Ingo Heilmann, second project
Prof. Ingo Heilmann
Institute of Biochemistry and Biotechnology/Cellular Biochemistry
Kurt-Mothes-Straße 3
06120 Halle (Saale)
phone: +49 (0) 345-55 24840
ingo.heilmann@biochemtech.uni-halle.de
Identification of protein kinases controlling Arabidopsis PI4P 5-kinases
The minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), acts as a lipid ligand to various target proteins in eukaryotic
cells. Because PtdIns(4,5)P2 is involved in the regulation of numerous cellular processes, its regulation must be tightly regulated. In the model plant Arabidopsis thaliana PtdIns(4,5)P2 is formed by a family of eleven PI4P 5-kinases, PIP5K1-11. In previous experiments we have shown that several PI4P 5-kinases can be phosphorylated in vitro by complex extracts from various Arabidopsis tissues, and phosphorylation sites have been identified by mass spectrometry and by peptide array analysis. The investigation of recombinant PI4P 5-kinases carrying amino acid substitutions in some of the indentified sites suggests an impact of phosphorylation on the catalytic function of the enzymes. Preliminary evidence suggests a number of Arabidopsis protein kinases as candidates that might phosphorylate PI4P 5-kinases.
It is the goal of the proposed work to identify Arabidopsis protein kinases that phosphorylate PI4P 5-kinases. The identification of candidate protein kinases will be achieved by a combination of methods, including in-gel-protein phosphorylation tests and yeast-two-hybrid (Y2H) tests, the review of known expression and localization patterns and the consideration of shared effects between protein kinases and PI4P 5-kinases on the same biological processes. Candidates will be recombinantly expressed and affinity-purified; purified recombinant PI4P 5-kinases will be tested as in vitro phosphorylation substrates for the protein kinase candidates. In cases of positive phosphorylation, effects of phosphorylation on the catalytic activity of PI4P 5-kinases will be assessed. Moreover, phosphorylation sites will be determined using mass spectrometry and peptide arrays. Upon successful identification of phosphosites, the corresponding residues will be substituted (A/D), and the recombinant PI4P 5-kinase substitution variants characterized in vitro. The data gathered will contribute to our understanding of PtdIns(4,5)P2-production in plants. The
evolutionary conservation of the PI-signaling system may enable the transfer of information gained to PI4P 5-kinases from other eukaryotic models.