Department of Molecular Biosciences and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, 78712, USA.
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, 210095, China.
New Phytol. 2021 Jun;230(6):2311-2326. doi: 10.1111/nph.17332. Epub 2021 Apr 6.
Elongated hypocotyl5 (HY5) is a key transcription factor that promotes photomorphogenesis. Constitutive photomorphogenic1 (COP1)-Suppressor of phytochrome A-105 (SPA) E3 ubiquitin ligase complex promotes ubiquitination and degradation of HY5 to repress photomorphogenesis in darkness. HY5 is also regulated by phosphorylation at serine 36 residue. However, the kinase responsible for phosphorylation of HY5 remains unknown. Here, using extensive in vitro and in vivo biochemical, genetic, and photobiological techniques, we have identified a new kinase that phosphorylates HY5 and demonstrated the significance of phosphorylation of HY5 in Arabidopsis thaliana. We show that SPA proteins are the missing kinases necessary for HY5 phosphorylation. SPAs can directly phosphorylate HY5 in vitro, and the phosphorylated HY5 is absent in the spaQ background in vivo. We also demonstrate that the unphosphorylated HY5 interacts strongly with both COP1 and SPA1 and is the preferred substrate for degradation, whereas the phosphorylated HY5 is more stable in the dark. In addition, the unphosphorylated HY5 actively binds to the target promoters and is the physiologically more active form. Consistently, the transgenic plants expressing the unphosphorylated form of HY5 display enhanced photomorphogenesis. Collectively, our study revealed the missing kinase responsible for direct phosphorylation of HY5 that fine-tunes its stability and activity to regulate photomorphogenesis.
长柱下胚轴 5（HY5）是一种促进光形态建成的关键转录因子。组成型光形态建成 1（COP1）-抑制光敏素 A-105（SPA）E3 泛素连接酶复合物促进 HY5 的泛素化和降解，以抑制黑暗中的光形态建成。HY5 还受丝氨酸 36 残基的磷酸化调节。然而，负责 HY5 磷酸化的激酶仍然未知。在这里，我们使用广泛的体外和体内生化、遗传和光生物学技术，鉴定了一种新的激酶，该激酶可以磷酸化 HY5，并证明了 HY5 在拟南芥中的磷酸化在光形态建成中的重要性。我们表明 SPA 蛋白是 HY5 磷酸化所必需的缺失激酶。SPAs 可以在体外直接磷酸化 HY5，并且在体内 spaQ 背景中不存在磷酸化的 HY5。我们还证明了未磷酸化的 HY5 与 COP1 和 SPA1 都有强烈的相互作用，并且是降解的首选底物，而在黑暗中，磷酸化的 HY5 更稳定。此外，未磷酸化的 HY5 积极结合到靶启动子上，并且是生理上更活跃的形式。一致地，表达未磷酸化形式的 HY5 的转基因植物显示出增强的光形态建成。总的来说，我们的研究揭示了负责直接磷酸化 HY5 的缺失激酶，该激酶通过调节其稳定性和活性来精细调节光形态建成。
