Biochemical evidence for a 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 microM Ca2+ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (approximately 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.
Pandey
S
S
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Sopory
S K
SK
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008274:Magnesium