Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalyzed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.     

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.     

Regulation of catalytic activities of HECT ubiquitin ligases

Studies in yeast and mammalian cells over the past decade have shown that HECT domain ubiquitin ligases (HECT E3 enzymes) are involved in diverse physiological pathways. Many substrates of specific HECT E3s have been identified, as well as many adaptor proteins that aid in defining substrate specificity or intra-cellular localization of HECT E3s. Here we review some recently discovered mechanisms for regulation of the catalytic activities of HECT E3s, including regulation at the level of E2 recruitment, phosphorylation-dependent relief of inhibitory intra-molecular interactions, and regulation by association with a deubiquitinating enzyme.     

Control of the activity of WW‐HECT domain E3 ubiquitin ligases by NDFIP proteins

HECT domain E3 ubiquitin ligases of the NEDD4 family control many cellular processes, but their regulation is poorly understood. They contain multiple WW domains that recognize PY elements. Here, we show that the small PY‐containing membrane proteins, NDFIP1 and NDFIP2 (NEDD4 family‐interacting proteins), activate the catalytic activity of ITCH and of several other HECT ligases by binding to them. This releases them from an autoinhibitory intramolecular interaction, which seems to be characteristic of these enzymes. Activation of ITCH requires multiple PY–WW interactions, but little else. Binding of NDFIP proteins is highly dynamic, potentially allowing activated ligases to access other PY‐containing substrates. In agreement with this, NDFIP proteins promote ubiquitination in vivo both of Jun proteins, which have a PY motif, and of endophilin, which does not.     

Animal HECT ubiquitin ligases: evolution and functional implications

Background  

The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs) are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described.     

The -4 phenylalanine is required for substrate ubiquitination catalyzed by HECT ubiquitin ligases

The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.     

WW domain interactions regulate the Hippo tumor suppressor pathway

The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis in Drosophila and mammalians. Although the signaling of the core kinases is relatively well understood, less is known about the upstream inputs, downstream outputs and regulation of the whole cascade. Enrichment of the Hippo pathway components with WW domains and their cognate proline-rich interacting motifs provides a versatile platform for further understanding the mechanisms that regulate organ growth and tumorigenesis. Here, we review recently discovered mechanisms of WW domain-mediated interactions that contribute to the regulation of the Hippo signaling pathway in tumorigenesis. We further discuss new insights and future directions on the emerging role of such regulation.     

HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation

Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation.

Structured summary

MINT-7894271: ING2 (uniprotkb:Q9H160) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull-down (MI:0096)MINT-7894319, MINT-7894339: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894301: Smurf1 (uniprotkb:Q9HCE7) physically interacts (MI:0915) with ING2 (uniprotkb:Q9H160) by anti bait co-immunoprecipitation (MI:0006)MINT-7894358: ING1b (uniprotkb:Q9UK53-2) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894249: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag co-immunoprecipitation (MI:0007)     

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family

There are 28 unique human members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family. Each member of the HECT E3 ubiquitin ligases contains a conserved bilobal HECT domain of approximately 350 residues found near their C-termini that is responsible for their respective ubiquitylation activities. Recent studies have begun to elucidate specific roles that each HECT E3 ubiquitin ligase has in various cancers, age-induced neurodegeneration, and neurological disorders. New structural models have been recently released for some of the HECT E3 ubiquitin ligases, but many HECT domain structures have yet to be examined due to chronic insolubility and/or protein folding issues. Building on these recently published structural studies coupled with our in-house experiments discussed in the present study, we suggest that the addition of ∼50 conserved residues preceding the N-terminal to the current UniProt defined boundaries of the HECT domain are required for isolating soluble, stable, and active HECT domains. We show using in silico bioinformatic analyses coupled with secondary structural prediction software that this predicted N-terminal α-helix found in all 28 human HECT E3 ubiquitin ligases forms an obligate amphipathic α-helix that binds to a hydrophobic pocket found within the HECT N-terminal lobe. The present study brings forth the proposal to redefine the residue boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.     

HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif-dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.     

An expanded WW domain recognition motif revealed by the interaction between Smad7 and the E3 ubiquitin ligase Smurf2

Smurf2 is an E3 ubiquitin ligase that drives degradation of the transforming growth factor-beta receptors and other targets. Recognition of the receptors by Smurf2 is accomplished through an intermediary protein, Smad7. Here we have demonstrated that the WW3 domain of Smurf2 can directly bind to the Smad7 polyproline-tyrosine (PY) motif. Of particular interest, the highly conserved WW domain binding site Trp, which interacts with target PY motifs, is a Phe in the Smurf2 WW3 domain. To examine this interaction, the solution structure of the complex between the Smad7 PY motif region (ELESPPPPYSRYPMD) and the Smurf2 WW3 domain was determined. The structure reveals that, in addition to binding the PY motif, the WW3 domain binds six residues C-terminal to the PY motif (PY-tail). Although the Phe in the WW3 domain binding site decreases affinity relative to the canonical Trp, this is balanced by additional interactions between the PY-tail and the beta1-strand and beta1-beta2 loop of the WW3 domain. The interaction between the Smurf2 WW3 domain and the Smad7 PY motif is the first example of PY motif recognition by a WW domain with a Phe substituted for the binding site Trp. This unusual interaction allows the Smurf2 WW3 domain to recognize a subset of PY motif-containing proteins utilizing an expanded surface to provide specificity.     

Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.     

Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase

Ubiquitin ligases (E3) select proteins for ubiquitylation, a modification that directs altered subcellular trafficking and/or degradation of the target protein. HECT domain E3 ligases not only recognize, but also directly catalyze, ligation of ubiquitin to their protein substrates. The crystal structure of the HECT domain of the human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. While the individual N and C lobes of WWP1 possess very similar folds to those of E6AP, the organization of the two lobes relative to one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes. Mutational analyses suggest that a range of conformations achieved by rotation about this hinge region is essential for catalytic activity.     

A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins

Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle. A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21. The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells. Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins. The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB.     

Regulation of the cell cycle by SCF-type ubiquitin ligases

Regulation of the cell cycle is dependent on protein degradation by the ubiquitin-proteasome system. Two major ubiquitin ligases, the anaphase-promoting complex or cyclosome (APC/C) and SCF complex, are responsible for the periodic proteolysis of many regulators of the cell cycle. The receptor component of the SCF complex is one of many F-box proteins, three of which--Skp2, Fbw7, and beta-TrCP--are well characterized and implicated in cell cycle regulation. We have generated mice deficient in Skp2, Fbw7, or beta-TrCP1 and have identified the roles of these proteins in both cell cycle regulation and mouse development. Clinical evidence also suggests that dysregulation of these F-box proteins contributes to human cancers.