PMC

Different forms of protein ubiquitylation. Protein substrates (grey) can be (a) monoubiquitylated with a single ubiquitin (ub, blue), (b) multiubiquitylated or (c) polyubiquitylated. (c) Ubiquitin chains can form (i) extended or (ii) closed conformations of one or more linkage type or (iii) even forked chains with multiple ubiquitins attached to a common moiety. (d) Ubiquitin is activated by an E1 activating enzyme (brown) and conjugated to its substrate (grey) through the coordinated action of E2 conjugating (orange) and E3 ligase (yellow) enzymes. Substrate recognition is typically conferred by E3s, whereas ubiquitin chain linkage specificity can be determined by E2, E3 or E2–E3 pairs. DUBs (purple) can trim ubiquitin moieties from chains to reverse the effects of substrate ubiquitylation and recycle the ubiquitin, or to edit the chain length or linkage type.

The diversity and specificity of ubiquitin signaling is relayed through different layers of coordinated protein–protein interactions (multivalency). (a) E3 ligases are tightly regulated to target a specific substrate at a specific time and location through multiple protein–protein interactions. Autoubiquitylation is a common way to regulate E3 ligase activity and is counteracted by a specific DUB. Phosphorylation is another mechanism used to activate or suppress E3 activity toward a substrate. Some E3 ligases also bind accessory proteins that can suppress their activity or mediate other interactions. (b) The moieties of a ubiquitin chain coordinate the simultaneous binding of multiple ubiquitin-binding domains (UBDs) from the same protein or from different ubiquitin receptors. This mode of multivalency can lead to preferences for chains of specific linkage type, to increased binding affinity and to the bridging of multiple ubiquitin receptors. (c) The consequence of a ubiquitin receptor binding to a ubiquitylated substrate is determined through multiple coordinated protein interactions. Ubiquitin receptors usually contain functional domains that direct localization to specific sites or complexes within a cell. They can also contain elements that interact with enzymes, such as kinases, DUBs or E3s. (d) Some ubiquitin receptors can be ubiquitylated and their UBDs can bind intramolecularly to ubiquitin. Such interactions relay an inhibitory effect such that the receptor no longer binds to its substrates; this effect is relieved by DUB activity. (e) In some cases, ubiquitylation is used to change binding affinities between protein–protein and protein–DNA interactions. In this example, interaction between covalently attached ubiquitin and a UBD leads to an increased binding affinity.

The multiple roles of protein ubiquitylation. (a) In the nucleus, ubiquitylation signals for proteasome-independent regulation of DNA repair. Histones and PCNA are examples of nuclear targets of ubiquitylation. (b) Ubiquitylation functions in kinase activation by acting as a scaffold to bring kinases and their substrates together to enable the activation of kinase cascades, as exemplified by the activation of IKK by TAK1. The E3 TRAF6 (TNF receptor-associated factor 6) ubiquitylates several proteins including NEMO and itself upon ligand stimulation. TAK1 and IKK complexes interact with each other upon binding these ubiquitin chains with their ubiquitin-binding subunits (TAB2 and NEMO, respectively). IKK is thus activated by TAK1 and competent to phosphorylate IκB, which leads to its ubiquitylation and proteasomal degradation (i). Iκ-B degradation releases NF-κB into nucleus, thereby activating gene transcription. (c) Proteasome-mediated degradation (i) regulates many cellular processes through targeted degradation of key regulatory proteins. (d) Ubiquitylation signals for protein quality control to degrade misfolded proteins. Protein quality control of ER proteins occurs through the ER-associated protein degradation (ERAD) pathway by proteasomal degradation (i), whereas some large protein aggregates are eliminated through the autophagy (Av, autophagic vesicles)-lysosome route (ii). Polyubiquitylation acts as a signal for autophagy targeting. (e) Ubiquitylation signals for vesicular trafficking between membrane compartments. (iii) Ligand-bound plasma membrane proteins internalized into early endosomes (EE). (iv) Proteins sorting into multivascular bodies (MVB). (v) Newly synthesized membrane proteins sorting from the trans-Golgi network (TGN) to the MVB. (f) Ubiquitin signaling regulates gene transcription.