Regulation of Arabidopsis PI4P 5-kinases by phosphorylation

磷脂酰肌醇-4，5-二磷酸(PtdIns(4，5)P2)是一种调节性磷脂，它特异性地控制具有看家功能或在压力适应过程中的靶蛋白的定位或活性。PtdIns(4，5)P2是由PI4P5-Kinase作用形成的，PI4P5-Kinase是一个由11个同工酶组成的家族。我们已经鉴定了几个在拟南芥中具有重要功能的PI4P5-激酶。在各种真核生物模型中，PI4P5-激酶都受到翻译后修饰的调节，已有研究表明不同的拟南芥PI4P5-激酶可以被磷酸化。然而，到目前为止，还不清楚：1)什么事件可能触发磷酸化；2)什么氨基酸被磷酸化；3)这些酶在植物中的定位和功能会产生什么后果。因此，将对拟南芥植物中PI4P5-激酶的磷酸化进行研究。将确定磷酸化的位置，并将相应的氨基酸改变为丙氨酸或天冬氨酸残基，分别构成地模拟去磷酸化或磷酸化状态。重组变异蛋白将在体外进行生化表征。荧光标记蛋白在异源系统(蚕豆保卫细胞和烟草花粉管)中瞬时表达和在拟南芥中稳定表达的亚细胞定位将被分析。生理功能将通过补充现有的拟南芥T-DNA突变体进行测试。

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) is a regulatory phospholipid that specifically controls localization or activity of target proteins with housekeeping functions or during stress-adaptation. PtdIns(4,5)P2 is formed by action of PI4P 5-kinases, which occur as a family of 11 isoenzymes in Arabidopsis. We have identified several PI4P 5- kinases with important functions in Arabidopsis. In various eukaryotic models PI4P 5-kinases are regulated by posttranslational modification and it has been shown that different Arabidopsis PI4P 5-kinases can be phosphorylated. However, so far it remains unclear i) what events might trigger phosphorylation, ii) what amino acids are phosphorylated, and iii) what consequences are regarding localization and functionality of the enzymes in the plants. Thus, the phosphorylation of PI4P 5-kinases in Arabidopsis plants will be investigated. The sites of phosphorylation will be determined and the respective amino acids changed to alanine or aspartate residues, constitutively mimicking the dephosphorylated or phosphorylated states, respectively. Recombinant variant proteins will be biochemically characterized in vitro. The subcellular localization of fluorescence-tagged proteins will be analyzed during transient expression in heterologous systems (Vicia faba guard cells and tobacco pollen tubes) and with stable expression in Arabidopsis. Physiological functionality will be tested by complementation of existing Arabidopsis T-DNA mutants.