Phospholipid-dependent Ca²⁺-activated protein kinase (C-kinase) in the pituitary: Further characterization and endogenous redistribution

Phospholipid-dependent, Ca²⁺-activated protein kinase (C-kinase) was recently shown to be expressed in rat pituitary. The enzyme is activated by Ca²⁺ and phosphatidylserine (PS). Diacylglycerol (DG), which is liberated during phosphoinositide turnover, and the potent tumor promoter 12-O-tretadecanoyl-phorbol-13-acetate (TPA) activate pituitary C-kinase in the presence of PS, even at resting levels of intracellular Ca²⁺ (10^-7 M), and increase the apparent affinity of the enzyme for Ca²⁺. While micromolar concentration of Ca²⁺ had no effect on the apparent affinity of the enzyme for PS (K_m ~ 15 μ/ml), elevation of Ca²⁺ to the millimolar range produced a sharp increase in the apparent affinity for PS (K_m ~ 5 μ/ml). Elevation of PS (up to 500 μ/ml) could not replace Ca²⁺ in supporting maximal enzyme activity even in the presence of DG. Cytosolic pituitary C-kinase (70% of total enzyme activity) is recovered in an inactive state and can be activated without further purification. The paniculate enzyme (30%) is recovered in a cofactors-insensitive form but can be activated after detergent-solubilization and anion exchange chromatography. Endogenous redistribution of soluble pituitary C-kinase to the membrane does not convert it to its proteolytic product which is insensitive to Ca²⁺, PS and DG. Pituitary C-kinase characterized here most likely plays a key role in signal transduction mechanisms involved in pituitary functions.