Mih1/Cdc25 is negatively regulated by Pkc1 in Saccharomyces cerevisiae

Kouitiro Yano, Yukifumi Uesono, Satoshi Yoshida, Akihiko Kikuchi, Jun Kashiwazaki, Issei Mabuchi, Yoshiko Kikuchi

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5 Citations (Scopus)

Abstract

Mitotic cyclin-dependent kinase (CDK) is activated by Cdc25 phosphatase through dephosphorylation at the Wee1-mediated phosphorylation site. In Saccharomyces cerevisiae, regulation of Mih1 (Cdc25 homologue) remains unclear because inactivation/degradation of Swe1 (Wee1 homologue) is the main trigger for G2/M transition. By deleting all mitotic cyclins except Clb2, a strain was created where Mih1 became essential for mitotic entry at high temperatures. Using this novel assay, the essential domain of Mih1 was identified and Mih1 regulation was characterized. Mih1(3E1D) with phosphomimetic substitutions of four putative PKC target residues in Mih1 had a reduced complementation activity, whereas Mih1(4A) with those nonphosphorylatable substitutions was active. The band pattern of Mih1 by SDS-PAGE was similar to that of Mih1(4A) only after inactivation of Pkc1 in a pkc1ts mutant. Over-expression of GFP-tagged Mih1 or GFP-Mih1(4A) accumulated as dot-like structures in the nucleus, whereas GFP-Mih1(3E1D) was localized in the cytoplasm. Over-expression of an active form of Pkc1 excluded GFP-Mih1 from the nucleus, but had minimal effect on GFP-Mih1(4A) localization. Furthermore, addition of ectopic nuclear localization signal to the Mih1(3E1D) sequence recovered complementation activity and nuclear localization. These results suggest that Mih1 is negatively regulated by Pkc1-mediated phosphorylation, which excludes it from the nucleus under certain conditions.

Original languageEnglish
Pages (from-to)425-441
Number of pages17
JournalGenes to Cells
Volume18
Issue number6
DOIs
Publication statusPublished - 2013 Jun

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

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    Yano, K., Uesono, Y., Yoshida, S., Kikuchi, A., Kashiwazaki, J., Mabuchi, I., & Kikuchi, Y. (2013). Mih1/Cdc25 is negatively regulated by Pkc1 in Saccharomyces cerevisiae. Genes to Cells, 18(6), 425-441. https://doi.org/10.1111/gtc.12047