Ubiquitylation on Canonical and Non-canonical Sites Targets the Transcription Factor Neurogenin for Ubiquitin-mediated Proteolysis

Abstract

Polyubiquitylation targets multiple proteins for degradation by the proteasome. Typically, the first ubiquitin is linked to lysine residues in the substrate for degradation via an isopeptide bond, although rarely ubiquitin linkage to the N-terminal residue has also been observed. We have recently shown that Neurogenin (NGN), a basic helix-loop-helix transcription factor that plays a central role in regulating neuronal differentiation, is degraded by ubiquitin-mediated proteolysis. We have taken a biochemical and mutagenesis approach to investigate sites of ubiquitylation of NGN, initially using extracts of eggs from the frog Xenopus laevis as a source of ubiquitylation and degradation components. NGN can be targeted for destruction by ubiquitylation via lysines or the N terminus. However, we see that a modified NGN, where canonical lysine ubiquitylation and N-terminally linked ubiquitylation are prevented, is nevertheless ubiquitylated and degraded by the proteasome. We show that polyubiquitin chains covalently attach to non-canonical cysteine residues in NGN, and these non-canonical linkages alone are capable of targeting NGN protein for destruction. Importantly, canonical and non-canonical ubiquitylation occurs simultaneously in the native protein and may differ in importance for driving degradation in interphase and mitosis. We conclude that native NGN is ubiquitylated on multiple canonical and non-canonical sites by cellular ubiquitin ligases, and all types of linkage can contribute to protein turnover.The selective degradation of proteins by ubiquitin-mediated proteolysis is a crucial regulator of diverse cellular processes in eukaryotes. Ub 4 is classically conjugated to the ⑀-amino group of a substrate lysine by an isopeptide bond (1). This serves as an anchor point to build a polyubiquitin chain that results in subsequent targeting to the proteasome for destruction. In a small number of proteins, Ub has been shown to be conjugated to the N-terminal ␣-amino group of a protein via a peptide linkage used as an anchor site for assembly of a lysine-linked polyubiquitin chain (2).The conjugation of ubiquitin to substrate proceeds when a nucleophilic substrate amino group attacks the labile E2-ubiquitin thioester bond. This mechanism does not exclude the possibility that ubiquitylation can occur on other substrate nucleophilic groups on amino acid side chains, such as the thiol groups present on cysteines. The resultant thioester-anchored ubiquitin chains would, however, be predicted to be less stable than the isopeptide/peptide bonds formed between ubiquitin and lysine/N-terminal amino groups (3, 4). Based on this, cysteine residues have generally been considered to be unlikely targets for ubiquitin modification (4).However, there is recent evidence that ubiquitylation can occur on substrate cysteine residues, albeit in a very limited number of cases, largely regulating intracellular protein movements and usually driven by viral E3 ligases (3, 5-7). Only one substrate, the N-terminal fragment of the Bcl2-relat...

Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation

“…We hypothesised that the Delta and NeuroD promoters might respond differentially to changes in Ngn2 un(der)phosphorylation; a differential response of the Delta and NeuroD promoters to increased promoter occupancy, resulting from Ngn2 post-translational modification changes occurring on cell cycle lengthening, could result in an alteration in the balance between progenitor maintenance and differentiation within a population of neural progenitors (Vosper et al, 2009;Ali et al, 2011). To test this hypothesis, we compared the response of NeuroD and Delta (Neurod1 and Dll1 in mouse) expression to overexpression of mouse Ngn2 (mNgn2) and a mutant version thereof, 9S-A mNgn2, in which the nine SP sites that can be phosphorylated by Cdks have all been mutated to alanine-proline (Ali et al, 2011) (Fig.…”

“…First, we investigated the half-life of these proteins in in vitro degradation assays in Xenopus egg extracts (Vosper et al, 2007;Vosper et al, 2009).…”

“…Acquisition of Xenopus laevis embryos, preparation and injection of synthetic mRNA, staging of embryos and in situ hybridisation were as described previously (Vernon et al, 2003;Vosper et al, 2007;Vosper et al, 2009;Ali et al, 2011). Degradation assays were performed as described (Ali et al, 2011).…”

The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro

“…Acquisition of Xenopus laevis eggs and embryos, preparation and injection of synthetic mRNA, staging of embryos, in situ hybridisation, pH3 analysis egg extract preparation and preparation and western blotting of embryo extracts were performed as described previously (Vernon et al, 2003;Vosper et al, 2007Vosper et al, , 2009Philpott and Friend, 1994;Richard-Parpaillon et al, 2004).…”

Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems

“…The sequential action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) results in isopeptide bond formation between the C terminus of ubiquitin and an amino group on the substrate protein. Typically, this isopeptide linkage occurs through the ⑀-amino group of one or more lysine residues in the substrate (1), although linkage through non-lysine residues has also been described, including esterification on cysteine, serine, and threonine residues as well as isopeptide bond formation with the ␣-amino group at the N terminus of substrates (2)(3)(4)(5)(6)(7)(8)(9)(10).…”