CHANGES IN CALCIUM-DEPENDENT PROTEIN-KINASE ACTIVITY DURING IN-VITRO TUBERIZATION IN POTATO

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogen eity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 mu M) and phosphor ylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 mu M). The kinase was inh ibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-am inohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibiti on = 0.25 mM). Polyclonal antibodies directed against the regulatory r egion of the soybean CDPK recognized a 53-kD polypeptide. In an autoph osphorylation assay, this same band was strongly labeled with [gamma-P -32]ATP in the presence of Ca2+. CDPK activity was high in nontuberize d plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had c ompleted formation. In the early stages of tuberization, Ca2+-dependen t phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberi zing plants or in completely formed tubers, indicating that this phosp horylation is a stage-specific event. In addition, dephosphorylation o f specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.