Structure and function of a HECT domain ubiquitin‐binding site

Kim, Hyung Cheol; Steffen, Alanna M; Oldham, Michael L.; Chen, Jue; Huibregtse, Jon M.

doi:10.1038/embor.2011.23

Cited by 109 publications

(153 citation statements)

References 20 publications

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“…1A)-have been more closely examined for their role in ubiquitination mediated by distinct E2 ubiquitin-conjugating enzymes and E3 enzymes (e.g., refs. [22][23][24][25][26][27][28][29][30][31]. Notably, patch II but not patch I was shown to be required for covalent attachment of ubiquitin to substrate proteins catalyzed by the HECT E3s Rsp5 and NEDD4L (22,30).…”

Section: Resultsmentioning

confidence: 99%

“…Notably, patch II but not patch I was shown to be required for covalent attachment of ubiquitin to substrate proteins catalyzed by the HECT E3s Rsp5 and NEDD4L (22,30). In addition, the HECT domains of Rsp5, NEDD4L, NEDD4, and SMURF2, which are all members of the NEDD4 subfamily of HECT E3s, harbor a noncovalent binding site for a second ubiquitin molecule (that is not in thioester complex with the HECT domain) (23,24,32,33). This interaction is mediated by patch I, and although patch I is not essential for isopeptide bond formation (30), it affects the processivity of ubiquitin chain formation (23,24,32,33).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the HECT domains of Rsp5, NEDD4L, NEDD4, and SMURF2, which are all members of the NEDD4 subfamily of HECT E3s, harbor a noncovalent binding site for a second ubiquitin molecule (that is not in thioester complex with the HECT domain) (23,24,32,33). This interaction is mediated by patch I, and although patch I is not essential for isopeptide bond formation (30), it affects the processivity of ubiquitin chain formation (23,24,32,33). In contrast to Rsp5 and NEDD4L, the HECT domain of E6AP does not appear to harbor a noncovalent interaction site for free ubiquitin (24).…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Mortensen

Schneider

Barbic

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein.I n eukaryotes, posttranslational modification of proteins by ubiquitin plays a pivotal role in the regulation of many cellular processes, including cell cycle, DNA metabolism (e.g., DNA repair, transcription), and various signal transduction pathways (1-4). The specificity of the ubiquitin-conjugation system is mainly ensured by E3 ubiquitin ligases, which mediate the recognition of target proteins. Based on the presence of distinct domains and their mode of action, E3 proteins can be grouped into three families, RING/RING-like E3s, RING-in-between-RING (RBR) E3s, and HECT E3s (5-7). All E3s have interaction sites for both substrate proteins and E2 ubiquitinconjugating enzymes. However, whereas in the case of RBR E3s and HECT E3s, ubiquitin is transferred from the E3 to substrates, RING/RING-like E3s function as adaptors between substrates and E2s (i.e., ubiquitin is transferred from the E2 to the substrate).E6AP, the founding member of the HECT E3 family, was originally identified as an interacting protein of the E6 oncoprotein of cancer-associated human papillomaviruses (HPVs) (8, 9). The E6-E6AP complex targets the tumor suppressor p53 and other proteins-which in the absence of E6 are not targeted by E6AP-for ubiquitination and degradation thereby contributing to HPV-induced cervical carcinogenesis (10, 11). In 1997, it was recognized that alterations in the UBE3A gene, which encodes E6AP, resulting in loss of E6AP expression or in the expression of E6AP variants with compromised E3 activity, are the cause of the Angelman syndrome (AS), a neurodevelopmental disorder (12-14). Recently, it was rep...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Mortensen

Schneider

Barbic

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Parkin does not contain a conventional ubiquitin-binding domain, but there are other examples in which ubiquitin binding has been observed in the absence of a conventional ubiquitin-binding motif. For example, the Rsp5 HECT domain E3 ligase contains a ubiquitin-binding site within its catalytic domain required for ubiquitin chain formation [39,40]. The tertiary structure of linear ubiquitin chains is similar to that of K63-linked ubiquitin chains, because K63 is spatially close to the N-terminal methionine of ubiquitin and both chains have highly extended conformations [41].…”

Section: Parkin Binds K63-linked Ubiquitin Chainsmentioning

confidence: 99%

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Zheng

Hunter

2013

Cell Res

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Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine~ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin's in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteinemediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.

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“…A short linker between the Nand C-lobes provides rotational flexibility, an essential architectural facet that is required for stepwise transfer of ubiquitin from the charged E2 to the E3 and then onto the target substrate. More recently, the structure of the Nedd4 HECT:Ubiquitin complex suggests that non-covalent association with the growing ubiquitin chain at key residues within the HECT domain is flexible enough to promote both ubiquitin transfer and polyubiquitin processivity 75,76 . Undoubtedly, it is likely that further such detailed mechanistic insight into the key catalytic and tripartite ubiquitin transfer step within E3 ligases will prove to be the 'holy-grail' in addressing the future challenge to rationally design and develop any new E3/substrate specific inhibitors.…”

Section: E3 Ubiquitin Ligasesmentioning

confidence: 99%

How ubiquitination regulates the TGF‐β signalling pathway: New insights and new players

Soond

Chantry

2011

BioEssays

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Subtitle:TGF-β regulation by NEDD4 E3s are discussed, while emphasising the potential role of isoform-derivatives of E1-E3 proteins in ubiquitination.Key Words: TGF beta, Smad, Ubiquitination, Uniquitinome, EMT, cancer Abstract Ubiquitination of protein species in regulating signal transduction pathways is universally accepted as fundamental during normal development and has been implicated in the progression of many human diseases, such as cancer. One particular pathway that has received much attention in this context is TGF-β signalling, particularly during the regulation of EMT and tumour progression. While E3-ubiquitin ligases offer themselves as potential therapeutic targets (based on their ability to confer substrate specificity), much remains to be unveiled regarding mechanisms that culminate in their regulation. With this in mind, the focus of this review highlights the significance of a recently described group of E3-ubiquitin ligase isoforms in the context of the TGF-β pathway regulation. Moreover, we now broaden these observations to incorporate a growing number of protein-isoforms within the ubiquitin ligase superfamily as a whole, and discuss what relevance they may have in defining a new 'iso-ubiquitinome'.

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Structure and function of a HECT domain ubiquitin‐binding site

Cited by 109 publications

References 20 publications

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

How ubiquitination regulates the TGF‐β signalling pathway: New insights and new players

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