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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1)

Takashi Shiromizu1, Hidemasa Goto, Yasuko Tomono

  • 1Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.

Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|April 25, 2006
PubMed

Insights

Checkpoint kinase 1 (Chk1) is phosphorylated during mitosis, with reduced phosphorylation at the metaphase-anaphase transition. Cyclin-dependent kinase 1 (Cdk1) regulates novel Chk1 phosphorylation sites, impacting mitotic progression.

Area of Science:

  • Cell biology
  • Molecular oncology
  • DNA damage response

Background:

  • Checkpoint kinase 1 (Chk1) is activated by ATR in response to DNA damage and replication stress, preventing premature mitosis.
  • The precise role and regulation of Chk1 during mitosis itself remain poorly understood.

Purpose of the Study:

  • To investigate the phosphorylation status and regulation of Chk1 during mitosis.
  • To identify novel Chk1 phosphorylation sites and their regulatory kinases in mitosis.

Main Methods:

  • Two-dimensional phosphopeptide mapping to identify Chk1 phosphorylation sites.
  • Treatment with specific kinase inhibitors (butyrolactone I, SB203580) and siRNA.
  • Cell cycle arrest using nocodazole followed by genotoxic stress (IR, UV).

Main Results:

  • Chk1 undergoes phosphorylation during mitosis, with a notable reduction at the metaphase-anaphase transition.
  • Novel phosphorylation sites, Ser286 and Ser301, were identified on Chk1.
  • Cyclin-dependent protein kinase 1 (Cdk1) inhibition reduced Chk1 phosphorylation at Ser301, suggesting Cdk1 as a key regulator.
  • Genotoxic stresses (IR, UV) did not induce canonical Chk1 phosphorylation at Ser317/Ser345 in mitotic cells.

Conclusions:

  • Mitotic Chk1 phosphorylation is regulated by novel sites, distinct from those activated by ATR during interphase.
  • Cyclin-dependent kinase 1 (Cdk1) plays a significant role in phosphorylating Chk1 during mitosis.
  • These findings suggest a Cdk1-mediated regulation of mitotic Chk1 function through novel phosphorylation sites.

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