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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation

Yong Chi1,2, John H Carter3, Jherek Swanger1

  • 1Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N. Seattle, WA 98109, USA.

Science Advances
|June 5, 2020
PubMed

Insights

Researchers identified 117 potential cell division targets of cyclin-dependent kinase 2 (CDK2) using an in situ method. This approach revealed new CDK2 substrates involved in chromatin regulation and epigenetic processes.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinase 2 (CDK2) plays a critical role in regulating the cell cycle.
  • CDK2 is implicated in various cancers due to its central role in oncogenic signaling pathways.

Purpose of the Study:

  • To identify novel substrates of CDK2 within the cell nucleus using an "in situ" approach.
  • To investigate the role of CDK2 in regulating chromatin, transcription, and DNA/RNA metabolism.

Main Methods:

  • Utilized an "in situ" phosphorylation method on isolated nuclei from cells engineered to express a modified CDK2.
  • Employed adenosine 5 omino-triphosphate analogs for specific labeling of CDK2 substrates.
  • Validated candidate substrates through biochemical assays.

Main Results:

  • Identified 117 candidate CDK2 substrates, with approximately 40% being previously known.
  • Validated novel substrates, including LSD1, DOT1L, and Rad54, demonstrating CDK2's involvement in epigenetic regulation.
  • Discovered that many identified substrates are chromatin-associated proteins involved in histone modification, transcription, and nucleic acid metabolism.

Conclusions:

  • The "in situ" phosphorylation technique is effective for identifying nuclear kinase substrates with high validation rates.
  • CDK2 substrates are involved in diverse cellular processes, including epigenetic regulation and nucleic acid metabolism, suggesting new links between cell division and these functions.
  • This methodology can be broadly applied to discover substrates for other nuclear kinases.

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