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RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch

Oncogene volume 34, pages 3377–3390 (2015)Cite this article

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Abstract

Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic.

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Acknowledgements

We would like to thank Dr Elena Romano and Dr Palma Mattioli of the Centre of Advanced Microscopy (CAM) of Tor Vergata University for their skillful assistance in the use of the facility. We thank Juan Bonifacino (National Institutes of Health, Bethesda, MD, USA) for the generous gifts of myc-GGA1, GGA2 and GGA3 expression vectors and Marino Zerial (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany) for providing the GFP constructs for Rab4, Rab5, Rab7 and Rab11. A special thanks to Natalia Speranzini for generating stable cell lines and to Gemma Biondo, Anita Palma and Daniela Posca for technical support. This work was partially supported by grants from MIUR and from AIRC—Associazione Italiana per la Ricerca sul Cancro (IG 10272), Italy.

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Author notes

  1. E Santonico and A Mattioni: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, Tor Vergata University of Rome, Rome, Italy,

    E Santonico, A Mattioni, M Mattei, G Cesareni & L Castagnoli

  2. Department of Cell Biology, University of Calabria, Cosenza, Italy,

    S Panni

  3. Department of Clinical and Molecular Medicine, Institute Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy,

    F Belleudi & M R Torrisi

  4. Azienda Ospedaliera S. Andrea, Rome, Italy

    M R Torrisi

  5. IRCCS Fondazione S. Lucia, Rome, Italy

    G Cesareni

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Santonico, E., Mattioni, A., Panni, S. et al. RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch. Oncogene 34, 3377–3390 (2015). https://doi.org/10.1038/onc.2014.256

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