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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
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Published on: July 25, 2019

Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

Toru Suzuki1, Minsoo Kim, Hiroko Kozuka-Hata

  • 1Division of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Japan. toru@ims.u-tokyo.ac.jp

Biochemical and Biophysical Research Communications
|May 10, 2011
PubMed

Insights

Tob/BTG proteins, crucial for cell cycle control, are regulated by ubiquitination. Monoubiquitination of Tob proteins prevents their degradation, revealing a new regulatory mechanism for protein stability.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Protein Biochemistry

Background:

  • Tob/BTG proteins are key regulators of cell proliferation.
  • Protein stability is tightly controlled via transcriptional and post-translational modifications, including ubiquitination.
  • Ubiquitin (Ub)/proteasome-dependent degradation is vital for cell cycle progression and DNA damage response.

Purpose of the Study:

  • To investigate the ubiquitination status of Tob family proteins.
  • To identify the specific ubiquitination sites on Tob proteins.
  • To elucidate the functional consequence of Tob protein ubiquitination on its stability and degradation.

Main Methods:

  • In vitro ubiquitination assays without E3 ligases.
  • Mass spectrometry to identify ubiquitination sites.
  • In vivo analysis of wild-type and mutant Tob protein ubiquitination and stability.

Main Results:

  • Tob proteins undergo monoubiquitination independently of E3 ligases in vitro.
  • Lysine residues 48 and 63 in the Tob/BTG homology domain are identified as ubiquitination sites.
  • A Tob mutant lacking Lys48 and Lys63 ubiquitination sites exhibits increased polyubiquitination and degradation in vivo.

Conclusions:

  • Monoubiquitination of Tob proteins acts as a protective mechanism against subsequent polyubiquitination and degradation.
  • This suggests a novel role for monoubiquitination in regulating protein stability.
  • The findings provide new insights into the post-translational regulation of Tob/BTG family proteins.

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