Control of PI4P 5-kinases by reversible phosphorylation in Arabidopsis thaliana

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) is a phospholipid that controls a diverse set of physiological functions by modulating localization or activity of target proteins. PtdIns(4,5)P2 is formed by PI4P 5-kinases, which occur as 11 isoenzymes in Arabidopsis. In various eukaryotic models PI4P 5-kinases are regulated by posttranslational modification and it has been shown that different PI4P 5-kinases from other model systems, including Arabidopsis PIP5K1, can be phosphorylated. Based on the solved X-ray structure of the human PIPKIIβ it has been proposed that phosphorylation at the membrane-oriented face of the protein impairs electrostatical interactions with the substrate membrane and thus, inhibits catalytic activity. For plant PI4P 5-kinases only little information is available and so far it remained unclear whether other Arabidopsis PI4P 5-kinases can be phosphorylated, what amino acids are phosphorylated, and what consequences arise for localization and functionality of the enzymes in plants. In the context of this thesis, phosphorylation of two Arabidopsis PI4P 5-kinases, PIP5K2 and PIP5K3 was investigated. Recombinantly expressed and affinity-purified PIP5K2 and PIP5K3 were found to be phosphorylated in vitro by a commercially available PKA and also by complex Arabidopsis extracts containing protein kinase activity. Phosphorylation of PIP5K2 and PIP5K3 decreased catalytic activity in an ATP dependent manner. Interestingly, treatment of PIP5K3 with plant extract without added ATP increased activity, whereas the addition of ATP resulted in a decrease in activity, indicating the presence of both activating and inhibiting factors in the plant extract. The sites of phosphorylation were determined by computer aided prediction, MS-based analysis of tryptic peptides and by peptide array analysis, and several phosphorylation sites were determined for PIP5K2 and PIP5K3. The respective amino acids were changed to alanine or aspartate residues, mimicking the dephosphorylated or phosphorylated states, respectively. The recombinant variant proteins were characterized in vitro for catalytic function and their subcellular localization was analysed during heterologous expression as fluorescence-tagged fusions in tobacco pollen tubes. Variant proteins were also expressed in relevant Arabidopsis mutant backgrounds and tested for complementation of known phenotypes. The investigations revealed PIP5K2 T430, PIP5K2 T500 and PIP5K3 T472 as strong candidates for phosphorylation sites. The location of the phosphorylation sites on a 3D-model of plant PI4P 5-kinases indicates that the control of PI4P 5-kinases by phosphorylation not only occurs by controlling membrane association but also includes phosphorylation sites not located in the protein-membrane interface.