation. Activation of RhoGTPases, and hence their potential to interact with downstream signalling molecules, is influenced by a range of auxiliary proteins. Guanine-nucleotide exchange factors (GEFs) catalyse the exchange of GDP for GTP, thus activating the RhoGTPase. By contrast, GTPase-activating proteins (GAPs) promote the intrinsic GTPase activity, thereby accelerating the hydrolysis of GTP to GDP and returning RhoGTPases to their inactive configuration. RhoGTPases can also be kept inactive by association with cytosolic chaperone proteins known as guanine-nucleotide dissociation inhibitors (GDIs), which block the action of GEFs, and extract GDP-bound RhoGTPases from membranes where they may otherwise become activated inappropriately. In addition to modulation by GEFs, GAPs and GDIs, RhoGTPases are subject to regulation via post-translational modification. For example, addition of lipidic groups to their C-termini enhance their interaction with membranes, while conjugation of Ub to RhoGTPases targets them for inactivation via proteasomal degradation (Nethe and Hordijk, 2010). By interrogating the role of IAP proteins in health and disease, Krishnaraj Rajalingam and colleagues noticed that silencing of the E3s XIAP and cIAP1 led to Rac1 stabilisation, elongated morphology and enhanced migration (Oberoi et al, 2012) (see Figure 1). Consistent with this notion, they demonstrate that XIAP and cIAP1 directly bind to Rac1 in a nucleotide-independent manner in vitro and in vivo, and that both these IAPs can conjugate polyubiquitin chains to Rac1, targeting it for proteasomal degradation. They further demonstrate that these IAPs are also required for Rac1 degradation following expression of RhoGDI, and exposure to cytotoxic necrosis factor 1 (CNSF1)—a toxin that is produced by urophathogenic Escherichia coli (UPEC) and targets members of the RhoGTPases, including Rac1, for proteasomal degradation. In the absence of XIAP and cIAPs, however, RhoGDI- and CNSF1-mediated depletion of Rac1 is prevented. IAPs appear not to be the only E3s that target Rac1 for proteasomal degradation following CNSF1 intoxication. Elegant work by Emmanuel Lemichez and colleagues demonstrate that the HECT-type E3 HACE1 is required for CNSF1-mediated depletion of Rac1 (Torrino et al, 2011) (see Figure 1). They further demonstrate that in the absence of HACE1, UPEC fail to efficiently invade endothelial cell monolayers, suggesting that HACE1 plays a major role in host defence against pathogens. The relative contributions of IAPs and HACE1 to Rac1 turnover remain to be clarified, but it is interesting to note that while HACE interacts specifically with active Rac1, IAPs seem to be indiscriminate about the activation state of Rac1. Figure 1 Schematic overviews of Ub-mediated regulation of Rac1. The activity status of Rac1 is determined by its association with GAPs, GEFs and GDIs as well as through post-translational modifications. Ubiquitylation ... IAPs also regulate migration of certain neuronal progenitor cells of the developing cerebellum of zebrafish. Normally, cells migrate from the upper rhombic lip region of the cerebellum to their final destination in several of the tegmental hindbrain nuclei and in the cerebellar cortex (Tahirovic et al, 2010). Strikingly, either expression of a dominant-negative form of Rac1, or overexpression of XIAP, in the upper rhombic lip cells impaired directional migration of these cells to their final destination. The phenotype caused by XIAP overexpression was partially rescued by co-expression of wild-type Rac1, indicating that XIAP exerts its effects through Rac1. The ability of mammalian IAPs to regulate migration via controlling Rac1 appears to be evolutionarily conserved, as the Drosophila IAP1 (DIAP1) has previously been reported to bind to Rac and regulate collective migration of a group of cells in the developing egg chamber (Geisbrecht and Montell, 2004). However, while the report by Oberoi et al, and previous data from the same group (Dogan et al, 2008), suggest that IAPs inhibit cell migration, the data from Drosophila (Geisbrecht and Montell, 2004), and other reports from mammalian systems (Mehrotra et al, 2010; Liu et al, 2011; Lopez et al, 2011), indicate that IAPs actually promote migration. Clarification of this discrepancy will be extremely important, not least because pharmacological inhibitors of IAPs (dubbed Smac mimetics) are undergoing clinical trials for the treatment of cancer (Dynek and Vucic, 2010). While Smac mimetics were developed to inactivate IAPs and induce tumour cell death, the study by Rajalingam and colleagues now suggests that treatment with IAP inhibitors may promote migration of surviving tumour cells and thus increase the risk of metastasis. Unravelling how IAPs regulate cell migration and invasion will be critically important, as metastasis represents the major clinical problem in cancer.