Authors: Taira Matsuo, Hiroyo Kuramoto, Tsutomu Kumazaki, Youji Mitsui and Tomoko Takahashi

Taira Matsuo

Corresponding author: t-matsuo@kph.bunri-u.ac.jp
Laboratory of Physiological Chemistry; Faculty of Pharmaceutical Sciences at Kagawa; Tokushima Bunri University; Kagawa, Japan

Hiroyo Kuramoto

Laboratory of Physiological Chemistry; Faculty of Pharmaceutical Sciences at Kagawa; Tokushima Bunri University; Kagawa, Japan

Tsutomu Kumazaki

Laboratory of Physiological Chemistry; Faculty of Pharmaceutical Sciences at Kagawa; Tokushima Bunri University; Kagawa, Japan

Youji Mitsui

Laboratory of Physiological Chemistry; Faculty of Pharmaceutical Sciences at Kagawa; Tokushima Bunri University; Kagawa, Japan

Tomoko Takahashi

Laboratory of Physiological Chemistry; Faculty of Pharmaceutical Sciences at Kagawa; Tokushima Bunri University; Kagawa, Japan

Abstract:
The mammalian LIN complex (LINC) plays important roles in regulation of cell cycle genes. LIN54 is an essential core subunit of the LINC and has a DNA binding region (CHC domain), which consists of two cysteine-rich (CXC) domains separated by a short spacer. We generated various LIN54 mutants, such as CHC deletion mutant, and investigated their subcellular localizations and effects on cell cycle. Wild-type LIN54 was predominantly localized in the nucleus. We identified two nuclear localization signals (NLSs), both of which were required for nuclear localization of LIN54. Interestingly, deletion of one CXC domain resulted in an increased cytoplasmic localization. The cytoplasmic LIN54 mutant accumulated in the nucleus after leptomycin B treatment, suggesting CRM1-mediated nuclear export of LIN54. Point mutations (C525Y and C611Y) in conserved cysteine residues of CXC domain that abolish DNA binding activity also increased cytoplasmic localization. These data suggest that DNA binding activity of LIN54 is required for its nuclear retention. We also found that LIN54^C525Y and LIN54^C611Y inhibited cell cycle progression and led to abnormal nuclear morphology. Other CXC mutants also induced similar abnormalities in cell cycle progression. LIN54^C525Y led to a decreased expression of some G₂/M genes, whose expressions are regulated by LINC. This cell cycle inhibition was partially restored by overexpression of wild-type LIN54. These results suggest that abnormal cellular localization of LIN54 may have effects on LINC activity.

Received: June 18, 2012; Accepted: July 20, 2012; Published Online: August 16, 2012

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