Nuclear-cytoplasmic shuttling of the oncogenic mouse UNP/USP4 deubiquitylating enzyme

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 ( ¹³³VEVYLLELKL¹⁴²) in Usp4. In addition, a functional nuclear import signal (⁷⁶⁶QPQKKKK⁷⁷²) was also identified, which was specifically recognized by importin α/β. 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.