Numamoto Minori, Sasano Yu, Hirasaki Masataka, Sugiyama Minetaka, Maekawa Hiromi, Harashima Satoshi
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan and Yeast Genetic Resources Laboratory, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan and Yeast Genetic Resources Laboratory, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
J Biochem. 2015 Jan;157(1):53-64. doi: 10.1093/jb/mvu055. Epub 2014 Oct 13.
The Saccharomyces cerevisiae Siw14, a tyrosine phosphatase involved in the response to caffeine, participates in regulation of the phosphorylation and intracellular localization of Gln3, a GATA transcriptional activator of nitrogen catabolite repression-sensitive genes. In Δsiw14 cells, the phosphorylation level of Gln3 is decreased and the nuclear localization of Gln3 is stimulated by caffeine. However, the mechanism by which Siw14 controls the localization and function of Gln3 remains unclear, although the nuclear localization of Gln3 is known to be induced by activation of the type 2A phosphatases (PP2As) Pph21 and Pph22, and the type 2A-related phosphatase Sit4. In this study, we show that the increased nuclear localization of Gln3 in response to caffeine caused by disruption of the SIW14 gene is dependent on the Sit4 and PP2A phosphatases. We also show that decreased phosphorylation of Gln3 caused by disruption of the SIW14 gene is completely suppressed by deletion of both PPH21 and PPH22, but only partially suppressed by deletion of SIT4. Taking these results together, we conclude that Siw14 functions upstream of Pph21 and Pph22 as an inhibitor of the phosphorylation and localization of Gln3, and that Sit4 acts independently of Siw14.
酿酒酵母Siw14是一种参与咖啡因应答的酪氨酸磷酸酶，它参与调控Gln3的磷酸化和细胞内定位，Gln3是氮分解代谢物阻遏敏感基因的GATA转录激活因子。在Δsiw14细胞中，Gln3的磷酸化水平降低，并且咖啡因可刺激Gln3的核定位。然而，尽管已知Gln3的核定位是由2A型磷酸酶（PP2As）Pph21和Pph22以及2A相关磷酸酶Sit4的激活所诱导，但Siw14控制Gln3定位和功能的机制仍不清楚。在本研究中，我们表明，SIW14基因破坏导致的Gln3对咖啡因应答时核定位增加依赖于Sit4和PP2A磷酸酶。我们还表明，SIW14基因破坏导致的Gln3磷酸化减少被PPH21和PPH22的缺失完全抑制，但仅被SIT4的缺失部分抑制。综合这些结果，我们得出结论，Siw14作为Gln3磷酸化和定位的抑制剂在Pph21和Pph22的上游发挥作用，并且Sit4独立于Siw14发挥作用。
