Phosphorylase Phosphatase Research Articles

Phosphorylase phosphatase and flash activation of skeletal muscle glycogen phosphorylase-A tribute to Edmond H. Fischer.

Glycogen is a polymerized form of glucose that serves as an energy reserve in all types of organisms. In animals glycogen synthesis and degradation, especially in liver and skeletal muscle, are regulated by hormonal and physiological signals that reciprocally control the opposing activities of glycogen synthase and glycogen phosphorylase. These enzymes are under allosteric control by binding of metabolites (e.g., ATP, AMP, G6P) and covalent control by reversible phosphorylation by kinase and phosphatase all assembled together on glycogen. More than 50 years ago Edmond Fischer and colleagues showed "flash activation" of phosphorylase in glycogen particles. This involved transient and extensive inhibition of protein phosphatase but even today the phenomenon is not understood. Phosphatase regulation is known to rely on regulatory subunits including glycogen binding subunits that serve as scaffolds, binding catalytic subunit, glycogen, and substrates. This tribute article to Edmond Fischer highlights his thoughts and ideas about the transient inhibition of phosphorylase phosphatase during flash activation of phosphorylase and speculates that phosphatase regulation in glycogen particles might involve a/b hybrids of phosphorylase.

Network pharmacology based investigation into the bioactive compounds and molecular mechanisms of Schisandrae Chinensis Fructus against drug-induced liver injury.

To investigate the against Drug-induced liver injury ingredients and their functional mechanisms in S. Chinensis Fructus. Liquid chromatograph-mass spectrometry analysis was performed on S. Chinensis Fructus extrac. The "Components-Target-Disease" network model was constructed by network pharmacology-based approaches. String analysis was performed to reveal enrichment of these target proteins, protein-protein interactions, pathways and related diseases. And experiment of APAP-induced drug-induced liver injury was to be verified. Cytoscape was used to determine the potential protein targets for these components in S. Chinensis Fructus, indicating that 17 against Drug-induced liver injury compounds in S. Chinensis Fructus regulate 52 diabetes-related proteins in 15 signal pathways and involve 14 core key targets. Verification experiment results that S. Chinensis Fructus prevented the elevation of serum biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), purine nucleoside phosphorylase (PNP) and alkaline phosphatase (ALP) against acute liver failure. Additionally, S. Chinensis Fructus reduced the content of malondialdehyde (MDA), increased the levels of the Superoxide dismutase (SOD) and Glutathione (GSH), and inhibited the production of proinflammatory cytokines in APAP-induced hepatotoxicity. The mechanisms of S. Chinensis Fructus against Drug-induced liver injury were involved in the regulation of multiple targets, especially affecting the ErbB signaling pathways. The active ingredients of S. Chinensis Fructus may play a role against Drug-induced liver injury by participating in the regulation of inflammatory factors, oxidative stress.

Diastereoselective Synthesis of Glycosyl Phosphates by Using a Phosphorylase–Phosphatase Combination Catalyst

Sugar phosphates play an important role in metabolism and signaling, but also as constituents of macromolecular structures. Selective phosphorylation of sugars is chemically difficult, particularly at the anomeric center. We report phosphatase‐catalyzed diastereoselective “anomeric” phosphorylation of various aldose substrates with α‐d‐glucose 1‐phosphate, derived from phosphorylase‐catalyzed conversion of sucrose and inorganic phosphate, as the phosphoryl donor. Simultaneous and sequential two‐step transformations by the phosphorylase–phosphatase combination catalyst yielded glycosyl phosphates of defined anomeric configuration in yields of up to 70 % based on the phosphate applied to the reaction. An efficient enzyme‐assisted purification of the glycosyl phosphate products from reaction mixtures was established.

A Novel Mechanism for Regulating Hepatic Glycogen Synthesis Involving Serotonin and Cyclin-Dependent Kinase-5

Hepatic autonomic nerves regulate postprandial hepatic glucose uptake, but the signaling pathways remain unknown. We tested the hypothesis that serotonin (5-hydroxytryptamine [5-HT]) exerts stimulatory and inhibitory effects on hepatic glucose disposal. Ligands of diverse 5-HT receptors were used to identify signaling pathway(s) regulating glucose metabolism in hepatocytes. 5-HT had stimulatory and inhibitory effects on glycogen synthesis in hepatocytes mediated by 5-HT1/2A and 5-HT2B receptors, respectively. Agonists of 5-HT1/2A receptors lowered blood glucose and increased hepatic glycogen after oral glucose loading and also stimulated glycogen synthesis in freshly isolated hepatocytes with greater efficacy than 5-HT. This effect was blocked by olanzapine, an antagonist of 5-HT1/2A receptors. It was mediated by activation of phosphorylase phosphatase, inactivation of glycogen phosphorylase, and activation of glycogen synthase. Unlike insulin action, it was not associated with stimulation of glycolysis and was counteracted by cyclin-dependent kinase (cdk) inhibitors. A role for cdk5 was supported by adaptive changes in the coactivator protein p35 and by elevated glycogen synthesis during overexpression of p35/cdk5. These results support a novel mechanism for serotonin stimulation of hepatic glycogenesis involving cdk5. The opposing effects of serotonin, mediated by distinct 5-HT receptors, could explain why drugs targeting serotonin function can cause either diabetes or hypoglycemia in humans.

N6-Acetyl-2′,3′,5′-tri-O-acetyladenosine; A Convenient, ‘Missed Out’ Substrate for Regioselective N6-Alkylations

A simple and efficient route to N 6 -acetyl-2′,3′,5′-tri- O-acetyladenosine ( 1)was developed based on selective N-deacetylation of pentaacetylatedadenosine 2 with methanol at room temperaturein the presence of imidazole. Preparative synthesis of 1 was elaborated utilizing a crude mixtureof 2 and 1 whichis produced by reaction of adenosine with acetic anhydride in pyridineat elevated temperatures. The total yield of 1 was80-85% starting with adenosine. It was shown that 1 is a convenient substrate for selectiveN 6 -alkylations. The study revealed the same regioselectivityin base-promoted reactions of 1 with activatedalkyl halides and Mitsunobu reactions of 1 withalcohols. A series of N 6 -alkyladenosines 5A- F wereprepared. Cytokinins 6B, D, E were prepared by enzymatic transformationof parent nucleoside derivatives 5B, D, E using a combinationof nucleoside phosphorylase and alkaline phosphatase.

Phosphorylase and the origin of reversible protein phosphorylation

This article presents a historical account of the discovery of reversible protein phosphorylation. The process was uncovered in the mid-1950s in a study designed to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. This enzyme was originally thought to be regulated by AMP now known to serve as an allosteric effector. By contrast, hormonal regulation was found to result from a phosphorylation of the protein triggered by Ca(2+) and ATP which activate phosphorylase kinase; the reverse reaction is catalyzed by a phosphorylase phosphatase. Although that reaction is extremely simple, it came nevertheless as a complete surprise because essentially nothing was known at that time about the structure and function of phosphoproteins. This study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes (carbohydrate metabolism and muscle contraction) could be regulated in concert. The regulation of phosphorylase was so straightforward that it was thought to represent the prototype for these kinds of interconversions. We know today that it was almost the absolute exception.

Excess Copper‐Induced Oxidative Damages and Changes in Radish Physiology

To identify the detrimental effects of excess copper in radish (Raphanus sativus) cv. Jaunpuri, plants were grown in refined sand at control (0.001) and 0.1 and 0.2 mM (excess) copper (Cu) supplied as Cu sulphate. Previously plants were maintained for 24 days in complete nutrient solution and on the 25th day, excess Cu was superimposed. After 6 days of metal supply, the visible effects of excess Cu appeared as retardation in growth and interveinal chlorosis of young leaves; chlorosis intensified later with the development of irregular brown spots on lamina. Excess Cu not only reduced the size of leaves but also affected adversely the root development. In radish, Cu toxicity decreased the fresh weight, biomass, root weight, concentrations of total and active iron, chlorophyll, activities of antioxidative enzymes catalase (CAT), and peroxidase (POX), starch phosphorylase (ST) and acid phosphatase (AP) with concomitant increase in Cu accumulation in different parts. These effects were more pronounced at 0.2 than 0.1 mM Cu.

Biochemical Changes, Yield, and Quality of Gram Under Boron Stress

Gram (Cicer arietinum L.) cv. Avrodhi was grown in refined sand at levels of boron (B) ranging from deficiency to excess (0.0033–6.6 mg B L−1). At low (<0.33 mg B L−1) B, apart from marked depression in growth at day 28, deficiency symptoms were initiated as chlorosis of young leaflets. With increase in age, these chlorotic leaflets turned necrotic and shed premature. Biomass weight of pods and seeds was maximum at 0.33 mg B L−1. At 6.6 mg B L−1, toxicity symptoms appeared as marginal necrosis of old leaflets along with growth depression. Later, the necrotic leaflets became completely dry and shed. In low B, activity of acid phosphatase, starch phosphorylase, and ribonuclease increased and that of polyphenol oxidase decreased in leaves of gram. Excess B at 6.6 mg B L−1 increased the activity of starch phosphorylase and acid phosphatase and decreased that of ribonuclease and polyphenol oxidase. Deficiency (<0.33 mg L−1) as well as excess (>0.33 mg L−1) of B deteriorated the quality of gram seeds by lowering seed yield, starch and protein concentration, and increasing the accumulation of phenols and sugars. The concentration of B increased in leaves and seeds with an increase in B supply. The values of deficiency, threshold of deficiency, threshold of toxicity and toxicity are, respectively, 13.5, 32, 190, and 310 µg B g−1 in leaves of gram.

Regulation of glycogen phosphorylase and glycogen synthase by adrenalin in soleus muscle of phosphorylase-kinase-deficient mice.

Mixed skeletal muscles of ICR/IAn mice contain 0.2% of the phosphorylase kinase activity found in C3H/He-mg mice at pH 8.2 in the presence of Ca2+. This lack of activity is caused by the absence of the normal phosphorylase kinase protein (Cohen, P. T. W. et al. (1976) Eur. J. Biochem. 66, 347–356). The trace residual activity in ICR/IAn mice has quite different properties from the normal enzyme. It has a much higher activity ratio (pH 6.8/8.2), its activity is less dependent on Ca2+ and it is not activated by cyclicAMP-dependent protein kinase or by trypsin. Its elution behaviour on Sepharose 413 also differs from the normal enzyme. A consequence of the higher activity ratio (pH 6.8/8.2) and decreased effect of Ca2+, is that mixed skeletal muscles of ICR/IAn mice contain 7% of normal activity at pH 6.8 in the absence of Ca2+. The activity of phosphorylase kinase in C3H/He-mg control mice is sixfold lower in red oxidative (soleus) muscle and 15-fold lower in cardiac muscle than in mixed skeletal muscle. In contrast, the trace residual activity in ICR/IAn mice is present at the same level in soleus muscle, cardiac muscle and mixed skeletal muscle. Soleus muscles of ICR/IAn mice therefore contain 1.5% of normal activity at pH 8.2 in the presence of Ca2+ and 30% of normal activity at pH 6.8 in the absence of Ca2+. Cardiac muscle contains 6% of normal activity at pH 8.2 in the presence of Ca2+ and 60% of normal activity at pH 6.8 in the absence of Ca2+. When isolated soleus muscles were incubated with adrenalin, the level of phosphorylase a in ICR/IAn mice rose from 2.8% in the absence to 5.7% in the presence of the hormone. In contrast, the level of phosphorylase a in normal mice varied from 2.4% to 11.1% in the absence of adrenalin, and increased to 17.7–23.9% in the presence of the hormone. It is concluded that the trace phosphorylase kinase activity in ICR/IAn mice is capable of phosphorylating phosphorylase b in vivo and that this enzyme is responsible for the very low level of phosphorylase a in resting soleus muscle of ICR/IAn mice, and perhaps even in normal mice. Since the residual enzyme cannot be activated by cyclic-AMP-dependent protein kinase, the elevation of phosphorylase a by adrenalin may be caused by an inhibition of phosphorylase phosphatase. The possibility that this occurs through the activation of protein phosphatase inhibitor 1 by cyclic-AMP-dependent protein kinase is discussed. Incubation of soleus muscles with adrenalin decreased the activity ratio (+ glucose 6-phosphate) of glycogen synthase from 0.24 to 0.08 in ICR/IAn mice and 0.31 to 0.14 in C3H/He-mg or Swiss albino mice. These results demonstrate that the inactivation of glycogen synthase by adrenalin in resting soleus muscle does not result from the activation of phosphorylase kinase by cyclic-AMP-dependent protein kinase. It is most likely mediated by a direct phosphorylation catalysed by cyclic-AMP-dependent protein kinase, although the activation of protein phosphatase inhibitor 1 may also contribute to the effect.

PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation

It has been known for over a decade that inhibition of protein phosphatase 1 (PP1) activity prevents entry into M phase, but the relevant substrate has not been identified. We report here that PP1 is required for dephosphorylation of the Cdc2-directed phosphatase Cdc25 at Ser287 (of Xenopus Cdc25; Ser216 of human Cdc25C), a site that suppresses Cdc25 during interphase. Moreover, PP1 recognizes Cdc25 directly by interacting with a PP1-binding motif in the Cdc25 N-terminus. We have also found that 14-3-3 binding to phospho-Ser287 protects Cdc25 from premature dephosphorylation. Upon entry into M phase, 14-3-3 removal from Cdc25 precedes Ser287 dephosphorylation, suggesting the existence of a phosphatase- independent pathway for 14-3-3 removal from Cdc25. We show here that this dissociation of 14-3-3 from Cdc25 requires the activity of the cyclin-dependent kinase Cdk2, providing a molecular explanation for the previously reported requirement for Cdk2 in promoting mitotic entry. Collectively, our data clarify several steps important for Cdc25 activation and provide new insight into the role of PP1 in Cdc2 activation and mitotic entry.

Glucose 6-phosphate regulates hepatic glycogenolysis through inactivation of phosphorylase.

High glucose concentration suppresses hepatic glycogenolysis by allosteric inhibition and dephosphorylation (inactivation) of phosphorylase-a. The latter effect is attributed to a direct effect of glucose on the conformation of phosphorylase-a. Although glucose-6-phosphate (G6P), like glucose, stimulates dephosphorylation of phosphorylase-a by phosphorylase phosphatase, its physiological role in regulating glycogenolysis in intact hepatocytes has not been tested. We show in this study that metabolic conditions associated with an increase in G6P, including glucokinase overexpression and incubation with octanoate or dihydroxyacetone, cause inactivation of phosphorylase. The latter conditions also inhibit glycogenolysis. The activity of phosphorylase-a correlated inversely with the G6P concentration within the physiological range. The inhibition of glycogenolysis and inactivation of phosphorylase-a caused by 10 mmol/l glucose can be at least in part counteracted by inhibition of glucokinase with 5-thioglucose, which lowers G6P. In conclusion, metabolic conditions that alter the hepatic G6P content affect glycogen metabolism not only through regulation of glycogen synthase but also through regulation of the activation state of phosphorylase. Dysregulation of G6P in diabetes by changes in activity of glucokinase or glucose 6-phosphatase may be a contributing factor to impaired suppression of glycogenolysis by hyperglycemia.

Validation of Interactions with Protein Phosphatase-1

This chapter presents an overview of the techniques that are available to study the interaction of protein phosphatase 1 (PP1) with other proteins. Most interactors of PP1 are inhibitors of the phosphorylase phosphatase activity of the catalytic subunit. A simple phosphatase assay can give an independent indication that the studied polypeptide is indeed an interactor of PP1. It discusses another technique known as “PP1 overlays,” based on the binding of PP1 to interactors that are associated with PVDF-membranes. PP1 overlays can be used to demonstrate a direct interaction between PP1 and a putative interactor in a complex mixture of proteins. The binding between PP1 and its interactors can also be explored by pull-down experiments when one of the two components is fused to gluthathione S-transferase (GST), a protein that binds with high affinity to glutathione-agarose. An occasional problem with GST pull-down experiments is the high background, because of the nonspecific binding of the putative PP1 interactor to GST. Another classical approach to demonstrate an interaction between two proteins is their coimmunoprecipitation from cell extracts. Some other tools are described in the chapter to study the interaction between PP1 and associated polypeptides.

Contents by Short Title

Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast. We found that the Glc7 protein has a half-life of over 180 min, which is sufficient for several generations. Glc7 protein stability was constant during the cell cycle and in batch culture growth. Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life. The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle. Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity. Glc7 activity was induced during stationary phase in a Glc8-dependent manner. In addition, extracellular glucose repressed the induction of Glc7 activity. These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator.

Phosphoprotein Inhibitors of Protein Phosphatase-1

The chapter discusses phosphoprotein inhibitors of protein phosphatase-1(PP1). Over 25 years ago, a search for proteins that could regulate phosphorylase phosphatase yielded heat-stable proteins that were called “inhibitor-1” (I-1) and “inhibitor-2” (I-2). I-1 potency as an inhibitor increased several hundred-fold after phosphorylation of Thr35 by cAMP-dependent protein kinase (PKA), whereas inhibitor-2 was effective without prior phosphorylation. Inhibitor-1 became the prototype for other phosphoprotein inhibitors of PP1. To distinguish protein phosphates in type-1 (PP1, sensitive to inhibitors) from type-2, these inhibitor proteins are made use of subsequently.. Several new PP1 inhibitor phosphoproteins, such as NIPP-1, inhibitor-4, CPI-17, PHI-1 and KEPI have been discovered in mammalian cells. Regulation of PP1 in cells incorporates the various inhibitor phosphoproteins to selectively target a subset of PP1 holoenzymes. This way the inhibitor proteins work in concert with the PP1 regulatory subunits. Study of the phosphorylation of the PP1 inhibitors takes on added significance for understanding signaling processes, and the chapter describes various methods developed for that purpose.

Glc8 is a glucose-repressible activator of Glc7 protein phosphatase-1

Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast. We found that the Glc7 protein has a half-life of over 180 min, which is sufficient for several generations. Glc7 protein stability was constant during the cell cycle and in batch culture growth. Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life. The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle. Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity. Glc7 activity was induced during stationary phase in a Glc8-dependent manner. In addition, extracellular glucose repressed the induction of Glc7 activity. These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator.

Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.

The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5'-[gamma-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was not affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1(667-1004)). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, G(L), R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, G(L)-PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and G(L) complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and G(L) were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6-PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and G(L) and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for G(L). The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of G(L)-PP1c, suggesting that R5-PP1c may function as a hepatic phosphorylase phosphatase, whereas G(L)-PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and G(L)

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, GL, R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, GL–PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and GL complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and GL were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6–PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and GL and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for GL. The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of GL–PP1c, suggesting that R5–PP1c may function as a hepatic phosphorylase phosphatase, whereas GL–PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and GL

The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1(C)). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.

Glc7p protein phosphatase inhibits expression of glutamine-fructose-6-phosphate transaminase from GFA1.

Inhibitor-1 (I-1) is a specific inhibitor of protein phosphatase-1 (PP1). We assayed the ability of I-1 to inhibit Saccharomyces cerevisiae PP1, Glc7p, in vivo. Glc7p like other PP1 catalytic subunits associates with a variety of noncatalytic subunits, and Glc7p holoenzymes perform distinct physiological roles. Our results show that I-1 inhibits Glc7p holoenzymes that regulate transcription and mitosis, but holoenzymes responsible for meiosis and glycogen metabolism were unaffected. Additionally, we exploited a genetic screen for mutants that were dependent on I-1 to grow. This scheme can identify processes that are negatively regulated by Glc7p-catalyzed dephosphorylation. In this paper I-1-dependent gfa1 mutations were analyzed in detail. GFA1 encodes glutamine-fructose-6-phosphate transaminase. One or more phosphorylated proteins activate GFA1 transcription because the pheromone response and Pkc1p/mitogen-activated protein kinase pathways positively regulate GFA1 transcription. Our findings show that an I-1-sensitive Glc7p holoenzyme reduces GFA1 transcription. Therefore, GFA1 is a member of a growing list of genes that are negatively regulated by Glc7p dephosphorylation.