The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch

doi:10.1073/pnas.0701773104

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch

*Biochemistry Laboratory, Istituto Dermopatico dell'Immacolata–Istituto di Ricovero e Cura a Carattere Scientifico, University of Rome “Tor Vergata,” 00133 Rome, Italy;
^†Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210;
^‡Toxicology Unit, Medical Research Council, University of Leicester, Leicester LE1 9HN, United Kingdom;
^§Department of Molecular and Cellular Biology, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, 28040 Madrid, Spain;
^¶Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD 21702; and
^‖Department of Molecular Cell Biology, Weizmann Institute of Science, 300 Herzl Street, Rehovot 76100, Israel

Edited by Webster K. Cavenee, University of California at San Diego School of Medicine, La Jolla, CA, and approved May 17, 2007 (received for review February 26, 2007)

Abstract

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin–protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73α, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73α for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73α and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1 ^−/− cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73α and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

Footnotes

^††To whom correspondence may be addressed at:
Biochemistry Laboratory, Istituto Dermopatico dell'Immacolata–Istituto di Ricovero e Cura a Carattere Scientifico, Room F-nord169, Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata,” 00133 Rome, Italy.
E-mail: bernasso{at}uniroma2.it
**To whom correspondence may be addressed at:
Comprehensive Cancer Center, Ohio State University, Wiseman Hall, Room 385K, 400 West 12th Avenue, Columbus, OH 43210.
E-mail: carlo.croce{at}osumc.edu
Author contributions: A.O. and M.M. contributed equally to this work; F.B. and G.M. designed research; A.O., M.M., M.R., and P. Sharma performed research; A.O., M.M., R.I.A., P. Salomoni, R.M., M.R.K., and M.O. contributed new reagents/analytic tools; A.O., M.M., R.I.A., P. Salomoni, M.O., C.M.C., F.B., and G.M. analyzed data; and C.M.C., F.B., and G.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0701773104/DC1.
Abbreviations:

C2,

Ca+2/lipid-binding;

E3,

E3 ubiquitin–protein ligase;

HECT,

homologous to E6AP C terminus;

IB,

immunoblot analysis;

IP,

immunoprecipitation;

MEF,

mouse embryonic fibroblast;

N4BP1,

Nedd4-binding partner-1;

WWn,

WW domain n.