Regulation of neurogenin stability by ubiquitin-mediated proteolysis

Vosper, Jonathan; Fiore-Heriche, Christelle S.; Horan, Ian; Wilson, Karen E.; Wise, Helen; Philpott, Anna

doi:10.1042/bj20070064

Cited by 50 publications

(70 citation statements)

References 31 publications

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“…Degradation Assays-Activated interphase and mitotic egg extracts were prepared as described previously (11). Extracts were supplemented with ubiquitin (from bovine erythrocytes, Sigma) at a final concentration of 1.25 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids and Constructs-NGN (also known as X-NGNR1a) and NeuroD constructs were described previously (11). NGNKO, NGNCO, and NGNKOCO mutants were generated using the QuikChange multisite-directed mutagenesis kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…Mitotic Extracts-Activated interphase egg extracts were prepared as described previously (11) and were then supplemented with 5 g of recombinant non-degradable cyclin B delta 90 per 100 l of extract. Extracts were incubated at room temperature for 20 min to enable extracts to enter mitosis.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously demonstrated that NGN is turned over extremely rapidly in Xenopus eggs and embryos via the UPS, whereas a related proneural protein NeuroD is stable (11). In an attempt to generate a stabilized form of NGN, its 8 lysine residues were mutated to arginines to produce a lysineless protein (NGNKO).…”

Section: Ngn Can Be Ubiquitylated On Both Lysines and The N Terminus-mentioning

confidence: 99%

“…Centrifugal extracts of Xenopus egg cytoplasm contain a large stockpile of enzymes required for early stages of embryogenesis including multiple components of the ubiquitin-proteasome system (UPS) and have been used extensively to explore the biochemistry of ubiquitylation (10). Using Xenopus eggs and embryos and mammalian cultured cells, we have demonstrated that NGN is a very short-lived protein whose rapid turnover is controlled by the UPS (11).…”

mentioning

confidence: 99%

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

Vosper

McDowell

Hindley

et al. 2009

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ngn Can Be Ubiquitylated On Both Lysines and The N Terminus-mentioning

confidence: 99%

mentioning

confidence: 99%

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

Vosper

McDowell

Hindley

et al. 2009

Journal of Biological Chemistry

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Neural Development: b HLH Genes

Philpott

2010

Encyclopedia of Life Sciences

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Transcription factors of the basic helix–loop–helix (bHLH) class are involved in the determination of precursor cells towards neural (proneural) and other cell fates. The proneural members of this class constitute a particular subset of bHLH proteins that are arranged in a cascade that affects different stages of neuronal commitment and differentiation, and act in a combinatorial code along with other transcription factors to specify neuronal identity. Proneural proteins themselves are a target of regulation by ID and HES proteins among others, and are involved in setting up a negative feedback pathway of lateral inhibition involving NOTCH and DELTA signalling. In addition, the activities of bHLHs are also subject to control by posttranslational modification and degradation. Finally, recent analyses have begun to identify the direct downstream targets of bHLH genes in the nervous system, and this is shedding light on the mechanisms ensuring specificity of the individual bHLH proteins. Key concepts: bHLH proneural transcription factors have multiple direct downstream transcriptional targets including other transcription factors, signal transducers and cytoskeletal modifiers. bHLH proneural transcription factors act in a combinatorial code to confer neuronal subtype identity. bHLH transcription factors are regulated posttranslationally by mechanisms including phosphorylation and ubiquitin‐mediated proteolysis. bHLH proneural transcription factors have multiple downstream targets that orchestrate neural differentiation. Negative regulators of bHLH proneural proteins such as HES and ID proteins act by dimerizing with and sequestering E protein partners. Lateral inhibition mediated by the NOTCH – DELTA pathway limits the number of neurons that differentiate from the pool of neuronal precursors.

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Progenitor cell maturation in the developing vertebrate retina

Yang

Silva

Koyano-Nakagawa

et al. 2009

Developmental Dynamics

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Progenitor cells in the developing retina initially divide so that each division produces two cells that divide again. Subsequently, progenitor cells change their mode of division so that one or both cells produced by a division can withdraw from the mitotic cycle and differentiate. We asked how these two progenitor cell stages differ molecularly and what controls the switch in the mode of division. We show that early preneurogenic progenitor cells express the transcription factor, Sox2, and the Notch ligand, Delta1. More mature neurogenic progenitor cells express Sox2 and the bHLH transcription factor, E2A, and not Delta1. Notch signaling maintains progenitor cells in the preneurogenic state. Sonic hedgehog expressed by newly differentiating cells initiates maturation of progenitor cells from preneurogenic to neurogenic at the neurogenic front, possibly by down-regulating Delta1 expression. Our results show that the preneurogenic-to-neurogenic transition is a highly organized unidirectional step made in unison by neighboring cells.

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Regulation of neurogenin stability by ubiquitin-mediated proteolysis

Cited by 50 publications

References 31 publications

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

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

Neural Development: b HLH Genes

Progenitor cell maturation in the developing vertebrate retina

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