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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization

Lina Herhaus1, Ana B Perez-Oliva1, Giorgio Cozza2

  • 1MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

Science Signaling
|April 16, 2015
PubMed

Insights

Casein kinase 2 (CK2) phosphorylates OTUB1, a deubiquitylating enzyme, promoting its nuclear entry. This nuclear translocation is crucial for OTUB1

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • OTUB1 is a deubiquitylating enzyme found in both cytosolic and nuclear compartments, targeting numerous substrates.
  • The localization and regulation of OTUB1 activity are critical for cellular processes, including DNA repair.

Purpose of the Study:

  • To investigate the role of casein kinase 2 (CK2) in regulating the cellular localization and function of OTUB1.
  • To determine the impact of CK2-mediated OTUB1 phosphorylation on DNA damage repair pathways.

Main Methods:

  • Utilized pharmacological inhibition and genetic ablation of CK2 in various cell types.
  • Employed Western blotting and immunofluorescence to detect OTUB1 phosphorylation and localization.
  • Performed in vitro assays to assess the catalytic activity and substrate binding of phosphorylated OTUB1.
  • Analyzed the formation of 53BP1 DNA repair foci in osteosarcoma cells following ionizing radiation exposure.

Main Results:

  • CK2 phosphorylates OTUB1 at Serine 16 (Ser16), a modification essential for its nuclear accumulation.
  • Inhibition or genetic deletion of CK2 prevents OTUB1 phosphorylation at Ser16, leading to its exclusion from the nucleus.
  • Phosphorylated OTUB1 exclusively localizes to the nucleus, while unphosphorylated OTUB1 is primarily cytosolic.
  • Both phosphorylated and non-phosphorylated OTUB1 exhibit similar in vitro catalytic activity, ubiquitin chain binding, and UBE2N interaction.
  • CK2-dependent nuclear translocation of OTUB1 enhances the formation of 53BP1 DNA repair foci in response to ionizing radiation.

Conclusions:

  • CK2-mediated phosphorylation of OTUB1 at Ser16 is a key regulatory mechanism controlling its nuclear import.
  • Nuclear localization of OTUB1, driven by CK2 activity, is necessary for efficient DNA damage repair.
  • These findings highlight a critical interplay between CK2, OTUB1, and DNA repair pathways.

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