Phospholipid-stimulated protein kinase in plants

In membrane fractions from zucchini (Cucurbita pepo L.) hypocotyls, catalytic properties of a platelet-activating factor (PAF)-activated protein kinase were investigated. In the presence of ethylenebis(oxyethylenenitrilo)]tetraacetic acid, phosphorylation of a 55-kDa membrane polypeptide and, to a lesser extent, several others, including a 120-kDa polypeptide, was stimulated by PAF. The phosphorylation of the 55-kDa polypeptide was used for quantification of the PAF-stimulated protein kinase. Stimulation of protein phosphorylation by PAF increased in a concentration range from 10-200 micrograms/ml (= 19-380 microM) PAF up to 10-fold above the control. Addition of Ca2+ ions in the micromolar range in the presence and in the absence of PAF increased the phosphorylation of the 55- and the 120-kDa polypeptide. Other phospholipids and lipids tested including phorbol ester, diglyceride, mono- and triglyceride, and oleic acid were ineffective. The same lipid specificity was previously observed for the activation of ATP-dependent H+ transport in microsomes (Scherer, G.F.E., Martiny-Baron, G., and Stoffel, B. (1988) Planta 175, 241-253). Lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were able to stimulate the phosphorylation of the same polypeptides as PAF and H+ transport but both to a lesser extent (PAF greater than LPC greater than LPE). In the presence of EGTA, PAF-stimulated phosphorylation of a 55- and a 57-kDa polypeptide was predominantly associated with vacuolar membranes and those of 42, 61, 63, and 120 kDa were predominantly associated with plasma membranes. Stimulation of ATP-dependent H+ transport by PAF was found in tonoplast vesicles whereas plasma membrane vesicles had only little transport activity and, therefore, an effect of PAF on plasma membrane H+ transport could not be measured. Stimulation of ATP hydrolysis by PAF was observed both in tonoplast- and plasma membrane-containing fractions.