A 67-kDa plasma-membrane-bound Ca²⁺-stimulated protein kinase active in sink tissue of higher plants

Abstract.

A novel 67-kDa protein kinase (p67^cdpk) was identified in the microsomal membrane fraction of apple (Malus domestica Borkh. cv. Braeburn) suspension cultures and subsequently found to be active in sink tissues. Microsomal proteins were blotted onto Nylon or polyvinylidenedifluoride membranes, and p67^cdpk assayed by in situ-labelling renatured proteins with [γ-³²P]ATP; thin-layer electrophoresis/thin-layer chromatography of acid hydrolysates of the ³²P-labelled protein band indicated that serine and threonine, but not tyrosine residues were phosphorylated. A detailed analysis of the ion-dependency of p67^cdpk revealed that it was a Ca²⁺-stimulated, Mg²⁺-dependent protein kinase. However, p67^cdpk was ten times more active in the presence of 10 mM Mn²⁺, and these assay conditions were used routinely to increase the sensitivity of the assay. Activity of p67^cdpk was found at high levels in the plasma membrane, and solubilisation experiments with a number of detergents suggested that p67^cdpk is an integral membrane protein. A homologous protein kinase with similar biochemical properties was also present in cell-suspension cultures of pear and maize. In maize (Zea mays L.) plants, sink tissues, such as young expanding leaves of both light-grown and etiolated plants, mature etiolated tissue and roots all had high levels of p67^cdpk activity. However, mature light-grown (source) tissues had barely detectable levels. In etiolated maize leaves and coleoptiles the kinase activity was highest in expanding tissue and decreased as the cells expanded. When etiolated maize plants were exposed to light, the activity of p67^cdpk was reduced to background levels after 8 h. Although p67^cdpk has biochemical properties similar to those of other plant calcium-dependent protein kinases, this is the first identification of a membrane-bound calcium-dependent protein kinase which is specifically active in sink tissues.