Ca2+ is known to undergo transient alterations of intracellular concentration in nerve and other cells in response to diverse stimuli and to exert many of its actions by binding to a protein termed calmodulin (CaM). Calmodulin alters the activity of many enzymes including several CaM-dependent protein kinases and a CaM-dependent protein phosphatase termed calcineurin (CN). Our laboratory has recently prepared in rabbits polyclonal antibodies to native bovine brain CN and its constituent subunits A and B. Antibodies to bovine brain CN B have been shown to cross-react with a 16,000 Mr protomer in extracts of PC-12 rat pheochromocytoma cells. Further, we have purified an apparently unique, narrow substrate specificity CaM-dependent myosin light chain kinase (MLCK) from bovine brain. We propose to characterize our antibody population in detail and to use these antibodies to effect a rapid purification of calcineurin by immune affinity chromatography and to compare the kinetic, physical and regulatory properties of this phosphatase with that generated by chromatography on immobilized CaM. The antibodies will be used to effect a selective removal of CN from brain and PC-12 cell extracts and to identify endogenous substrates by altered Ca2+-dependent dephosphorylation. Endogenous substrates of calcineurin will also be sought by identifying thiophosphorylated proteins from brain extract which bind to immobilized native calcineurin. The narrow substrate specificity CaM-dependent MLCK from bovine brain will be purified to homogeneity and characterized. The MLCK will be compared to that isolated from highly purified bovine cerebral cortex microvessels. The bovine brain MLCK will be tested for ability to phosphorylate the light chains of native bovine brain myosin and for the consequences of their phosphorylation with respect to actin-stimulated myosin ATPase activity. The calcineurin analog from PC-12 cells and a second CaM-dependent phosphatase form observed by our laboratory in this cell line will be characterized physically, chemically, and immunologically. The effect of Nerve Growth Factor on the immunocytochemical localization of PC-12 calcineurin will be determined. Finally, the effect of fusion of liposome-encapsulated inhibitory anti-CN antibodies with PC-12 cells on protein phosphorylation and expression of its differentiated functions will be sought.
