Nuclear-Cytoplasmic Shuttling of the Oncogenic Mouse UNP/USP4 Deubiquitylating Enzyme

Tatiana A Soboleva,David A Jans,Melanie Johnson-Saliba,Rohan T Baker

doi:10.1074/jbc.m401394200

Abstract

The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal ((133)VEVYLLELKL(142)) in Usp4. In addition, a functional nuclear import signal ((766)QPQKKKK(772)) was also identified, which was specifically recognized by importin alpha/beta. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed.

Highlights

The oncogenic deubiquitylating enzyme (DUB) Unp/ Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein
Involvement of the pRb Protein in Endogenous Usp4 Localization—Since Usp4 was recently shown to interact with the pRb family [18, 19], we examined the possibility that pRb may be involved in determining Usp4 subcellular localization
We show for the first time that Usp4 is a shuttling protein containing functional targeting signals for nuclear import and export, which are recognized by distinct members of the importin family of nuclear import and export receptors

Summary

Introduction

The oncogenic deubiquitylating enzyme (DUB) Unp/ Usp, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. DUBs constitute large gene families in all eukaryotes and can be divided into the ubiquitin-specific protease (UBP) and ubiquitin C-terminal hydrolase families based on amino acid sequence (6 – 8) This multiplicity is not required merely for the cleavage of ubiquitin precursors but is indicative of other regulatory roles in the ubiquitin pathway. In a separate study using cell lines rather than primary tissue, USP4 protein levels were shown to be slightly but consistently reduced in cell lines derived from small cell tumors, leading to the suggestion that USP4 may be a candidate tumor suppressor gene [17] Both mouse and human Usp have been shown to interact with the Rb family of tumor suppressor proteins, a possible mechanism for Usp4-mediated cell transformation [18, 19]

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