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Nek9, a novel FACT-associated protein, modulates interphase progression
Bertrand Chin-Ming Tan1, Sheng-Chung Lee
1Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
The Journal of Biological Chemistry
|December 9, 2003
Summary
The FACT complex interacts with Nek9, a novel partner crucial for cell cycle progression. This interaction, involving Nek9 phosphorylation, is essential for maintaining G1 and S phase, highlighting a new role for FACT in interphase regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The FACT complex (Spt16-Pob3/DUF/FACT) modulates chromatin structure, impacting DNA replication and transcription.
- The precise functions and molecular interactions of mammalian FACT beyond transcription elongation remain largely uncharacterized.
Purpose of the Study:
- To identify novel interacting partners and functional roles of the mammalian FACT complex.
- To elucidate the molecular mechanisms underlying FACT-mediated cellular processes during interphase.
Main Methods:
- Co-immunoprecipitation to identify interacting proteins.
- Electrophoretic mobility shift assays to assess complex formation and phosphorylation.
- RNA interference (dsRNAi) to analyze the functional impact of Nek9 depletion on cell cycle progression.
- Western blotting to detect protein phosphorylation at specific residues.
Main Results:
- A novel, stable ~600-kDa complex was identified between FACT and Nek9 in interphase nuclei.
- Active Nek9, indicated by phosphorylation at Thr(210), showed elevated phosphorylation when complexed with FACT.
- Depletion of Nek9 (Nek9(dsRNAi)) impaired G1 and S phase progression, correlating with phospho-Nek9-FACT complex formation.
Conclusions:
- Nek9 is a novel functional partner of the FACT complex.
- Nek9, potentially acting as an enzymatic partner, mediates FACT-associated processes vital for interphase progression.
- The phospho-Nek9-FACT complex plays a critical role in regulating cell cycle progression through G1 and S phases.