Phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic-AMP-dependent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle

The glycogen-bound form of protein phosphatase-1 (termed protein phosphatase-1G) is composed of the catalytic (C) subunit complexed to a glycogen-binding (G) subunit that anchors the enzyme to glycogen [Str?lfors et al. (1985) Eur. J. Biochem. 149, 295-303]. Incubation of purified protein phosphatase-1G with cyclic-AMP-dependent protein kinase and MgATP, which leads to stoichiometric phosphorylation of the G-subunit [Caudwell et al. (1986) FEBS Lett. 194, 85-90], was found to promote the release of the phosphatase from glycogen; similar observations were made using glycogen-protein particle preparations. An intravenous injection of adrenaline decreased protein phosphatase-1 activity associated with the glycogen-protein particles by 50% with a corresponding increase in the amount present in the cytosol. By contrast, adrenaline did not affect the distribution of glycogen synthase or glycogen phosphorylase which remained entirely bound to glycogen in these experiments. The specific release of protein phosphatase-1 from glycogen may facilitate its inactivation by inhibitor-1 in the cytosol, thereby preventing dephosphorylation of the glycogen metabolising enzymes. Translocation of protein phosphatase-1 may represent a novel mechanism for the activation of glycogenolysis and inhibition of glycogen synthesis by adrenaline.     

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.     

Phosphorylation of the glycogen-binding subunit of protein phosphatase-1G in response to adrenalin

The glycogen-binding (G) subunit of protein phosphatase-1 is phosphorylated in vivo. In rabbits injected with propranolol the serine residue termed site-1 was phosphorylated in 56% of the molecules isolated, and phosphorylation increased to 82% after administration of adrenalin. It is concluded that the G-subunit is a physiological substrate for cyclic AMP-dependent protein kinase. The G-subunit remained largely bound to glycogen even after injection of adrenalin, whereas half of the protein phosphatase-1 activity associated with glycogen was released into the cytosol. The results indicate that adrenalin induces dissociation of the catalytic subunit from the G-subunit in vivo.     

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.     

Multisite phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic AMP-dependent protein kinase and glycogen synthase kinase-3

The glycogen-binding (G) subunit of protein phosphatase-1G is phosphorylated stoichiometrically by glycogen synthase kinase-3 (GSK3), and with a greater catalytic efficiency than glycogen synthase, but only after prior phosphorylation by cyclic AMP-dependent protein kinase (A-kinase) at site 1. The residues phosphorylated are the first two serines in the sequence AIFKPGFSPQPSRRGS-, while the C-terminal serine (site 1) is one of the two residues phosphorylated by A-kinase. These findings demonstrate that (i) the G subunit undergoes multisite phosphorylation in vitro; (ii) phosphorylation by GSK3 requires the presence of a C-terminal phosphoserine residue; (iii) GSK3 can synergise with protein kinases other than casein kinase-2.     

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.     

Identification of three in vivo phosphorylation sites on the glycogen-binding subunit of protein phosphatase 1 from rabbit skeletal muscle, and their response to adrenaline

The in vivo phosphorylation stoichiometries of 4 serines on the glycogen-binding (G)-subunit of protein phosphatase 1 (PP1) have been determined. In fed rabbits injected with propranolol stoichiometries (mol/mol) were: site 1 (0.67 +/- 0.09), site 2 (0.20 +/- 0.07), site 3a (0.23 +/- 0.01) and site 3b (0). After injection with adrenalin they became: site 1 (0.90 +/- 0.02), site 2 (0.72 +/- 0.01), site 3a (0.23 +/- 0.02) and site 3b (0). These results, together with other data, establish that site 2 phosphorylation by cyclic AMP-dependent protein kinase triggers dissociation of PP1 from the G-subunit in vivo. They also demonstrate that a residue phosphorylated in vitro by glycogen synthase kinase 3 (site 3a) is phosphorylated in vivo.     

Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The gamma subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (gamma) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.     

Glycogen synthase association with the striated muscle glycogen-targeting subunit of protein phosphatase-1. Synthase activation involves scaffolding regulated by beta-adrenergic signaling

Glycogen-binding subunits for protein phosphatase-1 (PP1) target the PP1 catalytic subunit (PP1C) to glycogen particles, where the enzymes glycogen synthase and glycogen phosphorylase are concentrated. Here we identify sites within the striated muscle glycogen-binding subunit (G(M)) that mediate direct binding to glycogen synthase. Both PP1C and glycogen synthase were coimmunoprecipitated with a full-length FLAG-tagged G(M) transiently expressed in COS7 cells or C2C12 myotubes. Deletion and mutational analysis of a glutathione S-transferase (GST) fusion of the N-terminal domain of G(M) (residues 1-240) identified two putative sites for binding to glycogen synthase, one of which is the WXNXGXNYX(I/L) motif that is conserved among the family of PP1 glycogen-binding subunits. Either deletion of this motif or Ala substitution of Asn-228 in this motif disrupted the binding of glycogen synthase. Expression of full-length FLAG-G(M) in cells increased the activity of endogenous glycogen synthase, but protein disabled in either PP1 binding or glycogen synthase binding did not produce synthase activation. The results show that efficient activation of glycogen synthase requires a scaffold function of G(M) that involves simultaneous binding of both PP1C and glycogen synthase. Isoproterenol and forskolin treatment of cells decreased glycogen synthase binding to FLAG-G(M), thereby limiting synthase activation by PP1. This response was insensitive to inhibition by H-89, therefore probably not involving cAMP-dependent protein kinase, but did require inclusion of microcystin-LR during cell lysis, implying that phosphorylation was modulating binding of glycogen synthase. Phosphorylation control of binding to a scaffold site on the G(M) subunit of PP1 offers a new mechanism for regulation of muscle glycogen synthase in response to beta-adrenergic signals.     

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.     

Insulin-stimulated phosphorylation of the protein phosphatase-1 striated muscle glycogen-targeting subunit and activation of glycogen synthase

Protein phosphatase-1 (PP-1) in heart and skeletal muscle binds to a glycogen-targeting subunit (G(M)) in the sarcoplasmic reticulum. Phosphorylation of G(M) has been postulated to govern activity of PP1 in response to adrenaline and insulin. In this study, we used biochemical assays and G(M) expression in living cells to examine the effects of insulin on the phosphorylation of G(M), and the binding of PP-1 to G(M). We also assayed glycogen synthase activation in cells expressing wild type G(M) and G(M) mutated at the phosphorylation sites. In biochemical assays kinase(s) prepared from insulin-stimulated Chinese hamster ovary (CHO-IR) cells and C2C12 myotubes phosphorylated a glutathione S-transferase (GST) fusion protein, GST-G(M)(1-240), at both site 1 (Ser(48)) and site 2 (Ser(67)). Phosphorylation of both sites was dependent on activation of the mitogen-activated protein kinase pathway, involving in particular ribosomal protein S6 kinase. Full-length G(M) was expressed in CHO-IR cells and metabolic (32)P labeling at sites 1 and 2 was increased by insulin treatment. The G(M) expressed in CHO-IR cells or in C2C12 myotubes co-immunoprecipitated endogenous PP-1, and association was transiently lost following treatment of the cells with insulin. In contrast PP-1 binding to G(M)(S67T), a version of G(M) not phosphorylated at site 2, was unaffected by insulin treatment. Expression of G(M) increased basal activity of endogenous glycogen synthase in CHO-IR cells. Insulin stimulated glycogen synthase activity the same extent in cells expressing wild type G(M) or G(M) mutated to eliminate phosphorylation site 1 and/or site 2. Phosphorylation of G(M) is stimulated by insulin, but this phosphorylation is not involved in insulin control of glycogen metabolism. We speculate that other functions of G(M) at the sarcoplasmic reticulum membrane might be affected by insulin.     

Processivity and subcellular localization of glycogen synthase depend on a non-catalytic high affinity glycogen-binding site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.     

Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1(143-217), were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1(191-210) in the far-Western assay. NIPP-1(191-210) competed for PP-1 inhibition by full-length NIPP-1(1-351), inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1(143-217)-Sepharose but not from full-length NIPP-1(1-351)-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.     

Amino acid sequence of a region on the glycogen-binding subunit of protein phosphatase-1 phosphorylated by cyclic AMP-dependent protein kinase

The amino acid sequence of a region on the glycogen-binding (G)-subunit of protein phosphatase-1G that is phosphorylated by cyclic AMP-dependent protein kinase has been determined. The sequence is: (formula see text) This finding will facilitate studies of the effects of hormones on the phosphorylation state of the G-subunit in vivo.     

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).     

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Protein phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1_G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1_G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1_G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF- (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1_G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1_G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1_G subunit site-specific mutations will help clarify the exact role and regulation of PP-1_G site-specific phosphorylations on PP-1 catalytic function.