Autoubiquitination of BCA2 RING E3 Ligase Regulates Its Own Stability and Affects Cell Migration

Abstract

Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING-type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2-binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, the majority of which were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitin A-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the NH 2 terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared with wild-type BCA2. On the basis of these findings, we propose that BCA2 might be an important factor regulating breast cancer cell migration/metastasis. We put forward a novel model for BCA2 E3 ligase -mediated cell regulation. (Mol Cancer Res 2008;6(9):1385 -96)

Unique Requirements for Mono- and Polyubiquitination of the Peroxisomal Targeting Signal Co-receptor, Pex20

“…Among the peroxins, Pex2 and Pex10 possess a canonical RING domain in most species (31)(32)(33)(34), whereas in Pex12, only five of the eight conserved residues are present (35)(36)(37), suggesting that Pex12 can bind only one zinc atom per monomer. The RING peroxins are required for each other's stability (19,20,38), whereas the first two Cys residues of the RING domain are known to be essential for their E3 ligase activity (39).…”

The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal sorting of the Epidermal Growth Factor Receptor

“…1A). Rabring7 has previously been shown to have RINGfinger-dependent auto-ubiquitylation activity (Amemiya et al, 2008;Burger et al, 2005;Sakane et al, 2007). To determine whether RNF126 exhibits RING-finger-dependent E3 ligase activity, we used in vitro auto-ubiquitylation assays with the E2 enzyme UbcH5b.…”

“…In addition to a C-terminal RING finger domain, an N-terminal zinc finger domain in both RNF126 and Rabring7 has been predicted (Amemiya et al, 2008;Burger et al, 2006). To disrupt this domain, we employed site directed mutagenesis to substitute two cysteine residues that are predicted to chelate a zinc ion to alanines.…”

“…The zinc finger domain of RNF126 and Rabring7 bind directly to ubiquitin Recent structural studies have defined a role for zinc finger domains as ubiquitin interaction modules and Rabring7 has been shown to associate with ubiquitin (Amemiya et al, 2008;Burger et al, 2006;Dikic et al, 2009). This lead us to further characterize the ability of RNF126 and Rabring7 zinc finger domains to bind to ubiquitin.…”

The Pepper E3 Ubiquitin Ligase RING1 Gene,CaRING1, Is Required for Cell Death and the Salicylic Acid-Dependent Defense Response

“…Therefore, the reduced level of ubiquitination may be due to deletion of the TM domain (residues 1-78), and UbPred programs (http://ubpred.org/) reveal that this region of CaRING1 contains a predicted ubiquitination site (residue 15). BCA1, a RING-type E3 ubiquitin ligase in human cells, is known to regulate its own stability by autoubiquitination (Amemiya et al, 2008). Unexpectedly, however, the mutation of CaRING1 at the Lys-15 residue in the TM region did not abrogate the ubiquitination of CaRING1 K15R (Supplemental Fig.…”