The protein phosphatases involved in cellular regulation

Abstract

The phosphorylase phosphatases in rat and rabbit liver cytosol that are markedly stimulated by histone H1, protamine and polylysine were identified as protein phosphatases-2A_o, 2A₁ and 2A₂ by anion-exchange chromatography, gel-filtration and immunotitration experiments. Histone H1 and protamine also stimulated the dephosphorylation of phosphorylase kinase, glycogen synthase, fructose-1, 6-bisphosphatase, pyruvate kinase, acetyl-CoA carboxylase and phenylalanine hydroxylase by phosphatases-2A₁ and 2A₂, and with several of these substrates activation was even more striking (20–100-fold) than that observed with phosphorylase (∼ 5-fold). Activation by basic polypeptides did not involve dissociation of these phosphatases to the free catalytic subunit. The dephosphorylation of phosphorylase by protein phosphatase-1 was suppressed by basic polypeptides, protamine and polylysine being the most potent inhibitors. However, the dephosphorylation of glycogen synthase, pyruvate kinase and acetyl-CoA carboxylase were markedly stimulated by histone H1 and protamine (2–13-fold). Consequently, with the appropriate substrates, protein phosphatase-1 can also be regarded as a basic-polypeptide-activated protein phosphatase. Heparin stimulated (1.5–2-fold) the dephosphorylation of phosphorylase by phosphatases-2A_o and 2A₁, provided that Mn²⁺ was present, but phosphatase-2A₂ and the free catalytic subunit of phosphatase-2A were unaffected. Heparin, in conjunction with Mn²⁺, also stimulated (1.5-fold) the dephosphorylation of glycogen synthase (labelled in sites 3abc), phosphorylase kinase and phenylalanine hydroxylase by phosphatase-2A₁, but not by phosphatase-2A₂. By contrast, the dephosphorylation of phosphorylase and phosphorylase kinase by protein phosphatase-1 was inhibited by heparin. However, dephosphorylation of glycogen synthase and pyruvate kinase by phosphatase-1 was stimulated by this mucopolysaccharide. The studies demonstrate that basic proteins can be used to distinguish protein phosphatase-1 from protein phosphatase-2A, but only if phosphorylase is employed as substrate. Optimal differentiation of the two phosphatases is observed at 30 μ/ml protamine or at heparin concentrations < 150 μM.