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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

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

Tatiana A Soboleva1, David A Jans, Melanie Johnson-Saliba

  • 1Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 0200, Australia.

The Journal of Biological Chemistry
|October 21, 2004
PubMed

Insights

The deubiquitylating enzyme Usp4 (Unp) shuttles between the nucleus and cytoplasm. This study identified its nuclear import and export signals, clarifying its dynamic subcellular localization.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The deubiquitylating enzyme (DUB) Usp4 (Unp) is implicated in oncogenesis and interacts with tumor suppressors.
  • Previous studies reported conflicting subcellular localization for Usp4 (nuclear vs. cytoplasmic).
  • Distinguishing Usp4 from its paralog Usp15 has complicated localization studies.

Purpose of the Study:

  • To resolve the controversy surrounding Usp4's subcellular localization.
  • To identify the signals governing Usp4's nucleocytoplasmic transport.
  • To investigate cell-type-specific variations in Usp4 localization.

Main Methods:

  • Localization studies of exogenously expressed Usp4 (red fluorescent protein-Usp4).
  • Immunofluorescence using specific antibodies against endogenous Usp4.
  • Inhibition of nuclear export (leptomycin B) and nuclear import assays (Rev-GFP).

Main Results:

  • Both exogenous and endogenous Usp4 accumulate in the nucleus upon inhibition of nuclear export.
  • A nuclear export signal (NES) and a nuclear import signal (NLS) were identified in Usp4.
  • The NLS is recognized by importin alpha/beta, and Usp4 localization varies by cell type.

Conclusions:

  • Usp4 exhibits nucleocytoplasmic shuttling, a property previously unreported for DUBs.
  • Identified signals and cell-type specificity provide a framework for understanding Usp4's dynamic function.
  • This shuttling likely impacts Usp4's role in regulating tumor suppressor pathways.

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