Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

Abstract

Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of ∼1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed.

Figure 1

The ubiquitin-proteasome system. Conjugation of ubiquitin catalyzed by RING (a) or HECT (b) domain-containing ligases. (ai, bi) ATP-dependent activation of ubiquitin catalyzed by the ubiquitin-activating enzyme, E1. (aii, bii) Transfer of the activated ubiquitin to a ubiquitin-carrier protein (ubiquitin-conjugating enzyme), E2. In the case of a RING ligase, the ubiquitin-charged E2 binds to the E3 and transfers the activated ubiquitin moiety directly to the substrate that is also bound to the E3 (aiii). In the case of a HECT domain ligase, ubiquitin is transferred from the E2 to a Cys residue in the E3 (biii-a) and then to the substrate (biii-b). (iv and v) The conjugated substrate is degraded to short peptides by the 26S proteasome (iv) with release of free and reusable ubiquitin mediated by DUB(s) (v). Some of the ubiquitin is degraded in this process along with the substrate (iv)

Figure 2

Self-ubiquitination and degradation of E3 ligases occur in three substrate-related modes: (a) Self-ubiquitination and degradation of the ligase (i) are independent from ubiquitination of the substrate (ii). (b) Self-ubiquitination occurs concomitantly with ubiquitination of the substrate. (c) Inhibition of self-ubiquitination by the substrate

Figure 3

Schematic representation of RING1B regulation by self-ubiquitination and ubiquitination by an exogenous ligase. RING1B is target to ubiquitination by E6-AP, and/or other E3 ligase(s), generating Lys48-based ubiquitin chains that target RING1B for proteasomal degradation. Self-ubiquitination of RING1B results in the formation of Lys6-, Lys27-, and Lys48-based mixed and multiply branched ubiquitin chains that activate it as a ligase for histone H2A. The balance between these two types of ubiquitination is regulated in several ways. (i) Since the activating and degrading forms of ubiquitination target the same lysine residues in RING1B, the two modifications are mutually exclusive. (ii) The PRC1 subunit BMI1 inhibits both types of ubiquitination. (iii) The DUB USP7 reverses both self- and E6-AP-mediated ubiquitination of RING1B, thereby returning it to its native state, potentially allowing the balance between the types of ubiquitination to be shifted. (iv) BMI1 stimulates ubiquitination of H2A by RING1B

Figure 4

Schematic representation of the interplay between the ubiquitinating activities of APC/C and SCF complexes during cell cycle. (i) During G₁ phase, APC/C^CDH1 targets several F-box substrate receptors of SCF complexes, including SKP2 and TOME-1, and the SKP2 cofactor CKS1. In addition, CDC20 is also targeted to ubiquitination and degradation by APC/C^CDH1. While CDH1 is subject to self-ubiquitination during G₀ and G₁ phases within the context of the APC/C complex (ii), the degradation of the free released phosphorylated form is mediated by an SCF complex during S phase (iii). (iv) During S and G₂ phases, APC/C^CDC20 is inhibited by the F-box protein EMI1 (iv-a); however, in the M phase, EMI1 is targeted for destruction by SCF^β-TRCP alleviating the inhibition of APC/C^CDC20 (iv-b). (v) CDC20 is targeted in a self-catalytic manner during the spindle-associated checkpoint

Figure 5

Mechanisms for proteasomal targeting of E3 ligases. (a) Degradation mediated by self-ubiquitination. (b) Hierarchical linear control of ligases. Ligase B is controlled by autoubiquitination (i) or another proteolytic mechanism, e.g., lysosomal degradation; (ii). It modifies in trans ligases C and D (iii), which in turn can modify ligases E and F, and ligases G, H, respectively (iv and v), etc. (c) Two (or more) ligases target one another in a closed circular manner