Purification and molecular cloning of protein phosphatases of bovine adrenal medulla: An assessment of their physiological role in PC12 cells

When fractionated using an HPLC ion exchange column, three distinct peaks (peak I, II, and III) of phosphatase activity were observed in the supernatant of homogenized bovine adrenal medulla cells, suggesting the presence of at least three different phosphatases. These phosphatases showed different activities toward phosphocasein in the presence of Mg$\sp{2+}$ and Mn$\sp{2+}$. Peak III, which represents about 50% of the active enzyme activity when phosphocasein is used as substrate, showed a molecular weight of 140 KDa as determined by HPLC gel filtration and has been identified as a type 2A protein phosphatase. Okadaic acid, a phosphatase inhibitor (specific for type 2A and type 1) and tumor promoter, was employed to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. After 3 days cultured in the presence of 50 ng/ml NGF, 20-25% of the PC12 cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC$\sb{50}$ of about 7 nM. This inhibition was rapidly reversed after wash-out of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells indicating that continual phosphatase activity is required to maintain neurites. A 27-mer oligonucleotide was synthesized as a hybridization probe to isolate clones encoding the sequence of protein phosphatases from a bovine adrenal medulla cDNA library. A cDNA clone encoding the full length of the catalytic subunit of protein phosphatase type 2A has been isolated. The deduced protein sequence (309 residues, 35.63 KDa) is 99.7% identical to that of phosphatase 2A$\alpha$ form from rabbit skeletal muscle, human liver and porcine kidney and differs by only one amino acid (Arg-55 vs. Cys-55). At the nucleotide level, the clone showed 97% identity with that of the catalytic subunit of protein phosphatase type 2A$\alpha$ from human liver. Sequence comparison of bovine adrenal medulla clone with phosphatase type 1 from rabbit skeletal muscle and type 2B from mouse brain identifies six highly conserved domains in the three enzymes that are expected to be crucial for the catalytic activity of protein phosphatase