The -4 Phenylalanine Is Required for Substrate Ubiquitination Catalyzed by HECT Ubiquitin Ligases

Salvat, Catherine; Wang, Guangli; Dastur, Anahita; Lyon, Nancy; Huibregtse, Jon M.

doi:10.1074/jbc.m312201200

Cited by 70 publications

(79 citation statements)

References 28 publications

(42 reference statements)

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“…Alteration of the Ϫ4 F residue does not affect the transfer of ubiquitin from the E2 to the E3 but nearly completely blocks the transfer of ubiquitin from the E3 to the substrate. We proposed that the C-terminal portion of the HECT domain, and the Ϫ4 F residue in particular, was critical for orienting the thioester-bound ubiquitin molecule to provide access of the attacking amino group to the active-site cysteine (39). Therefore, both our current and previous results may point to a function of the C lobe in orienting the thioester-bound ubiquitin molecule.…”

Section: Discussionmentioning

confidence: 51%

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Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

Kim

Huibregtse

2009

Molecular and Cellular Biology

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222

221

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Polyubiquitination can mediate several different biochemical functions, determined in part by which lysine of ubiquitin is used to link the polyubiquitin chain. Among the HECT domain ubiquitin ligases, some, such as human E6AP, preferentially catalyze the formation of K48-linked polyubiquitin chains, while others, including Saccharomyces cerevisiae Rsp5 and human Itch, preferentially catalyze the formation of K63-linked chains. The features of HECT E3s that determine their chain type specificities have not been identified. We show here that chain type specificity is a function solely of the Rsp5 HECT domain, that the identity of the cooperating E2 protein does not influence the chain type specificity, that single chains produced by Rsp5 contain between 12 and 30 ubiquitin moieties, and that the determinants of chain type specificity are located within the last 60 amino acids of the C lobe of the HECT domain. Our results are also consistent with a simple sequentialaddition mechanism for polyubiquitination by Rsp5, rather than a mechanism involving the formation of either E2-or E3-linked polyubiquitin chain transfers.Ubiquitin can be covalently conjugated to proteins in several ways (20). Ubiquitin is sometimes conjugated via an isopeptide bond to a single lysine residue of a target protein and in other cases to multiple lysines. Less commonly, it is conjugated to the terminal amino group of a target (11) or even to cysteine side chains via a thioester bond (6). In all of these cases, a single ubiquitin might be conjugated at a given site (monoubiquitination), or multiple ubiquitins can be linked via one of the seven lysine residues of ubiquitin to form shorter oligoubiquitin chains (2-to 4-ubiquitin moieties) or longer polyubiquitin chains (Ͼ4-ubiquitin moieties). The chains might also be branched or linear, and if linear, either homogeneous or heterogeneous with respect to linkages (25). There are only a few cases for which we have a mechanistic understanding of how the enzymes of the ubiquitin system direct the generation of these distinct ubiquitin modifications. This is an important problem, because the different modes of ubiquitin conjugation have the potential to signal different biochemical fates. For example, lysine 29 (K29)-and K48-linked polyubiquitin chains are associated with proteasomal degradation, while K63-linked polyubiquitin chains have nonproteasomal functions in various signaling and trafficking pathways (4). All seven internal lysines of ubiquitin have been shown to be used for chain formation in vivo (34). The specific functions of some linkage types are uncharacterized, and there is the potential that some of these might mediate yet-to-be-discovered functions of polyubiquitin.For polyubiquitination reactions that involve RING and RING-like U-box ubiquitin ligases, the type of polyubiquitin chains formed appears to be directed primarily by the cooperating E2 enzyme. This is presumably because the E3 is functioning primarily as a docking protein, with the chemistry of ubiquitination occurring...

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Section: Discussionmentioning

confidence: 51%

“…E6AP and HPV type 33 (HPV33) E6 proteins were expressed using recombinant baculoviruses, as described previously (39). Briefly, both proteins were expressed as GST fusion proteins in High Five insect cells, affinity purified, and cleaved to remove the GST.…”

Section: Plasmidsmentioning

confidence: 99%

Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

Kim

Huibregtse

2009

Molecular and Cellular Biology

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222

221

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“…To test this hypothesis, we used E6AP mut/mut mice carrying a mutant form of the E6AP allele (Miura et al, 2002). The E6AP encoded by these E6AP mut/mut mice is defective in its ubiquitin ligase activity owing to a C-terminal mutation in the E6AP gene that eliminates a required C-terminal cysteine in addition to the last four amino acids, all of which are necessary for successful ubiquitin transfer (Talis et al, 1998;Salvat et al, 2004). K14E6 WT transgenic mice were crossed onto the E6AP mut/mut background and the resulting mice were subjected to ionizing radiation.…”

Section: E6's Inactivation Of Mouse P53 In the Absence Of E6apmentioning

confidence: 99%

HPV16 E6 confers p53-dependent and p53-independent phenotypes in the epidermis of mice deficient for E6AP

Shai

Wagstaff

et al. 2006

Oncogene

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High-risk human papillomaviruses are the causative agents of cervical and other anogenital cancers. In these cancers, two viral oncogenes, E6 and E7, are expressed. E6 is best known for its ability to inactivate the tumor suppressor p53, which is thought to arise through ubiquitin-mediated degradation of p53 and involve a ternary complex between E6, p53 and the E3 ligase, E6AP. In mice transgenic for wild-type HPV16 E6, its expression leads to epithelial hyperplasia and an abrogation of normal cellular responses to DNA damage. Whereas only the latter phenotype is dependent upon E6's inactivation of p53, both are reduced in transgenic mice expressing an E6 mutant severely reduced in its binding to E6AP and other cellular proteins that bind E6 through a shared a-helix motif. Here, we investigated whether E6AP is required for the induction of the above phenotypes through the use of both E6AP-mutant and E6AP-null mice. E6, in the absence of E6AP retains an ability to induce epithelial hyperplasia, abrogate DNA damage responses and inhibit the induction of p53 protein following exposure to ionizing radiation. We conclude that E6 is able to induce both p53-dependent and p53-independent phenotypes through E6AP-independent pathways in the mouse.

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“…As an initial test of this hypothesis, we determined whether a UbcH8-competent Ub E3 was capable of conjugating ISG15 to a substrate protein in vitro. Conjugation reactions were performed by using the Saccharomyces cerevisiae Rsp5p E3 ligase, which efficiently catalyzes the in vitro ubiquitination of human WBP2 (27). Two assays established that purified Rsp5p could function with UbcH8 to conjugate ISG15 to WBP2 in vitro.…”

Section: Isg15mentioning

confidence: 99%

The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-α/β-induced ubiquitin-like protein

Zhao

Beaudenon

Kelley

et al. 2004

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

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279

211

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Ubiquitin-(Ub) like proteins (Ubls) are conjugated to their targets by an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and in some cases an E3 ligase. ISG15 is a Ubl that is conjugated to cellular proteins after IFN-␣͞␤ stimulation. Although the E1 enzyme for ISG15 (Ube1L͞E1 ISG15 ) has been identified, the identities of the downstream components of the ISG15 conjugation cascade have remained elusive. Here we report the purification of an E2 enzyme for ISG15 and demonstrate that it is UbcH8, an E2 that also functions in Ub conjugation. In vitro assays with purified Ub E2 enzymes and in vivo RNA interference assays indicate that UbcH8 is a major E2 enzyme for ISG15 conjugation. These results indicate that the ISG15 conjugation pathway overlaps or converges with the Ub conjugation pathway at the level of a specific E2 enzyme. Furthermore, these results raise the possibility that the ISG15 conjugation pathway might use UbcH8-competent Ub ligases in vivo. As an initial test of this hypothesis, we have shown that a UbcH8-competent Ub ligase conjugates ISG15 to a specific target in vitro. These results challenge the concept that Ub and Ubl conjugation pathways are strictly parallel and nonoverlapping and have important implications for understanding the regulation and function of ISG15 conjugation in the IFN-␣͞␤ response. IFN-␣͞␤ play an essential role in innate immunity and are induced during many types of viral infections (1). Many genes are transcriptionally induced by IFN-␣͞␤, including ISG15 (IFNstimulated gene, 15 kDa) (2, 3). The ISG15 protein is a 15-kDa ubiquitin (Ub)-like protein (Ubl), consisting of two Ub-related domains, Ϸ30% (N-terminal domain) and 36% (C-terminal domain) identical to Ub. ISG15 becomes conjugated to a diverse set of cellular proteins after IFN-␣͞␤ stimulation (4). Although the biochemical consequences of ISG15 conjugation and the fate of the conjugated proteins are not known, it does not appear that ISG15 targets proteins for proteasomal degradation (5, 6).Conjugation of Ub to target proteins requires the cooperative activities of at least three classes of enzymes (7). The ATPdependent E1 enzyme activates Ub by C-terminal adenylation, followed by formation of a high-energy thioester bond between the terminal carboxylate of Ub and the active-site cysteine of E1. Ub is then transferred to the active-site cysteine of one of a number of related E2 enzymes. E3 enzymes then promote transfer of Ub from the E2 to the substrate, resulting in a stable amide bond between -amino groups of lysine side chains and Ub. E3 enzymes are the primary determinants of substrate specificity and can be divided into two classes based on mechanism. HECT E3s accept Ub from the E2 enzyme, again in the form of a thioester adduct, and transfer Ub from their active-site cysteine to the bound substrate (8). RING E3s consist of several subclasses and are either single or multisubunit enzymes that serve as docking proteins for both protein substrates and activated E2 enzymes, with transfe...

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The -4 Phenylalanine Is Required for Substrate Ubiquitination Catalyzed by HECT Ubiquitin Ligases

Cited by 70 publications

References 28 publications

Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

HPV16 E6 confers p53-dependent and p53-independent phenotypes in the epidermis of mice deficient for E6AP

The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-α/β-induced ubiquitin-like protein

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