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

Sayoko Sanada; Masashi Maekawa; Sota Tate; Hiroki Nakaoka; Yasuhiro Fujisawa; Koji Sayama; Shigeki Higashiyama

doi:10.1016/j.bbrc.2023.02.012

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

Biochem Biophys Res Commun. 2023 Apr 9:651:30-38. doi: 10.1016/j.bbrc.2023.02.012. Epub 2023 Feb 4.

Authors

Sayoko Sanada¹, Masashi Maekawa², Sota Tate³, Hiroki Nakaoka⁴, Yasuhiro Fujisawa⁴, Koji Sayama⁴, Shigeki Higashiyama⁵

Affiliations

¹ Department 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.
² Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan; Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime, 791-0295, Japan. Electronic address: maekawa-ms@keio.jp.
³ Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan; Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime, 791-0295, Japan.
⁴ Department of Dermatology, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan.
⁵ Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan; Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime, 791-0295, Japan; Department of Oncogenesis and Tumor Regulation, Osaka International Cancer Institute, Chuo-ku, Osaka, 541-8567, Japan. Electronic address: shigeki@m.ehime-u.ac.jp.

PMID: 36791496
DOI: 10.1016/j.bbrc.2023.02.012

Abstract

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, leads to the ubiquitination of >40 of its target substrates. Since a variety of point mutations in the substrate-binding domain of SPOP have been identified in cancers, including prostate and endometrial cancers, the pathological roles of those cancer-associated SPOP mutants have been extensively elucidated. In this study, we evaluated the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells expressing wild-type SPOP gene. SPOP knockdown using siRNA in HaCaT cells dramatically reduced cell growth and arrested their cell cycles at G1/S phase. The expression of DNA replication licensing factors CDT1 and CDC6 in HaCaT cells drastically decreased on SPOP knockdown as their translation was inhibited. CDT1 and CDC6 downregulation induced p21 expression without p53 activation. Our results suggest that SPOP is essential for DNA replication licensing in non-cancerous keratinocyte HaCaT cells.

Keywords: CDC6; CDT1; DNA replication licensing; SPOP; Translation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism
DNA Replication / genetics
Endometrial Neoplasms* / genetics
Female
HaCaT Cells* / metabolism
HaCaT Cells* / pathology
Humans
Male
Nuclear Proteins / genetics
Nuclear Proteins / metabolism
Ubiquitination

Substances

SPOP protein, human
Cell Cycle Proteins
CDC6 protein, human
Nuclear Proteins
CDT1 protein, human