Dephosphorylation of phosphoproteins by Escherichia coli alkaline phosphatase.

A purified commercial preparation of Escherichia coli alkaline phosphatase (EC 3.1.3.1) has been shown to dephosphorylate several phosphoproteins including bovine heart glycogen synthase D, mixed phosphohistones, and rabbit skeletal muscle phosphorylase kinase but not rabbit skeletal muscle glycogen phosphorylase. Alkaline phosphatase completely removed phosphate groups previously added during the preparation of glycogen synthase D, and completely converted the enzyme into the I form. The dephosphorylation reaction was reversed by a reaction catalyzed by cyclic AMP-dependent protein kinase. The activity of alkaline phosphatase on glycogen synthase 1) and on p-nitrophenyl phosphate was apparently due to the same enzyme since both activities showed identical sensitivity to heat and dithiothreitol, both activities co-chromatographed on DEAE-cellulose and on Sephadex G-200 columns, and both activities co-electrophoresed on polyacrylamide gels. A second alkaline phosphatase preparation isolated from E. coli K12 also had activity on glycogen synthase and on phosphohistone as well as p-nitrophenyl phosphate. Commercial E. coli alkaline phosphatase selectively dephosphorylated a mixture of phosphohistones, leaving the phosphorylated sites on the H2B-H3 fraction unhydrolyzed. The resistant sites in the phosphohistone mixture were dephosphorylated in the presence of urea. Phosphorylated phosphorylase kinase was actively dephosphorylated by alkaline phosphatase and the rate and extent of hydrolysis of protein phosphate bonds was related to the extent of phosphorylation of the substrate used. All of the phosphorylase kinase-bound phosphate could be hydrolyzed from aged enzyme. The rate of alkaline phosphatase action on heart glycogen synthase D was modulated by several substances. Activity on glycogen synthase D was specifically activated by divalent cations, especially Mn*+, and by sulfate. At the same time, activity on p-nitrophenyl phosphate was related only to changes in the ionic strength of the assay medium by various additions. Glycogen was a strong inhibitor of alkaline phosphatase-catalyzed glycogen synthase D dephosphorylation while having no effect on the dephosphorylation of p-nitrophenyl phosphate. Thus, chemicals known to regu-