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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Phosphorylation of the cyclin CaPcl5 modulates both cyclin stability and specific recognition of the substrate

Einav Simon1, Tsvia Gildor, Daniel Kornitzer

  • 1Department of Molecular Microbiology, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096, Israel.

Journal of Molecular Biology
|June 15, 2013
PubMed

Insights

The Candida albicans cyclin CaPcl5 activates Pho85 kinase, targeting transcription factor CaGcn4 for degradation. This cyclin also self-phosphorylates, leading to its own degradation and loss of substrate recognition, controlling its activity.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Fungal Pathogenesis

Background:

  • Cyclins and cyclin-dependent kinases (CDKs) regulate the cell cycle.
  • The Candida albicans CaPcl5/Pho85 complex targets transcription factor CaGcn4 for degradation.
  • Understanding substrate specificity in cyclin-CDK interactions is crucial for cell cycle control.

Purpose of the Study:

  • To investigate the determinants of substrate selectivity for the CaPcl5/Pho85 complex.
  • To elucidate the regulatory mechanisms controlling CaPcl5 activity and stability.
  • To explore novel mechanisms for limiting cyclin activity in Candida albicans.

Main Methods:

  • Mutational analysis of Candida albicans cyclin CaPcl5.
  • In vitro biochemical assays to assess substrate recognition.
  • In vivo studies using ubiquitin ligase assays to determine degradation pathways.

Main Results:

  • Specific residues in the N-terminal region and cyclin box helix I of CaPcl5 are critical for substrate recognition.
  • CaPcl5 undergoes SCF(CDC4)-mediated self-phosphorylation and subsequent degradation in vivo.
  • In vitro phosphorylation of CaPcl5 leads to a loss of specific substrate recognition.

Conclusions:

  • CaPcl5's substrate selectivity is determined by specific residues within its cyclin box.
  • CaPcl5 self-phosphorylation serves as a dual regulatory mechanism, triggering degradation and reducing substrate binding.
  • This study reveals a novel mechanism for limiting cyclin activity, impacting cell cycle control in Candida albicans.
Keywords:
3-AT3-aminotriazoleCDC4CDKCHXCandida albicansMBPPho85SCF ubiquitin ligaseWTcyclin-dependent kinasecycloheximidemyelin basic proteinwild type

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