Regulation of Smurf2 Ubiquitin Ligase Activity by Anchoring the E2 to the HECT Domain

Ogunjimi, Abiodun A.; Briant, Douglas J.; Pece-Barbara, Nadia; Roy, Christine Le; Guglielmo, Gianni M. Di; Kavsak, Peter A.; Rasmussen, Richele K.; Seet, Bruce T.; Sicheri, Frank; Wrana, Jeffrey L.

doi:10.1016/j.molcel.2005.06.028

Cited by 253 publications

(229 citation statements)

References 20 publications

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“…The domain of Nedd4-2 responsible for substrate binding and how it is affected by phosphorylation has not been identified. Smurf2, a HECT E3 ligase involved in TGF-␤ signaling, was shown to be regulated by a mechanism involving an interaction with phosphorylated Smad7, which in turn facilitates the recruitment of Ubc7 (24). In addition, the activity of the recently described HECT E3 ligase Mule͞ARF-BP1 is negatively regulated by its binding partner ARF (25), and we speculate that phosphorylation may modulate the substrate specificity of this enzyme, thereby explaining its diametrically opposed, substrate-dependent effects on cell survival (25)(26)(27).…”

Section: Discussionmentioning

confidence: 99%

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change

Gallagher

Gao

Liu

et al. 2006

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

250

237

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(2004) Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase itch. Science 306:271-275. Although the reference was included in the original text of the manuscript as ref. 7, the authors neglected to state in the figure legend that the blot used in Fig. 1F is the same blot (probed with a different antibody) shown in Fig. 3E of Gao et al. 2004. The figure and its corrected legend appear below.www.pnas.org/cgi/doi/10.1073/pnas.1002519107

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Section: Discussionmentioning

confidence: 99%

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change

Gallagher

Gao

Liu

et al. 2006

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

250

237

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“…The HECT C-lobe is very flexible in position, and its function requires oscillation between different orientations (32). One places the active site cysteine close to the E2 site, where it can receive ubiquitin (as exemplified by the crystal structure of WWP1) (33), and another places it far away, in a conformation that allows transfer of ubiquitin to a substrate lysine (as seen in the Smurf2 crystal structure) (21). The changes induced by heclin are unlikely to reflect direct contact with this small ligand but are, instead, consistent with movement of the C-lobe from a conformation similar to that in the WWP1 crystal to one more similar to that in the Smurf2 crystal (Fig.…”

Section: Significancementioning

confidence: 99%

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

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

129

133

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The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/ Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.inhibitor | ubiquitin | HECT ligase | phage display U biquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1-4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5-7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2,5,6,8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein-protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein-protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrat...

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“…The C2 domain of Smurfs also tethers the I-Smad/Smurf complex to lipid rafts at cell membranes where binding and ubiquitination of receptors are executed [28]. In this complex, the N-terminal part of Smad7 facilitates Smurf2 activity by helping recruit the E2 conjugating enzyme UbcH7 [29]. Recently, it was shown that the heat shock protein 90 (Hsp90) is a modifier of Smurf2-mediated receptor ubiquitination, as inhibiting Hsp90 activity led to an increased receptor ubiquitination [30].…”

Section: Regulation Of Tgfβ Receptor Stabilitymentioning

confidence: 99%

Regulating the stability of TGFβ receptors and Smads

et al. 2008

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Abbreviations: AIP4 (atrophin 1-interacting protein 4); BMP (bone morphogenetic protein); CHIP (carboxyl terminus of Hsc70-interacting protein); GSK3b (glycogen synthase kinase 3-b); HECT (homologous to E6-AP carboxyl terminus); MAPK (mitogen-activated protein kinase); NEDD4-2 (neural precursor cell expressed, developmentally downregulated 4-2); PIAS (protein inhibitor of activated Stat); Smurf (Smad ubiquitylation regulatory factor); STRAP (serine-threonine kinase receptorassociated protein); SUMO (small ubiquitin-like modifier); TbRI/TbRII (TGFb receptor type I and II); TGFb (transforming growth factor b); WWP1 (WW domain-containing protein 1); Ub (ubiquitin) npg Transforming growth factor β (TGFβ) controls cellular behavior in embryonic and adult tissues. TGFβ binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that together regulate gene expression. In this review, mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of TGFβ are presented. We discuss how the activity and duration of TGFβ receptor/Smad signaling can be regulated by post-translational modifications that affect the stability of key proteins in the pathway. We highlight links between these mechanisms and human diseases, such as tissue fibrosis and cancer. Regulating the stability of TGFβ receptors and Smads

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Regulation of Smurf2 Ubiquitin Ligase Activity by Anchoring the E2 to the HECT Domain

Cited by 253 publications

References 20 publications

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Regulating the stability of TGFβ receptors and Smads

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