Certain Pairs of Ubiquitin-conjugating Enzymes (E2s) and Ubiquitin-Protein Ligases (E3s) Synthesize Nondegradable Forked Ubiquitin Chains Containing All Possible Isopeptide Linkages

Abstract

It is generally assumed that a specific ubiquitin ligase (E3)linksIn eukaryotic cells, ubiquitination serves to target regulatory and misfolded proteins for rapid degradation by proteasomes (1-3), to trigger endocytosis of membrane proteins (4), and also to allow specific protein-protein associations important in signal transduction, DNA repair, and gene transcription (5-8). Protein ubiquitination involves formation of isopeptide linkages between the C-terminal carboxyl group of a ubiquitin (Ub) 4 and an ⑀-amino group on a lysine on the protein substrate or a preceding Ub to form a polyUb chain. To synthesize such linkages, the C-terminal carboxyl group of a Ub is first activated by formation of a thioester bond with a cysteine on the Ubactivating enzyme (E1), and the activated Ub is then transferred as a thioester to one of the 20 -40 Ub-conjugating enzymes (E2) of the cell. The formation of a Ub chain on the substrate is then catalyzed by a Ub ligase (E3), which binds the substrate and an E2. Several families of E3s exist that differ in structure and mechanism. If ubiquitination is catalyzed by a member of the Ring finger or the U-box E3 family, the activated Ub is transferred from the E2 directly to a lysine on the protein substrate or to a preceding Ub. The abundant Ring finger and the related U-box families are small monomeric proteins that bind the substrate at one end and then in that vicinity release the reactive Ub from the E2-Ub thioester (9, 10). If ubiquitination is catalyzed by an E3 of the HECT domain family, the activated Ub is transferred from the E2 first to a cysteine on the E3 to form another thioester bond and then to the substrate or to a preceding Ub * This work was supported by grants from the NIGMS, the High Q Foundation, the Fund for Innovation from Elan Corp. (to A. L. G.), the National Institutes of Health (to S. P. G.), and National Institutes of Health R01 Grant GM65267 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: Ub, ubiquitin; UPKn, ubiquitin peptide modified at lysine n by ubiquitination; Forked chain, Ub chain in which two Ub chains are linked to the adjacent lysines on the preceding Ub; E1, Ubactivating enzyme; E2, Ub-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; LC-MSMS liquid chromatography-tandem mass spectrometry; SIM, selective ion monitoring.

E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFα-induced JNK activation

“…Thus, Mule is a K48-specific E3 for Miz1. This is consistent with the general notion that HECT-domain-containing E3 ligases form only homogeneous ubiquitin chains, i.e., either K48-or K63-linked ubiquitin chains on their substrates (43). Indeed, Mule is the E3 ligase that ubiquitinates histone H2A (34), p53 (35), N-Myc (36), Mcl-1 (33) and Cdc6 (37) through K48-mediated linkage.…”

Regulation of death receptor signaling by the ubiquitin system

“…29 K63-linked polyubiquitin chains generally do not promote efficient proteasomal degradation, but instead regulate the activation of proinflammatory signaling [30][31][32][33][34] and other cellular processes. [35][36][37][38] Recent evidence suggests that K11-linked ubiquitin chains may promote substrate degradation and that linear ubiquitin chains may also have scaffolding functions. 39,40 Monoubiquitination is also an important modification and primarily serves to regulate the internalization of cell-surface proteins, intracellular protein sorting, and other cellular activities.…”

The ubiquitin landscape at DNA double-strand breaks

“…For example, the canonical lysine-48 (K48)-linked polyubiquitin targets the substrate protein for proteasomal degradation (Pickart and Cohen, 2004), whereas lysine-63 (K63)-linked polyubiquitin is often involved in localization or signaling events (Chen and Sun, 2009). Polyubiquitin can be linked through one residue to create a homogeneous chain, or through multiple residues, forming branched ubiquitin chains (Kim et al, 2007b).…”