Molecular Characterization of Ring Finger Protein 11

Abstract

Ring finger proteins serve many vital functions within the cell. We have identified RNF11, a novel 154-amino acid ring finger -containing protein, which is elevated in breast cancer. Within its ring finger domain, RNF11 contains an AKT phosphorylation site (T135) that is situated within a 14-3-3 binding domain. In WM239 cells with constitutively active AKT, RNF11 exhibits seven distinct phosphopeptides as measured using two-dimensional phosphopeptide mapping. Upon inhibition of the AKT pathway or mutation of T135, the phosphorylation at one of these sites is virtually eliminated, suggesting that AKT may phosphorylate RNF11 at T135. Moreover, RNF11 is phosphorylated by AKT in vitro and is recognized by phospho-AKT substrate antibodies.

Effects of partner proteins on BCA2 RING ligase activity

“…Phosphorylation has far reaching consequences for the modified proteins, amoung them are conformational changes which effect substrate affinity and specificity [28,29]. It had been previously demonstrated that BCA2 was phosphorylated in the presence of AKT [8]. Here, we showed that BCA2 was more stable when co-expressed with constitutively active AKT as opposed to kinase-dead AKT.…”

“…BCA2 was shown to be phosphorylated in the AKT domain [8]. To investigate the role of phosphorylation on BCA2 stability, HEK293T cells were co-transfected with a constitutively active variant of AKT or a kinase-dead AKT mutant, along with FLAG-tagged BCA2 (Figure 5A, lanes 1 and 2) or the S132, 133A mutant which abolished AKT phosphorylation (Figure 5A, lanes 3 and 4).…”

“…14-3-3σ construct was created by cutting the 14-3-3σ gene via restriction digest from a pre-existing bacterial expression vector pET100 containing the XPRESS-expression tag. AKT vectors were generously donated to our laboratory [8]. FLAG BCA2 vectors are as described [7].…”

“…BCA2 contains three domains, the amino-terminal B CA2 Z inc- F inger (BZF) domain, the AKT phosphorylation domain, and the carboxy-terminal RING H2 domain (Figure 1A) [7,8]. BCA2's RING domain confers autoubiquitination activity, consistent with other E3 ubiquitin ligases such as RING proteins MDM2 and SIAH1 [2,9,10].…”

Expansion and Diversification of BTL Ring-H2 Ubiquitin Ligases in Angiosperms: Putative Rabring7/BCA2 Orthologs

“…AKT is a serine/threonine kinase that regulates a diverse array of cellular functions. It has been demonstrated that AKT phosphorylates Rabring7/BCA2 within the AKT phosphorylation site located between the BZF and the RING-H2 zinc fingers [27]. Two Rabring7/BCA2 copies are usually detected in vertebrates [19].…”