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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation

Boris Sarcevic1, Amanda Mawson, Rohan T Baker

  • 1Cancer Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Darlinghurst, NSW, 2010, Australia. b.sarcevic@garvan.org.au

The EMBO Journal
|April 16, 2002
PubMed

Insights

Cyclin-dependent kinases (CDKs) regulate cell cycle progression. This study identifies hHR6A as a CDK substrate, showing its phosphorylation enhances ubiquitin-conjugating activity, crucial for cell proliferation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Cell cycle progression in eukaryotes is tightly regulated by cyclin-dependent kinases (CDKs) phosphorylating various protein substrates.
  • Ubiquitin-conjugating enzymes (UBCs) play critical roles in cellular processes, but their regulation is not fully understood.

Purpose of the Study:

  • To identify novel substrates of CDK2 involved in cell cycle regulation.
  • To investigate the functional consequences of hHR6A phosphorylation by CDKs.

Main Methods:

  • Screening of a cDNA library using solid-phase phosphorylation to identify CDK2 substrates.
  • In vitro phosphorylation assays using CDK-1 and CDK-2 on purified hHR6A.
  • Site-directed mutagenesis of Ser120 in hHR6A.
  • In vivo studies of hHR6A phosphorylation and histone H2B ubiquitylation during the cell cycle.
  • Genetic complementation studies in Saccharomyces cerevisiae.

Main Results:

  • hHR6A was identified as a novel substrate for CDK2.
  • Phosphorylation of hHR6A by CDK-1 and CDK-2 on conserved Ser120 increased its ubiquitin-conjugating activity fourfold.
  • hHR6A phosphorylation peaked during G2/M phase, correlating with increased histone H2B ubiquitylation.
  • Mutations at Ser120 affected hHR6A activity and its ability to support cellular proliferation in yeast.

Conclusions:

  • CDK-mediated phosphorylation of hHR6A on Ser120 is a key regulatory mechanism controlling its ubiquitin-conjugating activity.
  • This phosphorylation event is important for regulating cell cycle progression and cellular proliferation.
  • This study reveals a novel regulatory pathway for UBC family members through phosphorylation on a conserved residue.

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