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SPOP is essential for DNA replication licensing through maintaining translation of CDT1 and CDC6 in HaCaT cells

Sayoko Sanada1, Masashi Maekawa2, Sota Tate2

  • 1Department of Dermatology, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan; Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan.

Biochemical and Biophysical Research Communications
|February 15, 2023
PubMed

Insights

Speckle-type pox virus and zinc finger (POZ) protein (SPOP) is crucial for cell growth and DNA replication licensing in keratinocytes. Its absence halts cell cycle progression and inhibits key replication factors, highlighting its essential role.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Speckle-type pox virus and zinc finger (POZ) protein (SPOP) acts as a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, mediating ubiquitination of over 40 substrates.
  • Cancer-associated point mutations in SPOP's substrate-binding domain are linked to prostate and endometrial cancers, prompting extensive research into its pathological roles.

Purpose of the Study:

  • To investigate the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells.
  • To elucidate the role of SPOP in cell growth, cell cycle progression, and DNA replication in keratinocytes.

Main Methods:

  • SPOP was knocked down using small interfering RNA (siRNA) in HaCaT cells.
  • Cell growth, cell cycle phase distribution, and expression levels of DNA replication licensing factors (CDT1, CDC6) were analyzed.
  • p21 and p53 activation was assessed following SPOP knockdown.

Main Results:

  • SPOP knockdown significantly reduced cell growth and caused G1/S phase cell cycle arrest in HaCaT cells.
  • The expression of DNA replication licensing factors CDT1 and CDC6 decreased drastically due to inhibited translation upon SPOP knockdown.
  • Downregulation of CDT1 and CDC6 led to increased p21 expression without activating p53.

Conclusions:

  • Wild-type SPOP is essential for normal cell growth and proliferation in human keratinocytes.
  • SPOP plays a critical role in regulating DNA replication licensing by controlling the expression of CDT1 and CDC6.
  • These findings identify SPOP as a key regulator of DNA replication in non-cancerous keratinocytes.
Keywords:
CDC6CDT1DNA replication licensingSPOPTranslation

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