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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Cyclin E-CDK2 protein phosphorylates plant homeodomain finger protein 8 (PHF8) and regulates its function in the cell

Liping Sun1, Yan Huang1, Qian Wei1

  • 1From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, the University of the Chinese Academy of Sciences, Beijing 100101, China, and.

The Journal of Biological Chemistry
|December 31, 2014
PubMed

Insights

Cyclin E-CDK2 phosphorylates PHF8 at Ser-844, enhancing its demethylase activity. This promotes rRNA transcription and cell cycle progression, revealing a new regulatory mechanism for G1/S transition.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Epigenetics

Background:

  • Cyclin E-CDK2 is crucial for the G1/S phase transition.
  • PHF8 (plant homeodomain finger protein 8) was previously identified as a CDK2-interacting protein.

Purpose of the Study:

  • To investigate PHF8 as a novel substrate of cyclin E-CDK2.
  • To elucidate the functional consequences of PHF8 phosphorylation by cyclin E-CDK2.

Main Methods:

  • Mass spectrometry to identify phosphorylation sites.
  • Immunoblotting and flow cytometry to assess protein function.
  • Real-time PCR and ChIP assays to analyze gene transcription and promoter binding.
  • Luciferase reporter assays for transcriptional activity.

Main Results:

  • PHF8 Ser-844 was confirmed as a cyclin E-CDK2 phosphorylation site.
  • Phosphorylation enhanced PHF8's histone H3K9me2 demethylase activity.
  • WT PHF8 promoted S phase progression and transcription of cyclin E, E2F3, and E2F7 more effectively than the S844A mutant.
  • Phosphorylation stimulated PHF8-dependent rRNA transcription.

Conclusions:

  • Cyclin E-CDK2-mediated phosphorylation of PHF8 at Ser-844 activates its demethylase activity.
  • This activation promotes rRNA transcription and facilitates cell cycle progression.
  • PHF8 is a key effector linking cyclin E-CDK2 activity to gene expression and cell cycle control.
Keywords:
Cell CycleCell SignalingCyclinCyclin E-CDK2, PHF8, Phosphorylation, rRNA TranscriptionCyclin-dependent Kinase (CDK)Phosphorylation

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