Phosphorylation of signaling phospholipids in Coffea arabica cells

Membrane preparations of Coffea arabica suspension cells were incubated in the presence of 〚³²P〛γ-ATP. After lipid extraction and separation by thin layer chromatography, the following phosphorylated lipids were detected: phosphatidylinositol 4,5 bis-phosphate (PtdIns4,5P₂), lyso-phosphatidylinositol 4-phosphate (LPtdIns4P), phosphatidylinositol 4-phosphate (PtdIns4P), diacylglycerol pyrophosphate (DGPP), lyso-phosphatidic acid (LPA) and phosphatidic acid (PA). This suggests the presence of phosphatidylinositol (EC 2.7.1.67), phosphatidylinositol 4 phosphate (EC 2.7.1.68), diacylglycerol (EC 2.7.1.107) and monoacylglycerol (EC 2.1.1.94) kinases. The activities of these lipid kinases changed during the culture period of the C. arabica cells reaching peak at day 7 of culture; however, enzymatic activities were very low before and after day 7. The behavior of these lipid kinases in the presence of their respective substrates and exogenous substrates such as ATP was characterized. The apparent K_m values for ATP of all the lipid kinase activities were lower than 30 μM. All kinase activities assayed were totally dependent on the presence of Mg²⁺ and were unable to use Mn²⁺ or Ca²⁺ which produced a strong inhibition of all the lipid kinase activities. By using polyclonal antibodies against PtdIns 4-kinase and PtdInsP 5-kinase, we were able to identify at least two putative isoforms for the PtdIns 4-kinase and one for the PtdInsP 5-kinase. In both cases, the correlation of the amount of these proteins with their respective kinase activities depended on the culture cycle. The present work describes for the first time the characterization of the lipid kinases of C. arabica suspension cells, and the correlation of these activities with the culture cycle.