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Saccharomyces CDK1 phosphorylates Rad53 kinase in metaphase, influencing cellular morphogenesis

Laura Diani1, Claudia Colombelli, Benjamin Tamilselvan Nachimuthu

  • 1Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita' degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.

The Journal of Biological Chemistry
|October 6, 2009
PubMed

Insights

The protein kinase Rad53, crucial for DNA repair checkpoints, is phosphorylated by Clb-CDK1 at Ser774. This modification impacts cellular morphogenesis but not DNA damage or replication roles.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Rad53 is a key protein kinase regulating DNA damage and replication stress checkpoints in budding yeast.
  • Rad53 also plays a role in cellular morphogenesis.
  • Phosphorylation sites on Rad53 include SQ/TQ and SP/TP residues, targeted by PI3K-related kinases and CDKs.

Purpose of the Study:

  • To investigate the role of Clb-CDK1 in phosphorylating Rad53.
  • To determine the impact of Rad53 phosphorylation at Ser774 on its known functions.
  • To explore the connection between Rad53 phosphorylation and cellular morphogenesis.

Main Methods:

  • Mass spectrometry was used to identify phosphorylation sites on Rad53.
  • Site-directed mutagenesis was employed to create a Ser774-to-Asp mutation.
  • Sensitivity to calcofluor was assessed to evaluate cellular morphogenesis defects.

Main Results:

  • Clb-CDK1 phosphorylates Rad53 at Ser774 during metaphase.
  • This phosphorylation event is independent of the spindle assembly checkpoint.
  • Phosphorylation at Ser774 does not affect Rad53's DNA damage or replication checkpoint functions.
  • A Ser774-to-Asp mutation confers calcofluor sensitivity, indicating a role in morphogenesis.

Conclusions:

  • Clb-CDK1-mediated phosphorylation of Rad53 at Ser774 is a novel regulatory mechanism.
  • This specific phosphorylation event links Rad53 to cellular morphogenesis processes.
  • Rad53's role extends beyond DNA checkpoints to include cytoskeletal regulation.

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