Role of protein phosphatase 2A in the control of glycogen metabolism in yeast (original) (raw)
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Biochemical journal. Cellular aspects, 1983
The activity of glycogen synthase phosphatase in rat liver stems from the cooperation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. 2. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. 3. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. 4. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.
Journal of Biological Chemistry, 2001
We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mM glucose and by 2-fold at 20 mM glucose with a decrease in the concentration of glucose causing half-maximal rate (S 0.5 ) from 26 to 19 mM. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.
FEBS Letters, 2003
Changes in the glucosylation state of the glycogen primer, glycogenin, or its association with glycogen synthase are potential sites for regulation of glycogen synthesis. In this study we found no evidence for hormonal control of the glucosylation state of glycogenin in hepatocytes. However, using a modified glycogen synthase assay that separates the product into acid‐soluble (glycogen) and acid‐insoluble (proteoglycogen) fractions we found that insulin and glucagon increase and decrease, respectively, the association of glycogen synthase with an acid‐insoluble substrate. The latter fraction had a higher affinity for UDP‐glucose and accounted for between 5 and 21% of total activity depending on hormonal conditions. Phosphorylase overexpression mimicked the effect of glucagon. It is concluded that phosphorylase activation or overexpression causes dissociation of glycogen synthase from proteoglycogen causing inhibition of initiation of glycogen synthesis.
BMC Biochemistry, 2011
Background: PPP1R6 is a protein phosphatase 1 glycogen-targeting subunit (PP1-GTS) abundant in skeletal muscle with an undefined metabolic control role. Here PPP1R6 effects on myotube glycogen metabolism, particle size and subcellular distribution are examined and compared with PPP1R3C/PTG and PPP1R3A/G M . Results: PPP1R6 overexpression activates glycogen synthase (GS), reduces its phosphorylation at Ser-641/0 and increases the extracted and cytochemically-stained glycogen content, less than PTG but more than G M . PPP1R6 does not change glycogen phosphorylase activity. All tested PP1-GTS-cells have more glycogen particles than controls as found by electron microscopy of myotube sections. Glycogen particle size is distributed for all cell-types in a continuous range, but PPP1R6 forms smaller particles (mean diameter 14.4 nm) than PTG (36.9 nm) and G M (28.3 nm) or those in control cells (29.2 nm). Both PPP1R6-and G M -derived glycogen particles are in cytosol associated with cellular structures; PTG-derived glycogen is found in membrane-and organelle-devoid cytosolic glycogen-rich areas; and glycogen particles are dispersed in the cytosol in control cells. A tagged PPP1R6 protein at the C-terminus with EGFP shows a diffuse cytosol pattern in glucose-replete and -depleted cells and a punctuate pattern surrounding the nucleus in glucose-depleted cells, which colocates with RFP tagged with the Golgi targeting domain of β-1,4-galactosyltransferase, according to a computational prediction for PPP1R6 Golgi location.
Biochemical journal. Cellular aspects, 1977
Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAEcellulose, precipitation with (NH14)2S04 and gel filtration on Sephadex G}200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase bt, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the a-subunit or the fl-subunit), histone HI and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, H and III. Protein phosphatase'I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3 % of the phosphorylase kinase phosphatase and phosphorylaae phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-l, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95°% of the activity towards the a-subunit of phosphorylase kinase that was recovered from Sephadex G0200, It accounted for 10-15% of the glycogen synthase phosphataseo1 and glycogen synthase phosphatase2 activity, but less than 5 % of the activity against the 1subunit of phosphorylase kinase and 1-2 % of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95 % of the phosphorylase phosphatase and 1)-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5 % of the a-phosphorylase kinase phosphatase activity. Protein phosphatast-IH was sometimes eluted from Sephadex G-200 as a species of apparent mol.wt. 75000 (termed IIIA), sometimes as a species of mol.wtb 46000 (termed IuB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-4lA and IIIB were identical. These findings, taken with the observation that phosphorylase phosphatase, 1-phosphorylase kinase phosphatase, glycogen synthase phosphatasedI and glycogen synthase phos. phatase-2 activities co-purified up to the Sephadex G0200 step, suggest that a single protein phosphatase (protein phosphatase-HI) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G. A. & Antoniw, J. F. (1977) Btochem. J. 162, 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III. Ever since the discovery that cyclic AMP-skeletal-miuscle phosphorylase kinase (EC 2.7.1.37) dependent protein kinase (EC2.7.1.37) activates and inactivates skeletal-muscle glycogen synthase (EC 2.4.1.11) (Soderling et al., 1970), there has been
Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast
Cell Research, 2012
The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucosedeprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1∆, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.
Control of Liver Glycogen Synthase Activity and Intracellular Distribution by Phosphorylation
Journal of Biological Chemistry, 2009
Eukaryotic glycogen synthase activity is regulated by reversible phosphorylation at multiple sites. Of the two GS isoforms found in mammals, the muscle enzyme (muscle glycogen synthase) has received more attention and the relative importance of every known phosphorylation site in the control of its activity and intracellular distribution has been previously addressed. We have analyzed the impact of the dephosphorylation at the homologous sites of the glycogen synthase liver (LGS) isoform. Serine residues at these sites were replaced by non-phosphorylatable alanine residues, singly or in pairs, and the resultant LGS variants were expressed in cultured cells using adenoviral vectors. The sole mutation at site 2 (Ser 7) yielded an enzyme that was almost fully active and able to induce glycogen deposition in primary hepatocytes incubated in the absence of glucose and in FTO2B cells, a cell line that does not normally synthesize glycogen. Mutation at site 2 was also sufficient to trigger the aggregation and translocation of LGS from the cytoplasm to the hepatocyte cell cortex in the absence of glucose. However, this redistribution was not observed in hepatocytes incubated without glucose when an additional mutation (E509A), which renders the enzyme inactive, was introduced. This result suggests that LGS translocation is strictly dependent on glycogen synthesis.