Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes — Evidence for an autoregulatory mechanism

Abstract

RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo- and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.

Rho GTPases: Regulation and roles in cancer cell biology

“…6 For example, RhoBTB proteins have broad complex, tramtrack, bric a brac (BTB) domains that are able to interact with cullin3, a scaffold protein of ubiquitin ligase complexes involved in protein ubiquitination. 7 All Rho GTPases apart from the RhoBTB subfamily have been reported to alter cell morphology and/or the cytoskeleton when their expression is increased or reduced in cultured cells or model organisms. Here we describe how their activity is regulated, and their functions, particularly relating to cancer.…”

“…It has been shown so far that the BTB domains in RhoBTB proteins are important for the formation of Cullin-RING ubiquitin ligases (CRLs), the most prevalent class of E3 ubiquitin ligases. 7,123 Other BTB-containing proteins interact with cullin3, acting as adaptors that target proteins to the Cullin3-RING ubiquitin ligase complex (CRL3) for ubiquitination and hence degradation. 124 Indeed, RhoBTB2 has been shown to be a substrate for the cullin3 ubiquitin ligase complex.…”

Ursodeoxycholic acid-induced inhibition of DLC1 protein degradation leads to suppression of hepatocellular carcinoma cell growth

“…UDcA may participate in intramolecular interactions between Dlc1 and Usps. rhoBtB proteins, which are members of the rho family of small gtpases, were reported to interact with cullins, which function as scaffolding proteins that bring together the ubiquitin conjugating enzyme and substrate recognition component for ubiquitin mediated degradation by the 26s proteasome (27). However, more studies are required to determine the mechanism by which UDcA inhibits Dlc1 proteasomal degradation.…”

Atypical Rho Family Members

“…The only characterised interaction partner for RhoBTB1 and RhoBTB2 is cullin-3 (Berthold et al 2008a;Wilkins et al 2004) (Fig. 15.4).…”

“…Cullin-3 (Cul3) is a scaffolding protein that links RING E3 ligases to their substrates: BTB-containing proteins act as substrate adaptors in the Cul3 complexes, bringing substrates close to E3 ligases (Lydeard et al 2013). The first BTB domain of RhoBTB proteins interacts with the N-terminal region of Cul3 (Berthold et al 2008a). It is therefore possible that RhoBTBs bring substrates to the Cul3 E3 ligase complex, either through their second BTB domain or the GTP-binding domain ( Fig.…”