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.

scholarly journals Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

1993 ◽  
Vol 4 (7) ◽  
pp. 669-677 ◽  
Author(s):  
P Ferrigno ◽  
T A Langan ◽  
P Cohen
Keyword(s):  
Rat Liver ◽  
Protein Phosphatase ◽  
Human Fibroblasts ◽  
High Mobility ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Mobility Group Protein ◽  
Cell Extracts ◽  
Dependent Protein

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.

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 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.

Ca2+ and calmodulin-dependent protein phosphatase from Leishmania donovani

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 567-573 ◽  
Author(s):  
C. BANERJEE ◽  
D. SARKAR ◽  
A. BHADURI
Keyword(s):  
Cyclosporin A ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Leishmania Donovani ◽  
Protein Phosphatases ◽  
Cross Reactivity ◽  
Calmodulin Antagonists ◽  
Type Protein ◽  
Complete Obliteration ◽  
Dependent Protein

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

scholarly journals Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

1991 ◽  
Vol 275 (1) ◽  
pp. 233-239 ◽  
Author(s):  
A Takai ◽  
G Mieskes
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Enzyme Concentration ◽  
Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Type 1 Phosphatase

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

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 Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle

1986 ◽  
Vol 239 (1) ◽  
pp. 109-114 ◽  
Author(s):  
J Goris ◽  
E Waelkens ◽  
W Merlevede
Keyword(s):  
Skeletal Muscle ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Activity Ratio ◽  
Protein Phosphatases ◽  
Histone H1 ◽  
Rabbit Skeletal Muscle ◽  
Significant Activity ◽  
Purification Step ◽  
Apparent Homogeneity

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.

scholarly journals Sulphydryl agents modulate insulin- and epidermal growth factor (EGF)-receptor kinase via reaction with intracellular receptor domains: differential effects on basal versus activated receptors

1993 ◽  
Vol 292 (1) ◽  
pp. 217-223 ◽  
Author(s):  
S Clark ◽  
N Konstantopoulos
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Cytoplasmic Domain ◽  
Receptor Kinase ◽  
Egf Receptors ◽  
Receptor Structure ◽  
Epidermal Growth

Sulphydryl reagents have been shown to produce a variety of effects on insulin-receptor structure and function. However, localization of these effects to specific receptor domains has not been attempted. We have investigated this question with insulin- and epidermal growth factor (EGF)-receptors (both are receptor tyrosine kinases but have different sulphydryl/disulphide structures within the external domain), and the insulin receptor kinase (IRK) protein consisting solely of the insulin-receptor cytoplasmic domain and exhibiting constitutive kinase activity. Results showed a differential response between basal and activated receptors. The physiological reductant GSH stimulated basal receptor autophosphorylation, but was either without effect (EGF) or inhibited (insulin) activated receptors, and occurred without visible reduction of receptor structure. These results contrast with those obtained with dithiothreitol which appears to activate phosphorylation in association with reduction of the extracellular insulin-receptor disulphides, but is without effect on the EGF receptor or the IRK protein. Alkylating agents N-ethylmaleimide (NEM) and iodoacetamide (IAM) had opposing effects on receptor autophosphorylation. However, only in the basal state was IAM able to protect receptors from the inhibitory effect of NEM. Our results suggest that complex sulphydryl interactions can occur within the cytoplasmic domain of insulin- and EGF-receptors to alter receptor kinase activity. The basal and activated state of receptors is not the same with respect to sulphydryl reagent action, possibly due to conformational change in the receptor induced by ligand (insulin, EGF) or constitutive (IRK) activation.

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

1983 ◽  
Vol 111 (3) ◽  
pp. 912-918 ◽  
Author(s):  
Joseph Di Salvo ◽  
Donetta Gifford ◽  
Jackie R. Vandenheede ◽  
Wilfried Merlevede
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Dependent Protein
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