Regulation of protein phosphatase‐1_G from rabbit/skeletal muscle: 1. Phosphorylation by cAMP‐dependent protein kinase at site 2 releases catalytic subunit from the glycogen‐bound holoenzyme

The glycogen‐associated form of protein phosphatase‐1 (PP‐1_G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is: (Formula Presented.) At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1_G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1_G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1_G. At near physiological ionic strength, PP‐1_G and glycogen concentration, site 2 was autodephosphorylated by PP‐1_G with a t_0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t_0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t_0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca²⁺/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca²⁺, and represents an example of a protein phosphatase cascade.