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Molecular mechanisms of coupled monoubiquitination

Abstract

Many proteins contain ubiquitin-binding domains or motifs (UBDs), such as the UIM (ubiquitin-interacting motif) and are referred to as ubiquitin receptors. Ubiquitin receptors themselves are frequently monoubiquitinated by a process that requires the presence of a UBD and is referred to as coupled monoubiquitination. Using a UIM-containing protein, eps15, as a model, we show here that coupled monoubiquitination strictly depends on the ability of the UIM to bind to monoubiquitin (mUb). We found that the underlying molecular mechanism is based on interaction between the UIM and a ubiquitin ligase (E3), which has itself been modified by ubiquitination. Furthermore, we demonstrate that the in vivo ubiquitination of members of the Nedd4 family of E3 ligases correlates with their ability to monoubiquitinate eps15. Thus, our results clarify the mechanism of coupled monoubiquitination and identify the ubiquitination of E3 ligases as a critical determinant in this process.

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Figure 1: Analysis of eps15-UIM mutants.
Figure 2: Analysis of chimeric eps15-UIM proteins.
Figure 3: Coupled monoubiquitination in an in vitro ubiquitination assay.
Figure 4: In vitro ubiquitination assays with pretreated Nedd4.
Figure 5: Monoubiquitination is sufficient to render Nedd4 competent for coupled monoubiquitination.
Figure 6: Challenging the E3–isopeptide model.
Figure 7: In vivo ubiquitination assays with Nedd4-family members.

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Acknowledgements

We thank: L. Hicke for providing GST–ubiquitin (wild type), UbI44A and UbF4A constructs; S. Kumar for N4WBP5A construct; M. Sudol for γ-ENAC construct; P. Bienias for AIP4, WWP1 and WWP2 constructs. We thank I. Dikic for critical review of the manuscript. The authors' work is supported by grants from AIRC (Italian Association for Cancer Research) and The European Community (RUBICON, VI Framework) to S.P. and P.P.D.F.; AICR (Association for International Cancer Research) to S.P.; Human Science Frontier Program, the Italian Ministries of Health and of Education and University (MIUR), and the Monzino Foundation to P.P.D.F. B.O. is recipient of a fellowship from RUBICON.

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Correspondence to Pier Paolo Di Fiore or Simona Polo.

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Woelk, T., Oldrini, B., Maspero, E. et al. Molecular mechanisms of coupled monoubiquitination. Nat Cell Biol 8, 1246–1254 (2006). https://doi.org/10.1038/ncb1484

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