Despite the fact that the

Despite the fact that the precise relevance of Ube2E self-ubiquitination in vivo is still elusive, findings of Sarkari et al. do establish Ube2E1 ubiquitination in GSK-923295 [36]. In vivo self-ubiquitination of Ube2E1 is further supported by the high-throughput study aimed to identify all ubiquitinated proteins that resulted in identification of multiple ubiquitination sites in the N-terminus of this E2 [37]. Identification of ubiquitinated Ube2E1 upon inhibition of the proteasome with bortezomib further suggests that self-ubiquitination may, at least partially, act as a mechanism for E2 turnover [37] in contrast to the findings of Sarkari et al. [36]. It thus seems that future studies would be necessary to identify all possible roles of Ube2E self-ubiquitination in vivo. Our experiments show that the N-terminal ubiquitination results in an overall slowing down of Ube2Es toward the E3/substrate rather than affecting their chain-building capacity. The fact that Ube2E1wt activity is not restricted toward mono-ubiquitination is also evident from Fig. 4a, where the full-length enzyme displayed robust self-poly-ubiquitination. Thus, the apparent “unhindered” mono-ubiquitination of E3 by Ube2E1wt observed by Schumacher et al. [28] originated from their use of UbK0, which prohibited extension of the Ub chains onto the E2 itself leading to incomplete inhibition. However, partial inhibition of Ube2E1wt upon mono-ubiquitination was still evident from the amount of unmodified cIAP2 left in the reactions containing Ube2E1wt as the E2 compared to the “core UBC domain” (see Fig. 3 in Ref. [28]). Ube2E1 ubiquitination in vivo[36], [37] indicates that not only the E3 ligases tested by us but also presumably many of those found to interact with it in yeast two-hybrid screens enhance the self-ubiquitination. Thus, self-ubiquitination may act to prevent “hyper-ubiquitination” of those E3s or their cognate substrates thereby regulating ubiquitination. It is also possible that the N-terminal lysine residues may undergo other post-translational modifications in response to various environmental cues to prevent their ubiquitination and subsequent activity reduction. Ube2Es also apparently appear to be redundant as most of the E3s that interact with Ube2Es are also capable of recruiting Ube2Ds. However, the conserved nature of all three Ube2Es across metazoans and the embryonic lethality of homozygous Ube2E3 knockout mice [38] suggests specific non-redundant biological roles of each of these three E2s. It is therefore also possible that self-ubiquitination of Ube2Es controls unwanted activity of these E2s with “false” cognate E3s such as RNF4 that interact with Ube2Es solely due to their Ube2D-like UBC domains while “true” cognate E3s engage in selective interactions with the N-terminal extensions of specific Ube2Es and thereby inhibit the self-ubiquitination-mediated activity reduction. However, extensive studies encompassing all potential E3 partners of Ube2Es are required in order to establish this proposition. Nonetheless, the self-ubiquitination-mediated regulation of Ube2Es remains undeniably relevant for controlling ubiquitination of proteins in vitro and also possibly in vivo. Though auto-ubiquitination of various ubiquitin-conjugating E2 enzymes has been observed for more than two decades [30], [39], it has mostly been perceived to facilitate degradation of the E2 itself. Only exception to this widely perceived role of E2 auto-ubiquitination was the mono-ubiquitination of Ube2T at the conserved Lys91 that seems to negatively regulate its activity [32]. However, Ube2T being studied in conjunction to the Fanconi anemia group L protein FANCL almost exclusively, the ubiquitination-mediated down-regulation of E2s has not caught widespread attention. Given the fact that a large number of E2s are capable of ubiquitinating themselves [40], it therefore remains to be seen if other “auto-positive” E2s, if not all, are regulated by self-ubiquitination and if there are DUbs that would rescue such ubiquitinated E2s from proteolysis and maintain E2 homeostasis. Our study, therefore, not only establishes self-ubiquitination as a novel regulatory mechanism of Ube2Es but also paves the path to future investigations into the role of self-modification of ubiquitin E2 enzymes.