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RING-type E3s and their substrates There is enormous diversity in substrate ubiquitination and its regulation, as the targets of RING-type E3s are incredibly varied. RING-type E3s are implicated as tumor suppressors, oncogenes, and mediators of endocytosis, and play critical roles in complex multi-step processes such as DNA repair and activation of NF-κB signaling. A RING-type E3 may have multiple substrates and several E3s can target the same substrate. Not surprisingly, the mechanisms of substrate recognition by RING-type E3s are highly varied, and occur in the context of networks of interactions that often also include HECT E3s and deubiquitinating enzymes (DUBs). Substrates may bind directly to a RING-type E3 or may associate indirectly. The capacity of RING-type E3s for self-ubiquitination, first utilized as a means of assessing their potential to function with E2s , frequently occurs in vivo, as does ubiquitination of RING E3s by heterologous RING or HECT-type E3s as part of regulatory networks . E3-substrate interactions may be constitutive, and, in such cases, regulation can occur at the level of E3 transcription or degradation. The complexity of such trans-regulation is illustrated by the interplay of SCF and APC/C E3s during the NSC74859 australia where, for example, APC/CCdh1 targets the F-box protein Skp2 for degradation in early G1, thereby stabilizing p27 and preventing premature G1-S transition, and SCFβTrCP targets the ‘pseudo-substrate’ and suppressor of APCCdc20, EMI1, for degradation in late G2  (reviewed in this issue by Bassermann et al.). Similarly, SCFFBXO11 ubiquitinates and targets Cdt2, the conserved substrate recognition subunit of CRL4Cdt2, for degradation, stabilizing its substrates, such as p21 and Set8, and allowing for cells to properly exit the cell cycle , . An emerging theme is a role for metabolites in substrate recognition and E3 activity. As above, the effect may be via a direct interaction between a metabolite and a RING-type E3, or may be indirect, for example via interaction with the substrate. In the latter category, sterols serve as feedback regulators of their own synthesis by regulating the association of Insig-1 with the ER-resident RING E3 gp78 and hence the stability of the former, which is critical to the regulation of cholesterol biosynthesis . The plant hormones auxin and jasmonic acid are examples of regulation by a direct metabolite:E3 interaction , . These hormones bind directly to SCF complexes and target transcriptional repressors for ubiquitination and degradation. This strategy provides a way to de-repress gene expression and alter the transcriptional profile in response to environmental factors in plants. Another intriguing example of regulation by a metabolite is a report that the RING E3 TRAF-2 is inactive due to its RING structure being unsuitable for E2 interactions, but is activated by its association with sphingosine-1-phosphate . A structural understanding of how this occurs awaits further studies. Nevertheless, as nature rarely uses a good idea just once, it seems likely that additional examples of small molecule or metabolite activation (or inhibition) of E3s will be uncovered in the future. The most common means of regulating substrate ubiquitination is by post-translational modifications that alter either ligase activity or substrate recognition by RING-type E3s. Examples of regulation via protein phosphorylation are widespread. Regulated substrate phosphorylation on either Ser or Thr residues allows for the recognition of numerous substrates by SCFβTrCP and SCFFbw7. Tyrosine phosphorylation of activated receptor tyrosine kinases (RTKs) facilitates their recognition by the Cbl family of RING E3s (Cbl, Cbl-b and Cbl-c) . Cbl-c phosphorylation also modulates the dynamic interaction of its RING with E2~Ub (see below). The net result of activation of Cbl family members includes the ubiquitination of RTKs, leading to their lysosomal degradation and an attenuation of signaling. Both phosphorylation of the core RING subunit and dephosphorylation of Cdc20 play roles in the activation of the APC/CCdc20. The level of complexity that phosphorylation offers is exemplified by p53 and Mdm2. Regulated phosphorylation of specific residues on either p53 or Mdm2 can either inhibit or enhance their interaction. Also, Mdm2 degradation as a consequence of self-ubiquitination is enhanced by phosphorylation, which prevents interaction of Mdm2 with the DUB HAUSP/USP7. The failure of Mdm2 to be deubiquitinated by USP7 leads to degradation of Mdm2 and increased p53 activity under conditions of genotoxic stress  (reviewed in this issue by Vousden et al).