Plant Phosphoinositide Kinases. A Study of PtdIns 4-kinases and a PtdIns 4-phosphate 5-kinase

Sammanfattning: Phosphoinositides are of particular interest in signal transduction processes as they can be sequentially phosphorylated and used as precursors for the intracellular messengers inositol trisphosphate (InsP3) and diacylglycerol (DAG) that in turn affects calcium levels and protein kinase cascades, eventually leading to a physiological response. The activity and regulation of PtdIns 4-kinases and PtdInsP kinase was studied. Two distinct PtdIns 4-kinase activities were detected in spinach plasma membranes. They differed in size and response to metal ions. The larger one (120 kDa) has striking similarities to PtdIns 4-kinase from Arabidopsis thaliana. The smaller one (65 kDa) is so far unique. The PtdInsP kinase from A. thaliana, AtPIP5K1p, was shown to be regulated by phosphorylation in vitro, which represents the first indication of regulation of plant PtdIns metabolism by phosphorylation-dephosphorylation. Further characterisation of this enzyme, which is responsible for increased turnover of PtdIns(4,5)P2, revealed dual substrate specificity. It catalyses the formation of both PtdIns(4,5)P2 and PtdIns(3,4)P2 from PtdIns4P and PtdIns3P, respectively. Drought and salinity are among the most usual and hence significant of the stresses a plant must endure in nature and these stress factors are therefore also the most detrimental for crop yields. Phospholipids in suspension cultured A. thaliana cells were identified and characterised. Pulse-radiolabelling experiments revealed a dramatic turnover of several phospholipids including PtdIns(4,5)P2 diacylglycerolpyrophosphate and phosphatidylcholine in response to salinity and osmotic shock. Diacylglycerolpyrophosphate and phosphatidylcholine turnover in response to sorbitol as an osmotic agent, was much less dramatic than their response to salt. PtdIns(4,5)P2 turnover, on the other hand, was highly increased for both sorbitol and salt, indicating that plant cells can discern different osmotic stresses.