scholarly journals The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen

1988 ◽  
Vol 250 (3) ◽  
pp. 659-663 ◽  
Author(s):  
M Bollen ◽  
W Stalmans
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Soluble Fraction ◽  
Glycogen Synthase ◽  
Gel Filtration ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Phosphatase Activities ◽  
Glycogen Particles

1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase activity (measured after incubation with trypsin) from glycogen to the soluble fraction. The degree of inhibition of phosphatase G corresponded closely to the extent to which the phosphorylase phosphatase activity was released from the glycogen particles. Incubation of glycogen-free protein phosphatase G with modulator did not change the affinity of the enzyme for added glycogen, but decreased the amount of phosphatase that could be bound to glycogen. 3. The phosphorylase phosphatase activity that was released from the glycogen particles by modulator migrated on gel filtration as a complex (Mr 106,000) of the catalytic subunit with modulator. Phosphorylase phosphatase activity could be transferred from glycogen-bound protein phosphatase G to modulator that was covalently bound to Sepharose. After elution from the column, the enzyme was identified as the free catalytic subunit (Mr 37,000).

scholarly journals Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 423-433 ◽  
Author(s):  
J F Antoniw ◽  
H G Nimmo ◽  
S J Yeaman ◽  
P Cowen
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Substrate Specificities ◽  
Phosphatase Activities

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 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 b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II 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 phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, 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 alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit 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 beta-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 alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) 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) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

scholarly journals Insulin-receptor phosphotyrosyl-protein phosphatases

1988 ◽  
Vol 256 (3) ◽  
pp. 893-902 ◽  
Author(s):  
M J King ◽  
G J Sale
Keyword(s):  
Insulin Receptor ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Egf Receptor ◽  
Soluble Fraction ◽  
Protein Phosphatases ◽  
Egf Receptors ◽  
Cell Extracts ◽  
Dependent Protein

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.

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

2001 ◽  
Vol 360 (2) ◽  
pp. 449-459 ◽  
Author(s):  
Gareth J. BROWNE ◽  
Mirela DELIBEGOVIC ◽  
Stefaan KEPPENS ◽  
Willy STALMANS ◽  
Patricia T. W. COHEN
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Diabetic Rats ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Hepatic Glycogen ◽  
Insulin Dependent ◽  
Insulin Dependent Diabetic

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

scholarly journals Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity

1986 ◽  
Vol 235 (2) ◽  
pp. 351-357 ◽  
Author(s):  
M F Lin ◽  
G M Clinton
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Acid Phosphatase Activity ◽  
High Performance ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Peptide Sequence ◽  
Phosphatase Activities ◽  
Nitrophenyl Phosphate

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.

Induction of hepatic glycogenesis in the fetal rat

1989 ◽  
Vol 256 (1) ◽  
pp. E49-E54 ◽  
Author(s):  
P. A. Gruppuso ◽  
D. L. Brautigan
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Hepatic Glycogen ◽  
Phosphatase Activities ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

We have performed an in vivo study to test the hypothesis that induction of fetal hepatic glycogenesis is stimulated by insulin and involves activation of protein phosphatase type-1. Control animals and the following two experimental groups were studied: maternal fasting for 48 h prior to term and chronic maternal hyperinsulinemia for 5 days prior to term. Maternal fasting led to decreased fetal hepatic glycogen content and fetal growth retardation. In contrast, no decrease in fetal hepatic glycogen content or fetal weight occurred with maternal hyperinsulinemia despite fetal hypoglycemia and fetal hypoinsulinemia. In neither model were fetal hepatic synthase phosphatase or phosphorylase phosphatase activities affected. In control fetuses, the appearance of hepatic glycogen from days 17 to 21 of gestation correlated with induction of glycogen synthase. Phosphorylase phosphatase and synthase phosphatase activities already were present on day 17 of gestation and changed little through term. However, phosphatase catalytic protein reactive with anti-phosphatase type-1 antibodies did increase approximately fivefold from day 18 to 21. In adult animals fasted for 48 h, 50% of hepatic glycogen synthase phosphatase activity was lost, whereas phosphorylase phosphatase activity was stimulated fourfold. The apparent size of protein phosphatase type-1 catalytic subunit as detected by Western immunoblotting was altered by fasting in the adult but not by substrate restriction (maternal fasting) in the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Competition between two phosphatases fine-tunes Hedgehog signaling

2020 ◽  
Vol 220 (2) ◽  
Author(s):  
Min Liu ◽  
Aiguo Liu ◽  
Jie Wang ◽  
Yansong Zhang ◽  
Yajuan Li ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Hedgehog Signaling ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Feedback Mechanisms ◽  
Hh Signaling ◽  
High Level ◽  
Signaling Activity

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. How its signaling activity is fine-tuned in response to fluctuated Hh gradient is less known. Here, we identify protein phosphatase V (PpV), the catalytic subunit of protein phosphatase 6, as a homeostatic regulator of Hh signaling. PpV is genetically upstream of widerborst (wdb), which encodes a regulatory subunit of PP2A that modulates high-level Hh signaling. We show that PpV negatively regulates Wdb stability independent of phosphatase activity of PpV, by competing with the catalytic subunit of PP2A for Wdb association, leading to Wdb ubiquitination and subsequent proteasomal degradation. Thus, regulated Wdb stability, maintained through competition between two closely related phosphatases, ensures graded Hh signaling. Interestingly, PpV expression is regulated by Hh signaling. Therefore, PpV functions as a Hh activity sensor that regulates Wdb-mediated PP2A activity through feedback mechanisms to maintain Hh signaling homeostasis.

scholarly journals Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver

1985 ◽  
Vol 225 (3) ◽  
pp. 665-670 ◽  
Author(s):  
G Mieskes ◽  
H D Söling
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Liver Protein ◽  
Key Enzyme ◽  
Phosphatase 2A

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.

scholarly journals The split protein phosphatase system

2018 ◽  
Vol 475 (23) ◽  
pp. 3707-3723 ◽  
Author(s):  
Anne Bertolotti
Keyword(s):  
Protein Phosphatase ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Post Translational Modification ◽  
Antagonistic Action ◽  
Cellular Processes ◽  
Depth Study ◽  
Split Protein ◽  
Phosphatase System ◽  
Physiological Substrates

Reversible phosphorylation of proteins is a post-translational modification that regulates all aspect of life through the antagonistic action of kinases and phosphatases. Protein kinases are well characterized, but protein phosphatases have been relatively neglected. Protein phosphatase 1 (PP1) catalyzes the dephosphorylation of a major fraction of phospho-serines and phospho-threonines in cells and thereby controls a broad range of cellular processes. In this review, I will discuss how phosphatases were discovered, how the view that they were unselective emerged and how recent findings have revealed their exquisite selectivity. Unlike kinases, PP1 phosphatases are obligatory heteromers composed of a catalytic subunit bound to one (or two) non-catalytic subunit(s). Based on an in-depth study of two holophosphatases, I propose the following: selective dephosphorylation depends on the assembly of two components, the catalytic subunit and the non-catalytic subunit, which serves as a high-affinity substrate receptor. Because functional complementation of the two modules is required to produce a selective holophosphatase, one can consider that they are split enzymes. The non-catalytic subunit was often referred to as a regulatory subunit, but it is, in fact, an essential component of the holoenzyme. In this model, a phosphatase and its array of mostly orphan substrate receptors constitute the split protein phosphatase system. The set of potentially generalizable principles outlined in this review may facilitate the study of these poorly understood enzymes and the identification of their physiological substrates.

scholarly journals A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling

2016 ◽  
Vol 291 (33) ◽  
pp. 17360-17368 ◽  
Author(s):  
Tanvir Khatlani ◽  
Subhashree Pradhan ◽  
Qi Da ◽  
Tanner Shaw ◽  
Vladimir L. Buchman ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Protein Interactions ◽  
Protein Phosphatase ◽  
Thrombus Formation ◽  
Protein Phosphatase 2A ◽  
Adaptor Protein ◽  
Fibrin Clot ◽  
Catalytic Subunit ◽  
Clot Retraction ◽  
Phosphatase 2A

The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block (396PAIPPKKPRP405) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395–407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity.

scholarly journals Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

2000 ◽  
Vol 20 (21) ◽  
pp. 8143-8156 ◽  
Author(s):  
Haifeng Yang ◽  
Wei Jiang ◽  
Matthew Gentry ◽  
Richard L. Hallberg
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cell Types ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Phosphatase 2A

ABSTRACT CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p incdc55-null cells is the result of ectopic stabilization of this protein during G2 and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

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