Institute of Botany, Heinrich-Heine University, 40225, Düsseldorf, Germany.
Department of Biosciences-Plant Biology, Saarland University, 66123, Saarbruecken, Germany.
New Phytol. 2020 Jan;225(1):250-267. doi: 10.1111/nph.16168. Epub 2019 Oct 13.
The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.
铁（Fe）摄取的关键基本螺旋-环-螺旋（bHLH）转录因子 FER-样缺铁诱导转录因子（FIT）受多种信号通路控制，这对于调整 Fe 摄取以适应生长和环境限制非常重要。FIT 蛋白存在于活性和非活性蛋白池中，FIT 的调节域 C 末端丝氨酸 Ser272 的磷酸化提供了 FIT 激活的触发因素。在这里，我们使用磷酸化突变体活性测定法，并研究了三个另外预测的磷酸化位点（Ser221 以及 Tyr238 和 Tyr278 上的酪氨酸）的磷酸模拟和磷酸化失活突变，除了 Ser272 之外。这些位点的磷酸化突变会影响酵母、植物和哺乳动物细胞中的 FIT 活性。在细胞定位、核迁移、同源二聚化和与 FIT 激活伴侣 bHLH039 的二聚化、启动子转录激活和蛋白质稳定性等方面，观察到了多样化的细胞表型。FIT 的磷酸化模拟 Tyr 突变扰乱了 fit 突变体植物的互补。总之，我们提供的证据表明，FIT 通过 Ser 位点磷酸化而被激活，通过 Tyr 位点磷酸化而被失活。因此，我们提出 FIT 活性受替代磷酸化途径调节。
