Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging

Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 42.35). 07/2007; 447(7148):1135-8. DOI: 10.1038/nature05902
Source: PubMed

ABSTRACT Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s.

  • Source
    • "Such degradation seems to depend both on ubiquitin-activating enzymes and on the 26S proteasome pathway. In the human ubiquitylation pathway, ubiquitin is adenylated either by UBA1 or UBA6, the two only ubiquitin-activating enzymes (Jin et al., 2007). Because the degradation of XPB depends both on ubiquitin-activating enzymes and on the 26S proteasome, we conclude that XPB undergoes rapid ubiquitylation after SP-treatment that leads to its quick degradation by the proteasome pathway. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nucleotide excision repair (NER) removes DNA lesions resulting from exposure to UV irradiation or chemical agents such as platinum-based drugs used as anticancer molecules. Pharmacological inhibition of NER is expected to enhance chemosensitivity but nontoxic NER inhibitors are rare. Using a drug repositioning approach, we identify spironolactone (SP), an antagonist of aldosterone, as a potent NER inhibitor. We found that SP promotes a rapid and reversible degradation of XPB, a subunit of transcription/repair factor TFIIH. Such degradation depends both on ubiquitin-activating enzyme and on the 26S proteasome. Supplementation of extracts from SP-treated cells with purified TFIIH restored TFIIH-dependent repair and transcription activities in vitro, demonstrating the specific impact of SP on two fundamental functions of TFIIH. Finally, SP potentiated the cytotoxicity of platinum derivatives toward tumor cells, making it a potential therapeutic and research tool.
    Chemistry & biology 02/2014; 21(3). DOI:10.1016/j.chembiol.2013.12.014 · 6.59 Impact Factor
  • Source
    • "However, owing to the unique topology of UbL73P, we postulated that not all ubiquitin conjugation pathways will utilize it equally. Thus, we asked whether the two human E1 enzymes, Ube1 and Uba6, which have been shown to dictate downstream ubiquitination events in vivo (Jin et al., 2007), showed preference between wild-type and UbL73P. In an in vitro E1 charging reaction, Ube1 and Uba6 differ only slightly in UbL73P charging (Figure 2A); however, Uba6 cannot use UbL73P in charging UbcH7 in an E2-charging reaction (Figure 2B). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.
    Cell Reports 11/2013; 5(3). DOI:10.1016/j.celrep.2013.10.008 · 8.36 Impact Factor
  • Source
    • "The only E2 known to be specifically activated by Uba6 is Use1 (Jin et al., 2007). Depletion of Use1 with three independent siRNAs resulted in a 1.6-to 2.4-fold increase in the abundance of Ube3a (Figure 6D, lanes 1–4), independent of its mRNA (Figure 6B). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Uba6 (E1)-Use1 (E2) ubiquitin transfer cascade is a poorly understood alternative arm of the ubiquitin proteasome system (UPS) and is required for mouse embryonic development, independent of the canonical Uba1-E2-E3 pathway. Loss of neuronal Uba6 during embryonic development results in altered patterning of neurons in the hippocampus and the amygdala, decreased dendritic spine density, and numerous behavioral disorders. The levels of the E3 ubiquitin ligase Ube3a (E6-AP) and Shank3, both linked with dendritic spine function, are elevated in the amygdala of Uba6-deficient mice, while levels of the Ube3a substrate Arc are reduced. Uba6 and Use1 promote proteasomal turnover of Ube3a in mouse embryo fibroblasts (MEFs) and catalyze Ube3a ubiquitylation in vitro. These activities occur in parallel with an independent pathway involving Uba1-UbcH7, but in a spatially distinct manner in MEFs. These data reveal an unanticipated role for Uba6 in neuronal development, spine architecture, mouse behavior, and turnover of Ube3a.
    Molecular cell 03/2013; 50(2). DOI:10.1016/j.molcel.2013.02.014 · 14.46 Impact Factor
Show more

Similar Publications


1 Download
Available from