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

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 insitu-labelling renatured proteins with [gamma-P-32]ATP; thin-layer electrophoresis/thin-layer chromatography of acid hydrolysates of the P-32-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 a, 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.