Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells

Yuki Komamura-Kohno, Kumiko Karasawa-Shimizu, Takako Saitoh, Michio Sato, Fumio Hanaoka, Shoji Tanaka, Yukio Ishimi

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26 Citations (Scopus)

Abstract

MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.

Original languageEnglish
Pages (from-to)1224-1239
Number of pages16
JournalFEBS Journal
Volume273
Issue number6
DOIs
Publication statusPublished - 1 Mar 2006

Keywords

  • CDK
  • Cell cycle
  • DNA replication
  • MCM proteins
  • Phosphorylation

Cite this

Komamura-Kohno, Y., Karasawa-Shimizu, K., Saitoh, T., Sato, M., Hanaoka, F., Tanaka, S., & Ishimi, Y. (2006). Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells. FEBS Journal, 273(6), 1224-1239. https://doi.org/10.1111/j.1742-4658.2006.05146.x