Role of Phosphorylase Kinase and Cyclic AMP-dependent Protein Kinase in the Regulation of Phosphorylase Phosphatase

Abstract

The enzymes of glycogen metabolism are regulated by interconversion between phosphorylated and dephosphorylated forms. In contrast with our well-defined knowledge of the phosphorylation processes of different enzymes and their role in glycogen breakdown and synthesis, the phosphatases catalysing the reverse processes are only now being understood. The regulation of dephosphorylation reactions is still unclear. Phosphorylase phosphatase converts active phosphorylase a (EC 2.4.1 . l) into inactive phosphorylase b by cleavage of the phosphate groups from the serine residues. For the control of phosphorylase phosphatase (EC 3.1.3.17) some mechanisms are possible, namely ligands that affect the substrate phosphorylase a or phosphatase; proteins that affect phosphorylase a (or phosphatase); or enzymic modification of phosphatase. It is known that different proteins can influence the phosphorylase phosphatase reaction. The activation of phosphorylase with synchronous inhibition of phosphorylase phosphatase was observed in the protein-glycogen complex isolated from rabbit skeletal muscle (Haschke et af., 1970). No clear explanation has been offered for the transient inhibition of phosphorylase phosphatase during the ‘flash activation’. Haschke et af. (1972) attributed the inhibition to a protein-protein interaction caused by an unknown protein component of the complex. This assumption was supported by frontal gel filtration of muscle extract and purified enzymes showing a strong association between phosphorylase, phosphorylase kinase and phosphatase (Gergely et al., 1974, 1975). A heat-stable protein isolated from different mammalian tissues also inhibits the phosphatase reaction (Brandt et af., 1974, 1975~). This heat-stable inhibitor protein is phosphorylated by cyclic AMP-dependent protein kinase (Cohen rt al., 1977). Toth et al. (1977) demonstrated the reversible phosphorylation and dephosphorylation of this heat-stable inhibitor protein in vivo. Huang & Glinsmann (1975, 1976a,h) demonstrated the existence of two heat-stable inhibitor proteins for phosphatase. In the present study the effect of phosphorylase kinase and cyclic AMP-dependent protein kinase on the regulation of phosphorylase phosphatase has been investigated. Rabbit skeletal-muscle phosphorylase a was prepared and assayed as previously described (Bot et al., 1977). The specific activity of phosphorylase a was 55 units/mg in the presence of 0.016~-gh1cose 1-phosphate and in the absence of AMP. Non-activated phosphorylase kinase was prepared from rabbit skeletal muscle, and its activity was measured, as described by Cohen (1973). Thiophosphate-activated phosphorylase kinase was prepared by the method of Gergely et al. (1976). Cyclic AMP-dependent protein kinase was prepared from rabbit skeletal muscle by the method of Beavo e ta / . (1974). The separation of regulatory and catalytic subunits of protein kinase was carried out on a Blue Dextran/Sepharose column (Witt & Roskoski, 1975). The isolated protein kinase holoenzyme, the separated regulatory and catalytic subunits, phosphorylase a and phosphorylase kinase were homogeneous by sodium dodecyl sulphate (0.1 %)/ polyacrylamide-gel (7.5 %) electrophoresis. The gel-electrophoresis experiments were carried out as described by Weber & Osborn (1969). Phosphorylase phosphatase was prepared by method of Brandt et a/. (197%). Inhibition of the phosphorylase phosphatase reaction by phosphorylase kinase and cyclic AMP-dependent protein kinase was assayed in the following manner. Phosphorylase a (1 .Omg/ml) was incubated in 0.04~-Tris/2m~-EDTA/O.5m~-dithiothreito~ buffer (pH7.4) with phosphorylase phosphatase in the presence of various concentrations of phosphorylase kinase or cyclic AMP-dependent protein kinase or its regulatory/ catalytic subunit. The reaction mixtures were incubated at 30T , portions (50~1) were removed at various times, and the reaction was stopped by the addition of 0.1 M-NaF/