BIOCHEMICAL-EVIDENCE FOR A CALMODULIN-STIMULATED CALCIUM-DEPENDENT PROTEIN-KINASE IN MAIZE

We provide biochemical evidence for the presence of a Ca2+-dependent c almodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purif ied to homogeneity by means of ammonium sulphate precipitation, DEAE-S ephacel chromatography, CaM-Sepharose chromatography and gel purificat ion. The purified kinase required 5 mM Mg2+ for activity and had an op timum pH of 7.5. The kinase is a Ca2+-binding protein, as was evident by Ca-45(2+)-binding and Ca2+ mobility-gel-shift assays. 1 mu M Ca2+ s timulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (approximate to 100 nM). Addition of Ca2+ and CaM antagonists decreased the kinase activity. Under in vitr o assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylat ed at serine residues. showing that the kinase belongs to the serine/t hreonine family of protein kinases. Autophosphorylation of CCaMK occur red on threonine residue(s) and was Ca2+ dependent. Addition of exogen ous CaM had no effect on autophosphorylation. The properties of the ma ize kinase suggests that it is a CCaMK that shows dual stimulation wit h Ca2+ and CaM for substrate phosphorylation and only Ca2+ requirement for autophosphorylation. Antibodies raised against the kinase cross-r eacted with maize total proteins to give a single band of 72 kDa and p recipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation stu dies with antibodies showed that the kinase is ubiquitous.