The polycation-stimulated protein phosphatases: regulation and specificity

Abstract

Four classes of protein phosphatases are presumed to play an important role in dephosphorylating the major proteins involved in the control of general metabolism. Based on the enzyme-directed regulation of activity they have been classified as ATP,Mg-dependent-, polycation-stimulated-, Mg 2+-dependent protein phosphatases and calcineurin. We have recently purified from rabbit skeletal muscle four distinct PCS protein phosphatases, classified according to the apparent molecular weight of the native enzymes in gel filtration at an early stage of the purification as: PCS H (390 kDa), PCS M (250 kDa) and PCS L (200 kDa) phosphatases. The PCS H phosphatase could be resolved into a 3(65 : 55 35 kDa)-subunit PCS H1 phosphatase and a 2(65 : 35 kDa)-subunit PCS H2 enzyme probably derived from the PCS H1 phosphatase, both characterized as specific deinhibitor phosphatases. PCS M phosphatase, a 3(72 : 65 35 kDa)-subunit enzyme, shows a high degree of stimulation with a low concentration optimum of polycations and is sensitive to a Ca 2+-dependent protease, which brings about a five- to ten-fold increase in inhibitor-1 phosphatase activity. PCS L phosphatase is characterized by a 2(65 : 35 kDa)-subunit structure, a low intrinsic deinhibitor phosphatase activity and a low degree of stimulation of phosphorylase phosphatase activity requiring high concentrations of polycations. At low concentrations of polycations the stimulation of phosphorylase phosphatase activity of the PCS enzymes is enzyme-directed, since it occurs at concentrations far below the substrate concentration. The degree of stimulation is also typical for each type of enzyme (PCS M > PCS H1 > PCS H2 > PCS L > PCS C) and dependent on the polycation used; at the optimum concentration the most effective polycations (polylysine, protamine, histone H1) stimulate the phosphorylase phosphatase activity to about the same extent. Polycation concentrations above the optimum are less effective on phosphorylase phosphatase activity and can even become inhibitory to the basal activity. Whether this effect is enzyme- or substrate-directed (or both) is not known. The stimulation by polycations could be completely lost following preincubation of the PCS phosphatase with polycations. This deactivation is time-, temperature- and concentration-dependent. However the polycations did not affect the basal phosphorylase phosphatase activity. In addition to phosphorylase a and inhibitor-1, casein, myosin light chains and phosphorylase b kinase (α-subunit) are choice substrates for these enzymes. Protamine, histone H1 and polylysine also stimulate the dephosphorylation of phosphocasein, phosphorylated myosin light chains and the α-subunit of phosphorylase kinase by the PCS phosphatases. The polycation stimulation of inhibitor-1 dephosphorylation was always less prominent (up to two-fold) and absent with the deinhibitor or synthetic peptides as substrates. Paranitrophenylphosphate phosphatase activity of the PCS phosphatases is extremely low and cannot be stimulated by polycations. Unlike the catalytic subunit of the ATP,Mg-dependent protein phosphatase, the PCS phosphatases exhibit a remarkable preference for the phosphothreonyl- over the phosphoseryl peptides. The catalytic subunits isolated from the three basic types of PCS phosphatases are apparently identical. The migration of the catalytic subunits obtained from the different types of PCS phosphatases in sucrose density gradient centrifugation was identical (37–41 kDa). In contrast with the holoenzymes they are less sensitive to polycation stimulation, showing an identical concentration optimum and the stimulation by metal ions is lost.