Plant Molecular Biology 12: 453-461, 1989 © 1989 Kluwer Academic Publishers. Printed in Belgium 453 The phosphorylation site of Ca 2 +-dependent protein kinase from alfalfa Zoltan Olah, Laszlo Bogre, Csaba Lehel, Anna Farago ~, Janos Seprodi ~ and Denes Dudits* Institute of Genetics, BiologicalResearch Center, Hungarian Academy of Sciences, P.O. Box 521, 6 7 01-Szeged, Hungary (*authorfor correspondence) 11 st Institute of Biochemistry, Semmelweis University, Medical School, P.O. Box 260, 1444-Budapest 8, Hungary Received 21 June 1988; accepted in revised form 19 January 1989 Key words: alfalfa, Ca 2 + -dependent protein kinase (CDPK), oligopeptide substrate, peptide competition, protein kinase C, substrate specificity Abstract A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H 1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA, AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca2+-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can. be used as a tool to study substrates of plant kinases in crude cell extracts. Introduction In higher plants, as in animals [16], Ca 2+ may serve as an intracellular signal transducer for external stimuli, while protein kinases with dif- ferent Ca 2 + sensitivity are considered as possible effector enzymes [37]. Initially, Ca2+-cal - modulin-activated protein kinases were dis- covered in plants [15,35,36,42]. Later, Ca 2+- -activated protein kinases were detected in the plant cytosol; these kinases need acidic phospho- lipids for activity [10,29,32,43]. Recently a new type of protein kinases has been purified from soybean and from alfalfa using bovine histone H 1 as a substrate. These Ca2+-dependent protein kinases (CDPK) share common characteristics [3,14]. The bovine histone H1 polypeptide contains several phosphorylation sites for different protein kinases. The use of peptide substrates offers a powerful tool to investigate specificity determi- nants of various protein kinases. Phosphorylation of the histone H2B-related peptide GKKRKRSRKA by the animal histone kinase II [38,39] and the cyclic nucleotide-dependent pro- tein kinases has been reported [39,41]. For fur- ther characterization of the substrate specificity of histone kinase II, the nonapeptide (AAASFKAKK) has been designed [40,41]. It was shown that the enzyme referred to as histone