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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples
09:45

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Published on: May 10, 2015

A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division

S Y Cai1, R W Babbitt, V T Marchesi

  • 1Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.

Proceedings of the National Academy of Sciences of the United States of America
|March 17, 1999
PubMed

Insights

A novel ubiquitin-processing protease, Ubp-M, is crucial for cell division. Its activity and localization are regulated by phosphorylation, impacting mitotic chromosome condensation and preventing apoptosis.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Ubiquitin modification plays a critical role in regulating protein function and cellular processes.
  • Mitosis involves complex molecular events, including protein phosphorylation and deubiquitination, that ensure accurate chromosome segregation.
  • The precise roles of specific ubiquitin-processing proteases during cell division are not fully understood.

Purpose of the Study:

  • To identify and characterize a novel ubiquitin-processing protease involved in mammalian cell division.
  • To investigate the regulatory mechanisms, enzymatic activity, and cellular localization of this protease.
  • To determine the functional significance of this protease in mitotic progression and cell survival.

Main Methods:

  • Identification and cloning of a novel ubiquitin-processing protease (Ubp-M) from mammalian cells.
  • In vitro phosphorylation assays using mitotic cell extracts and purified cdc-2/cyclin B.
  • In vitro deubiquitination assays using recombinant Ubp-M and histone H2A.
  • Expression of wild-type and mutant Ubp-M as green fluorescent protein (GFP) fusion proteins in cultured cells.
  • Analysis of cellular localization and cell division progression in transfected cells.

Main Results:

  • A new ubiquitin-processing protease, Ubp-M, was identified, showing phosphorylation during mitosis and dephosphorylation during the metaphase/anaphase transition.
  • The carboxyl-terminal domain of Ubp-M can be phosphorylated by mitotic cell extracts and cdc-2/cyclin B.
  • Recombinant Ubp-M deubiquitinates histone H2A in vitro, with the phosphorylated form retaining enzymatic activity.
  • Wild-type Ubp-M localizes to the cytoplasm, while active-site mutants associate with mitotic chromosomes and persist in the nucleus post-mitosis.
  • Expression of mutant Ubp-M leads to cell cycle arrest, apoptosis, and defects in cell division.

Conclusions:

  • Ubp-M is a novel mitotic phosphoprotein regulating cell division.
  • Its enzymatic activity and localization are cell cycle-dependent, suggesting a role in mitotic progression.
  • Ubp-M may deubiquitinate key proteins involved in mitotic chromosome condensation, potentially targeting histones H2A and H2B.
  • Dysregulation of Ubp-M function can lead to cell division failure and apoptosis, highlighting its critical role in maintaining genomic stability.

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