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Ubiquitin Chain Analysis by Parallel Reaction Monitoring
08:33

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Published on: June 17, 2020

Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains

Abdallah K Al-Hakim1, Anna Zagorska, Louise Chapman

  • 1MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.

The Biochemical Journal
|February 8, 2008
PubMed

Insights

AMPK-related kinases NUAK1 and MARK4 are regulated by the deubiquitinating enzyme USP9X. This study reveals unusual Lys(29)/Lys(33)-linked polyubiquitin chains control these key cell signaling proteins.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • AMPK-related kinases, including NUAK1 and MARK4, are crucial regulators of cell polarity and proliferation.
  • These kinases are activated by the LKB1 tumor suppressor kinase.
  • The precise regulatory mechanisms, particularly post-translational modifications, are not fully understood.

Purpose of the Study:

  • To investigate the role of ubiquitination and deubiquitination in the regulation of NUAK1 and MARK4.
  • To identify the specific ubiquitin linkages involved in the regulation of these kinases.
  • To explore the functional consequences of ubiquitination on NUAK1 and MARK4 activity.

Main Methods:

  • In vivo polyubiquitination assays.
  • Co-immunoprecipitation to study protein interactions.
  • Knockdown and overexpression studies of USP9X.
  • Analysis of ubiquitin chain topology (Lys(29)/Lys(33) linkages).
  • Site-directed mutagenesis to identify non-binding mutants.

Main Results:

  • NUAK1 and MARK4 were found to be polyubiquitinated in vivo and interact with USP9X.
  • USP9X deubiquitinates NUAK1 and MARK4, with knockdown of USP9X increasing their polyubiquitination.
  • Ubiquitin chains linked by Lys(29) and/or Lys(33) were identified on NUAK1 and MARK4.
  • Non-USP9X-binding mutants exhibited hyper-ubiquitination and reduced LKB1-mediated phosphorylation.
  • AMPK and other AMPK-related kinases also showed polyubiquitination.

Conclusions:

  • NUAK1 and MARK4 are substrates of the deubiquitinating enzyme USP9X.
  • The study provides the first evidence for regulation of AMPK family kinases by Lys(29)/Lys(33)-linked polyubiquitin chains.
  • Polyubiquitination, mediated by these unusual linkages, may serve as an inhibitory mechanism for NUAK1 and MARK4 activity.

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