Biochemical evidence fora calmodulin-stimulated calcium-dependent protein kinase in maize

We provide biochemical evidence for the presence of a Ca²⁺-dependent calmodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE-Sephacel chromatography , CaM-Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg²⁺ for activity and had an optimum pH of 7.5. The kinase is a Ca²⁺-binding protein, as was evident by ⁴⁵Ca²⁺-binding and Ca²⁺ mobility-gel-shift assays. 1 μM Ca²⁺ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (~100 nM). Addition of Ca²⁺ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca²⁺ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca²⁺ and CaM for substrate phosphorylation and only Ca²⁺ requirement for autophosphorylation. Antibodies raised against the kinase cross-reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.