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，具有管家功能或在应激适应期间特异性控制靶蛋白的定位或活性。PtdIns（4，5）P2是由磷脂酰肌醇4 β 5-激酶（PI 4P 5-kinases）作用而形成的，该激酶在拟南芥中作为11种同工酶的家族而存在。我们已经确定了几个PI 4P 5-激酶在拟南芥中具有重要的功能。在各种真核生物模型中，PI 4P 5-激酶通过翻译后修饰来调节，并且已经表明不同的拟南芥PI 4P 5-激酶可以被磷酸化。然而，到目前为止，仍然不清楚i）什么事件可能触发磷酸化，ii）什么氨基酸被磷酸化，以及iii）关于酶在植物中的定位和功能的后果是什么。因此，将研究拟南芥植物中PI 4P 5-激酶的磷酸化。将确定磷酸化位点，并将相应的氨基酸改变为丙氨酸或天冬氨酸残基，分别组成性模拟去磷酸化或磷酸化状态。重组变体蛋白将在体外进行生化表征。荧光标记蛋白的亚细胞定位将在异源系统（蚕豆保卫细胞和烟草花粉管）中瞬时表达和拟南芥中稳定表达期间进行分析。将通过现有拟南芥T-DNA突变体的互补来测试生理功能。

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.