Regulation of protein phosphatase-1G from rabbit skeletal muscle. 2. Catalytic subunit translocation is a mechanism for reversible inhibition of activity toward glycogen-bound substrates

The glycogen-associated form of protein phosphatase-1 (PP-1G) comprises a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit. In the preceding paper in this issue of the journal we showed that the C subunit is released from PP-1G in response to phosphorylation of the G subunit by cAMP-dependent protein kinase. We now show that at 0.15-0.2 M KCl the phosphorylase phosphatase activity of glycogen-bound PP-1G is 5-8 times higher than that of released C subunit or unbound PP-1G, which are strongly inhibited at these ionic strengths. The activity of glycogen-bound PP-1G towards glycogen synthase was about 5-fold higher than that of released C subunit at 0.15M KCl. Studies with glycogen-bound substrates and myosin P-light chain (which does not interact with glycogen) indicated that PP-1G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP-1G and substrate are glycogen-associated. The inhibition by increasing ionic strength and enhanced activity upon binding to glycogen reflected changes in K'm, but not Vmax. From the determined specificity constant, k'cat/K'm approximately 4 x 10(6) s-1 M-1, it was calculated that at physiological levels of glycogen-bound PP-1G (200 nM) and phosphorylase (70 microM), dephosphorylation of the latter could occur with a half time of 15 s, sufficient to account for inactivation rates in vivo. The much higher catalytic efficiency of glycogen-bound PP-1G toward the glycogen-metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP-1G in the regulation of these substrates, and establishes a novel mechanism for selectively regulating their phosphorylation states in response to adrenalin and other factors affecting phosphorylation of the G subunit.     

Regulation of protein phosphatase-1G 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-1G) 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: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 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-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 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 t0.5 values less than those toward the alpha 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.     

Identification of a third form of protein phosphatase 1 in rabbit skeletal muscle that is associated with myosin

A third form of protein phosphatase 1 has been identified in skeletal muscle which is distinct from the species composed of the catalytic subunit complexed to the glycogen-binding subunit (protein phosphatase 1G) or inhibitor-2 (protein phosphatase 1I). The third form has an apparent molecular mass of 110 kDa, is not immunoprecipitated by antibody prepared against the glycogen-binding subunit, does not interact with glycogen and is devoid of inhibitor-2. It is tightly bound to myosin and is therefore termed protein phosphatase 1M.     

A myofibrillar protein phosphatase from rabbit skeletal muscle contains the beta isoform of protein phosphatase-1 complexed to a regulatory subunit which greatly enhances the dephosphorylation of myosin.

A form of protein phosphatase-1 (PP1M), which possesses 25-fold higher activity towards the P light chain of myosin (in heavy meromyosin) than other forms of protein phosphatase-1, was purified over 200,000-fold from the myofibrillar fraction of rabbit skeletal muscle. PP1M, which eluted from Superose 12 with an apparent molecular mass of 60 kDa, was dissociated by LiBr into two subunits. One of these displayed enzymic properties identical to those of the catalytic subunit of protein phosphatase-1 (PP1C) and was identified as the beta isoform of PP1C by amino acid sequencing. The second subunit had no intrinsic protein phosphatase activity, but greatly increased the rate at which PP1C dephosphorylated skeletal-muscle heavy meromyosin and decreased the rate at which it dephosphorylated glycogen phosphorylase. The properties of PP1M, together with those of smooth muscle PP1M [Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035] and the previously characterised glycogen-associated form of protein phosphatase-1 (PP1G), indicate that the subcellular localisation and substrate specificity of PP1 is determined by its interaction with specific targetting subunits.     

Latent forms of type-1 protein phosphatase in rabbit skeletal muscle

We have examined the characteristics of partially purified forms of rabbit skeletal muscle type-1 protein phosphatase (PP-1). Over 90% of PP-1 in unfractionated extracts and in glycogen particles was inactive, but could be activated by the divalent cations, Mn2+ or Co2+ (Me2+) plus trypsin. Gel filtration of muscle extracts revealed two inactive forms of PP-1; one activated by Me2+ alone or Me2+ plus trypsin, and a second containing inhibitor-2 and activated only by Me2+ plus trypsin. The kinetics of Me2+ plus trypsin activation revealed that after DEAE-chromatography, PP-1 was altered from the forms in crude extracts, gel filtered extracts or glycogen particles. The results indicate that the purified form of PP-1 catalytic subunit has properties which distinguish it from the forms of the enzyme in muscle extracts.     

Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban.

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.     

The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Separation and identification of type 1 and type 2 protein phosphatases from rabbit reticulocyte lysates

Protein phosphatase type 1 and type 2 activities (designated PP-1 and PP-2, respectively) from rabbit reticulocyte lysates have been identified and characterized based on criteria previously established for similar activities in rabbit skeletal muscle and rabbit liver. These include (a) chromatographic separation on DEAE-cellulose, (b) substrate specificity toward glycogen phosphorylase a and the alpha- and beta-subunits of phosphorylase kinase, (c) differential sensitivity to the heat-stable protein phosphatase inhibitors-1 and -2, and (d) sensitivity to MgATP. When total lysate phosphatases are assayed in the presence of 1 mM MnCl2, protein phosphatase type 2 represents 84% of lysate phosphorylase phosphatase activity. However, when phosphatase assays are carried out with MgATP concentrations similar to those in the lysate, type 2 activity is diminished, and the levels of type 1 (41%) and type 2 (59%) phosphatase activities are comparable. A small proportion (6%) of total lysate phosphatase is tightly bound to the ribosomes, where type 1 phosphatase predominates. At least five species of protein phosphatases can be identified in lysates. These constitute two forms of protein phosphatase type 1, one of which (designated FC) is dependent on MgATP and a lysate activator protein FA; both FC and FA have been identified previously in skeletal muscle. Three species of protein phosphatase type 2 have been identified and designated PP-2B, PP-2A1, and PP-2A2 based on criteria recently established for rabbit skeletal muscle and rabbit liver phosphatases, which display similar phosphatase profiles. Lysate protein phosphatases types 1, FC, 2A1, and 2A2 can all act on phosphorylase a and the alpha- (type 2) or beta-(type 1) subunit of phosphorylase kinase. PP-2B, a Ca2+/calmodulin-dependent phosphatase, specifically dephosphorylates the alpha-subunit of phosphorylase kinase, but does not act on phosphorylase alpha. The heat-stable protein phosphatase inhibitor-2 from skeletal muscle completely blocks the activity of the two type 1 phosphatases (PP-1, FC), but has no effect on the three species of type 2 protein phosphatase. A preliminary assay of the two heat-stable phosphatase inhibitors in lysates indicates significant levels of inhibitor-2, but little or no detectable inhibitor-1.     

Protein Phosphatases 1 and 2A Dephosphorylate B-50 in Presynaptic Plasma Membranes from Rat Brain

The protein B-50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phosphatase type 1 and 2A (PP-1 and PP-2A)-like activities. The present studies further demonstrate that B-50 is dephosphorylated not only by a spontaneously active PP-1-like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 micrograms of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor-2 and by high concentrations (microM) of okadaic acid, identifying it as a latent form of PP-1. In the presence of inhibitor-2 to block PP-1, histone H1 (16-64 micrograms/ml) and spermine (2 mM) increased B-50 dephosphorylation. This sensitivity to polycations and the reversal of their effects on B-50 dephosphorylation by 2 nM okadaic acid are indicative of PP-2A-like activity. PP-1- and PP-2A-like activities from SPM were further displayed by using exogenous phosphorylase alpha and histone H1 as substrates. Both PP-1 and PP-2A in rat SPM were immunologically identified with monospecific antibodies against the C-termini of catalytic subunits of rabbit skeletal muscle PP-1 and PP-2A. Okadaic acid-induced alteration of B-50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity-purified anti-B-50 immunoglobulin G. These results provide further evidence that SPM-bound PP-1 and PP-2A-like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B-50.     

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.     

The glycogen-binding subunit of protein phosphatase-1G from rabbit skeletal muscle. Further characterisation of its structure and glycogen-binding properties

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Str?hlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.     

The structure, role, and regulation of type 1 protein phosphatases.

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.     

Effect of an Asp905Tyr mutation of the glycogen-associated regulatory subunit of protein phosphatase-1 on the regulation of glycogen synthesis by insulin and cyclic adenosine 3',5'-monophosphate agonists.

The glycogen-associated regulatory subunit of protein phosphatase-1 (PP-1G) plays a major role in insulin-stimulated glycogen synthesis and thus the regulation of nonoxidative glucose disposal in skeletal muscle. In a general population of Caucasians a polymorphism at codon 905 of PP-1G from an aspartate to tyrosine has been reported to be associated with insulin resistance and hypersecretion. In this report functional studies were performed on rat skeletal muscle L6 cells stably transfected with an Asp905Tyr mutant PP-1G to evaluate the impact of this mutation on cellular responsiveness to insulin and cAMP. Although transfection resulted in a 3-fold increase in mutant PP-1G subunit expression, basal and insulin-stimulated PP-1 catalytic activities were decreased when compared with L6 cells transfected with wild-type PP-1G. The Asp905Tyr mutation resulted in an increase in cellular sensitivity to cAMP agonist, resulting in an inhibition of insulin's stimulatory effect on glycogen synthesis. More importantly, low concentrations of (Bu)2cAMP completely reversed insulin's stimulatory effects on glycogen synthesis when added to insulin-treated cells expressing mutant PP-1G. This was due to a rapid activation of glycogen phosphorylase a and a simultaneous inactivation of glycogen synthase via cAMP-mediated reductions in insulin-stimulated PP-1 catalytic activities. We conclude that an Asp905Tyr mutation of PP-1G is accompanied by a relative increase in sensitivity to cAMP agonists as well as a diminished capacity of the mutant PP-1G to effectively mediate the inhibitory effects of insulin on glycogen breakdown via PP-1 activation.     

Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. Free catalytic subunit appears to mediate s6 dephosphorylation

The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Olivier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide containing the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatography of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phosphatase in peak A (Mr 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of Mr 26,000 and 48,000 that co-immunoprecipitate with PP-1C, while peak C has a single additional protein of Mr 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this increase is associated with changes in the phosphorylation state of the Mr 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mechanisms which are thought to modulate the activity of type 1 phosphatase.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits.

The alpha form of the A subunit of human protein phosphatase 2A was expressed in insect cells following infection with a recombinant baculovirus. A alpha was expressed as a soluble protein that comprised approximately 10% of total cellular protein. The expressed A alpha subunit was purified by chromatography on amino-hexyl-Sepharose and Mono Q with a yield of 2 mg/500-ml culture. The recombinant protein had the same apparent molecular mass as the bovine cardiac protein and was devoid of myosin light chain phosphatase activity. Biological activity of expressed A was assessed by assays of complex formation with the catalytic (C) and B subunits, purified from bovine cardiac tissue, and by inhibition of phosphatase activity. Purified A alpha had a high apparent affinity for C (IC50 = 0.10 nM) and bound with a stoichiometry of 1 mol of A/mol of C. Interaction of A alpha with the catalytic subunit caused a maximal inhibition of myosin light chain and phosphorylase phosphatase activities of 50 and 79%, respectively. The AC complex prepared by reconstitution of recombinant A alpha with C had the same electrophoretic mobility in nondenaturing polyacrylamide gels and the same elution volume when chromatographed on a size exclusion column as the native AC complex purified from cardiac muscle. Similar chromatographic profiles were also observed for the heterotrimer reconstituted from recombinant A alpha, purified B and C, and the native bovine cardiac heterotrimeric holoenzyme. Cross-linking of the native enzyme and the reconstituted heterotrimer generated the same pattern of high molecular weight species. Immunological analyses of these complexes demonstrated that distinct cross-linked forms composed of ABC, AC, AB, and BC were obtained. These results suggest that each of the three subunits of protein phosphatase 2A forms direct contacts with both of the others.     

The protein phosphatases involved in cellular regulation. Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle

A type-1 protein phosphatase (protein phosphatase-1G) was purified to homogeneity from the glycogen-protein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1C) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels. Protein phosphatase-1G was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-1C, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-1G was inactivated very rapidly by inhibitor-1. Protein phosphatase-1G interacted with inhibitor-2 (I-2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3. The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-1G by inhibitor-1. The relationship of the G subunit to the 'deinhibitor protein' is discussed.     

Inhibition of myogenesis by depletion of the glycogen-associated regulatory subunit of protein phosphatase-1 in rat skeletal muscle cells

In this study, we examined the role of the glycogen-associated regulatory subunit of protein phosphatase-1 (PP-1(G)) in L6 rat skeletal muscle cell myogenesis. The level of PP-1(G) was depleted by transfection with an inducible antisense-oriented PP-1(G) gene. Western blot analysis of the PP-1(G)-depleted cell line revealed a >90% depletion of PP-1(G) protein and a 45% reduction in cellular PP-1 activity and abolished the ability of L6 myoblasts to differentiate into multinucleated myotubes. PP-1(G)-depleted cells also exhibited a marked reduction in the expression of the differentiation marker myogenin as well as creatine kinase. After 7 days in culture, PP-1(G)-depleted cells sustained myoblast levels of inhibitor of differentiation-2, whereas control L6 cells had a severely lower inhibitor of differentiation-2 level and progressed into myotubes. Myoblasts were unable to exit the cell cycle, as measured by the impaired induction of p27 cyclin-dependent kinase inhibitor, a >2-fold increase in DNA synthesis, and elevated levels of phosphorylated retinoblastoma protein (pRb). Replacement of the PP-1(G) gene restored PP-1(G) protein expression, PP-1 enzymatic activity, and the ability to differentiate into myotubes. We conclude that PP-1(G) plays a definite role in L6 myogenesis via its regulation of PP-1 catalytic activity.