Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32P-labeled phosphorylase a by decreasing the Km of PP1 for phosphorylase 5-fold, while having no effect on the Vmax of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.
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PMID:Role of protein targeting to glycogen (PTG) in the regulation of protein phosphatase-1 activity. 924 97

A complementary DNA encoding a novel human protein phosphatase 1 (PP1) glycogen-targetting subunit of molecular mass 33 kDa has been sequenced. PPP1R6 is 31% identical to the glycogen-targetting subunit (G(L)) of PP1 from rat liver, 28% identical to the N-terminal region of the glycogen-targetting subunit (G(M)) from human skeletal muscle and 27% identical to glycogen-targetting subunit PPP1R5. Unlike human PPP1R5 and its murine homologue PTG, whose mRNAs are most abundant in skeletal muscle, heart and liver, PPP1R6 is present at similar levels in a wide variety of tissues. The PPP1R6 is associated with glycogen in muscle but is not subject to the same modes of covalent and allosteric regulation as G(M) and G(L).
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PMID:PPP1R6, a novel member of the family of glycogen-targetting subunits of protein phosphatase 1. 941 28

Protein targeting to glycogen (PTG), also known as PPP1R5, is a widely expressed member of a growing family of proteins that target protein phosphatase-1 (PP-1) to glycogen particles. Because PTG also binds to glycogen synthase and phosphorylase kinase, it has been suggested that it serves as a "scaffold" for efficient activation of glycogen synthesis. However, very little is known about the metabolic effects of PTG. In this study, we have used recombinant adenovirus to overexpress PTG in primary rat hepatocytes, a cell type with high glycogenic capacity. We find that overexpression of PTG potently activates glycogen synthesis in cultured hepatocytes. Surprisingly, the glycogenic effect of PTG is observed even in the complete absence of carbohydrates or insulin in the culture medium. Furthermore, glycogenolytic agents such as forskolin or glucagon are largely ineffective at activating glycogen degradation in PTG overexpressing hepatocytes, even though large increases in cAMP levels are demonstrated. These metabolic effects of PTG overexpression are accompanied by a 3.6-fold increase in glycogen synthase activation state and a 40% decrease in glycogen phosphorylase activity. Our results are consistent with a model in which PTG overexpression "locks" the hepatocyte in a glycogenic mode, presumably via its ability to promote interaction of enzymes of glycogen metabolism with PP-1.
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PMID:Overexpression of protein targeting to glycogen (PTG) in rat hepatocytes causes profound activation of glycogen synthesis independent of normal hormone- and substrate-mediated regulatory mechanisms. 975 75

The skeletal muscle glycogen-binding subunit (GM) of protein phosphatase-1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S-transferase (GST) fusions with the N-terminal domain of GM (GST-GM(1-240)) and with the full length R5 protein (GST-R5) both bound to glycogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-GM(1-240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60-70%) in glycogen binding, showing these hydrophobic residues influenced the protein-glycogen interaction. The wild type and V150A/ F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-GM proteins as well as GST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch.
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PMID:A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits. 984 3

Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-(13)C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [(13)C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes.
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PMID:Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen. 1068 77

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.
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PMID:Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes. 1086 64

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.
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PMID:Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels. 1099 19

Insulin is the most-potent physiological anabolic agent known, promoting the synthesis and storage of carbohydrates and lipids and inhibiting their degradation and release into the circulation. This action of the hormone is due in part to the acute regulation of metabolic enzymes through changes in their phosphorylation state. In fat, liver, and muscle, insulin stimulates the dephosphorylation of a number of enzymes involved in glycogen and lipid metabolism via activation of protein phosphatases. Numerous studies have indicated that protein phosphatase-1 (PP1) is the primary phosphatase involved in insulin action. Although PP1 is a cytosolic protein, the phosphatase is compartmentalized in cells by discrete targeting subunits. These proteins confer substrate specificity to PP1 and mediate the specific regulation of intracellular pools of PP1 by a variety of extracellular signals. Four proteins have been described that target the phosphatase to the glycogen particle. G(M) and GL are expressed exclusively in striated muscle and liver, while protein targeting to glycogen (PTG) and R6 are more widely expressed. Despite a common targeting function, these four proteins are not highly conserved, suggesting profound differences in the mechanisms by which they contribute to the hormonal regulation of PP1 activity. Overexpression studies in cell lines or animals have revealed major differences among these proteins regarding basal glycogen levels and hormonal responsiveness. Furthermore, alterations in the expression or function of PP1 glycogen-targeting subunits may contribute to the onset of insulin resistance and type 2 diabetes.
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PMID:The role of protein phosphatase-1 in insulin action. 1123 11

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.
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PMID:Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes. 1160 Apr 96

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. Expression of one family member, PTG, in the liver of normal rats improves glucose tolerance without affecting other plasma variables but leaves animals unable to reduce hepatic glycogen stores in response to fasting. In the current study, we have tested whether expression of other targeting subunit isoforms, such as the liver isoform G(L), the muscle isoform G(M)/R(Gl), or a truncated version of G(M)/R(Gl) termed G(M)DeltaC in liver ameliorates glucose intolerance in rats fed on a high fat diet (HF). HF animals overexpressing G(M)DeltaC, but not G(L) or G(M)/R(Gl), exhibited a decline in blood glucose of 35-44 mg/dl relative to control HF animals during an oral glucose tolerance test (OGTT) such that levels were indistinguishable from those of normal rats fed on standard chow at all but one time point. Hepatic glycogen levels were 2.1-2.4-fold greater in G(L)- and G(M)DeltaC-overexpressing HF rats compared with control HF animals following OGTT. In a second set of studies on fed and 20-h fasted HF animals, G(M)DeltaC-overexpressing rats lowered their liver glycogen levels by 57% (from 402 +/- 54 to 173 +/- 27 microg of glycogen/mg of protein) in the fasted versus fed states compared with only 44% in G(L)-overexpressing animals (from 740 +/- 35 to 413 +/- 141 microg of glycogen/mg of protein). Since the OGTT studies were performed on 20-h fasted rats, this meant that G(M)DeltaC-overexpressing rats synthesized much more glycogen than G(L)-overexpressing HF rats during the OGTT (419 versus 117 microg of glycogen/mg of protein, respectively), helping to explain why G(M)DeltaC preferentially enhanced glucose clearance. We conclude that G(M)DeltaC has a unique combination of glycogenic potency and responsiveness to glycogenolytic signals that allows it to be used to lower blood glucose levels in diabetes.
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PMID:Reversal of diet-induced glucose intolerance by hepatic expression of a variant glycogen-targeting subunit of protein phosphatase-1. 1170 47


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