E3 Ligases Determine Ubiquitination Site and Conjugate Type by Enforcing Specificity on E2 Enzymes

Abstract

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono-and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.Targeting of most substrates to the 26 S proteasome requires covalent marking with polyubiquitin chains. Protein ubiquitination is a multistep process accomplished by the concerted action of three enzymes. The reaction begins with the ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin and then forms a thioester bond between the activated glycine residue and a cysteine residue in its active site. Subsequently, a ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a trans-thioesterification reaction. Finally, a ubiquitin-protein ligase (E3) recruits a target protein and guides the transfer of the activated ubiquitin from the E2 to the substrate (1-3). Ubiquitin transfer from the E2 enzyme to the substrate is catalyzed directly by really interesting new gene (RING) 3 finger-containing E3s or indirectly when a homologous to E6-AP C terminus (HECT) domain E3 is mediating the transfer (4). Several forms of ubiquitination have been identified (5). Monoubiquitination or multiple monoubiquitinations are referred to as the conjugation of single or multiple ubiquitin moieties to distinct lysine residues on the substrate. These forms of ubiquitination were implicated in various cellular pathways, which include endocytosis and sorting of proteins to different cellular compartments (6, 7), as well as in several cases of proteasomal activity, such as the processing of the p105 precursor of the transcription regulator NF-B (8). However, polyubiquitination is the most common post-translational modification of proteins destined for degradation (9).In polyubiquitination assembly, ubiquitin conjugation was originally thought to be repeated in a cyclic manner whereby in each step a new moiety of ubiquitin is linked to one of the lysine residues of the previously conjugated ubiquitin. However, in view of recent findings, several alternative mechanisms have been proposed (10). Li et al. (11) demonstrated in a recon...

A Comparative Analysis of the Ubiquitination Kinetics of Multiple Degrons to Identify an Ideal Targeting Sequence for a Proteasome Reporter

“…The goal of many E3 ligases is to bring the Ub∼E2 complex into close proximity of the target protein to allow for direct transfer of the ubiquitin from the E2 to the target protein. Recent publications have found that the E3 ubiquitin ligase only enhances substrate ubiquitination, but is not absolutely required [50]. Further, it is distinctly possible that the sequence of the p53-based substrate is similar enough to the sequence on Hdm2 (and Mdm2) to allow for ubiquitination.…”

Limiting the power of p53 through the ubiquitin proteasome pathway

“…E1 enzymes initiate the ubiquitin reaction by ATPdependent activation of ubiquitin and tether it to an E2. The E3 ligases ascertain the specificity of the substrate and facilitate the transfer of this activated complex to the target protein (David et al 2011). Ubiquitin chains established by sequential K48 linkage (polyubiquitination) lead to protein degradation via 26S proteasome, while K63-linked ubiquitin chains regulate signaling (Thrower et al 2000).…”

Regulation of NUB1 Activity through Non-Proteolytic Mdm2-Mediated Ubiquitination

“…As expected, the polyubiquitin-like profile of NUB1 obtained with WT ubiquitin expression was lost with K0 ubiquitin which displayed only the monoubiquitinated form of NUB1 (Fig 3A). Mdm2 is known to catalyze the ligation of K48-linked and K11-linked polyubiquitin chains to p53 [15]. To determine which kind of polyubiquitin chain Mdm2 uses to polyubiquitinate NUB1, we have used K48R and K11R mutants of ubiquitin.…”