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PubMed1982

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells.

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.

Alkaline Phosphatase↗
PubMed1988

Inhibition of phosphatase and sulfatase by transition-state analogues.

The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase. Vanadate was a potent inhibitor of all three enzymes. Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-). The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase. Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions. Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase. Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol. It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes. The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed.

Acid Phosphatase↗
PubMed1988

Protein phosphotyrosine phosphatase purified from the particulate fraction of human placenta dephosphorylates insulin and growth-factor receptors.

Protein phosphatase activity specific for Tyr(P) (phosphotyrosine) residues (PTP-phosphatase) was found in the cytosol and particulate fractions of human placenta. In the particulate fraction, half of the PTP-phosphatase activity could be extracted with 1% Triton X-100. The PTP-phosphatase remaining in the Triton-insoluble residue was solubilized with 0.6 M-KCl plus 1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propane-1-sulphonate) and was purified 1850-fold by adsorption to DEAE-Sepharose, affinity chromatography on Zn2+-iminodiacetate-agarose, phosphocellulose adsorption, Fractogel filtration and Mono Q chromatography. The cytoskeleton-associated PTP-phosphatase was distinguished from acid, alkaline and other protein Ser(P) (phosphoserine)/Thr(P) (phosphothreonine) phosphatases by its neutral pH optimum, activity in the presence of EDTA, inhibition by Zn2+, vanadate, or molybdate, and low activity with either [Ser(P)]phosphorylase a or p-nitrophenyl phosphate. The PTP-phosphate displayed a Km of 0.15 microM with [Tyr(P)]serum albumin as substrate, 10-100-fold lower than the Km for previously described protein phosphatases. The cytoskeleton-associated PTP-phosphatase catalysed the dephosphorylation of receptors for insulin, insulin-like growth factor-1 and epidermal growth factor labelled by autophosphorylation. The properties of this PTP-phosphatase suggest that it plays a role in the regulation of hormone receptors and cytoskeleton proteins by reversible phosphorylation on tyrosine residues.

Cell Fractionation↗
PubMed2001

Protein tyrosine phosphatase genes downregulated in melanoma.

Phospho-tyrosine levels are increased in melanoma, apparently consistent with reports of elevated protein tyrosine kinase activity. Some protein tyrosine kinases are encoded by oncogenes and have been implicated in melanoma genesis. Decreased protein tyrosine phosphatase activity may also increase phospho-tyrosine. Protein tyrosine phosphatase genes are candidate tumor suppressors and loss of expression may contribute to melanoma genesis. Here we survey protein tyrosine phosphatase expression in pigment cells. Protein tyrosine phosphatase genes were cloned by reverse transcriptase polymerase chain reaction using degenerate primers based upon conserved sequences within the phosphatase catalytic domain. Reaction products were cloned and sequenced: 118 and 113 partial protein tyrosine phosphatase products were isolated from normal melanocytes and melanoma cells, respectively. Northern blotting analysis was used to study expression of 15 protein tyrosine phosphatase genes. Expression of PTP-kappa and PTP-pi was absent or downregulated in more than 20% of melanoma cell lines and in some unmanipulated melanoma biopsies. These closely related enzymes are members of the 2B receptor protein tyrosine phosphatase family previously implicated in contact inhibition. Loss of protein tyrosine phosphatase expression may contribute to the abnormal tyrosine phosphorylation seen in melanoma; these genes are candidate tumor suppressors.

Blotting, Northern↗
PubMed1996

Activation of protein phosphatase 1. Formation of a metalloenzyme.

The recombinant catalytic subunit of protein phosphatase 1 is produced as an inactive enzyme which can be activated by Mn2+ (Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M. F., and Lee, E. Y. C. (1992) J. Biol. Chem. 267, 1484-1490). In this report, we have investigated the effects of divalent cations on the activity of recombinant catalytic subunit of protein phosphatase 1. Latent phosphatase 1 can be activated by Co2+ or Mn2+, whereas other metal ions tested including Fe2+, Zn2+, Mg2+, Ca2+, Cu2+, or Ni2+ were not effective or were only weakly effective in activating the enzyme. The Mn(2+)-stimulated activity was susceptible to inactivation by EDTA; however, the Co(2+)-activated phosphatase was stable after dilution and chelation of the Co2+ with excess EDTA. After stable activation of phosphatase 1 using 57Co2+, a stoichiometric amount of 57Co2+ was shown to be tightly bound to phosphatase 1. These findings demonstrate for the first time the generation of a stable metalloenzyme form of phosphatase 1. Fe2+ reversibly deactivated the Co(2+)-stimulated activity, but did not displace the bound Co2+. Interestingly, treatment of the enzyme with a combination of Fe2+ and Zn2+ (but not the individual metal ions) significantly activated phosphatase 1. These results suggest that at least two metal binding sites exist on the enzyme and that protein phosphatase 1 may be an iron/zinc metalloprotein in vivo.

Enzyme Activation↗
PubMed1997

The effects of sodium butyrate on transcription are mediated through activation of a protein phosphatase.

In this study we have investigated the molecular mechanism by which sodium butyrate modulates gene expression when added to cultured cells. As a model system we used hepatoma tissue culture cells in which sodium butyrate treatment increases histone H1(0) mRNA level and decreases c-myc mRNA level. Because we observed that stimulation of histone H1(0) gene expression could take place in the absence of protein neosynthesis, we hypothesized that sodium butyrate induced a post-translational modification of a factor involved in the transcription process. Using different types of well known kinase and phosphatase inhibitors, we studied the implication of kinase or phosphatase activity in this pathway. Interestingly, cell treatment with potent serine-threonine-phosphatase inhibitors, calyculin A or okadaic acid, prevented the regulation of both histone H1(0) and c-myc gene expressions by sodium butyrate. On the other hand, the tyrosine phosphatase inhibitor, vanadate, or the protein kinase C inhibitor, staurosporine, did not significantly modify sodium butyrate effects. Using protein phosphatase 1 and 2A for in vitro assays, we found a 45% increase of phosphatase activity after cell treatment by sodium butyrate, possibly due to a protein phosphatase 1-type protein phosphatase. These data strongly suggest that signaling pathway(s) triggered by sodium butyrate to modulate gene expression involve(s) a serine-threonine-phosphatase activity.

Butyrates↗
PubMed2001

Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation.

We investigated whether myosin light chain phosphatase activity changes during nitric oxide-induced relaxation of contracted intact carotid media and how changes in phosphatase activity mediate this relaxation. We also investigated one mechanism for regulating this phosphatase. Myosin phosphatase activity, myosin light chain phosphorylation, guanosine 3',5'-cyclic monophosphate (cGMP) concentration, and phosphorylation of the inhibitory protein CPI-17 were all assayed in homogenates of one carotid media ring at each time point during nitric oxide-induced relaxation. The application of sodium nitroprusside to histamine-contracted media caused rapid declines in light chain phosphorylation and force. These were temporally correlated with a rapid elevation of cGMP and a large transient increase in myosin phosphatase activity. During the early response to nitroprusside, when force declined, increases in myosin phosphatase activity, concurrent with cGMP-mediated decreases in calcium and myosin light chain kinase activity, could accelerate light chain dephosphorylation. CPI-17 was dephosphorylated upon application of nitroprusside at the same time that myosin phosphatase activity increased, suggesting that the removal of inhibition by phospho-CPI-17 contributed to the increase in myosin phosphatase activity. After 20 min of nitroprusside, myosin phosphatase activity had declined to basal levels, however low force was sustained. Additional light chain phosphorylation-independent mechanisms may be involved in sustaining the relaxation.

Animals↗
PubMed1997

Prognostic factors in patients with prostate cancer refractory to endocrine therapy: univariate and multivariate analyses including doubling times of prostate-specific antigen and prostatic acid phosphatase.

Although several prognostic factors have been discussed, multivariate analysis that includes tumor marker doubling time has not yet been examined in patients with prostate cancer refractory to endocrine therapy. A number of conventional prognostic factors including doubling times of prostate-specific antigen and/or prostatic acid phosphatase were examined in 56 prostate cancer patients who were refractory to endocrine therapy, using univariate and multivariate analyses. On univariate analysis, 6 parameters (doubling times of prostate-specific antigen and prostatic acid phosphatase at the time of refractory status, performance status, duration from beginning of endocrine therapy to prostate-specific antigen/prostatic acid phosphatase failure, mode of recurrence, the presence or absence of prostate-specific antigen/prostatic acid phosphatase normalization, and alkaline phosphatase) were shown to be significant prognostic factors. On multivariate analysis, only performance status and doubling times of prostate-specific antigen and prostatic acid phosphatase were significant. These observations showed that the doubling times of prostate-specific antigen and prostatic acid phosphatase, calculated at the time of prostate-specific antigen/prostatic acid phosphatase failure by estimating serial prostate-specific antigen or prostatic acid phosphatase, were a valuable prognostic factor in patients with prostate cancer refractory to endocrine therapy.

Acid Phosphatase↗
PubMed1993

Micrococci demonstrate a phosphatase activity which is repressed by phosphates and which can be differentiated from that of staphylococci.

The phosphatase activities of 114 micrococcal strains belonging to seven different species and of an additional 150 unspeciated micrococcal strains were evaluated on solid media at various pHs containing or not containing phosphates. In the presence of phosphates, only nine strains (five unspeciated strains, one Micrococcus luteus strain, and three Micrococcus varians strains) yielded a positive reaction on plates at pH 8. In media (at pH 8) deprived of phosphates, in contrast, all but 15 strains demonstrated clear-cut phosphatase activity. Acid phosphatase could not be evaluated on solid media since none of the strains grew satisfactorily on plates at pH 5. The phosphatase activities of seven (one or two for each species, which included phosphatase-negative strains) of the strains whose colonies proved phosphatase negative at pH 8 and of 18 (two or three strains per species) of those with phosphatase-positive colonies were evaluated at pH 5 and 8.5 in toluene-treated cells which had been grown in liquid media at pH 7 containing or not containing phosphates. All strains demonstrated distinct phosphatase activity at both pHs when grown in media not containing phosphates. In contrast, when strains were grown in the presence of such substances, virtually no activity was observed at pH 8.5, and, generally, a much reduced activity was observed at pH 5. The phosphatase activity of micrococci of the various species (three to eight strains per species) was also compared with that of staphylococci of different species (5 to 10 strains per species) by the methyl green-phenolphthalein diphosphate method, the sensitivity of which can be varied by using different enzyme substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1993

Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A.

A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.

Animals↗
PubMed1996

Regulation of p42 mitogen-activated-protein kinase activity by protein phosphatase 2A under conditions of growth inhibition by epidermal growth factor in A431 cells.

Epidermal growth factor (EGF), which plays an important role in the growth regulation of a large variety of normal and tumor cells, has been shown to display an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. In a previous study aimed at dissecting the biochemical events leading to this dual action in A431 cells which over express EGF receptor, we have reported a relationship between the dual stimulator/inhibitor effect of EGF and the activity of the serine/threonine p42 mitogen-activated protein (MAP) kinase. Indeed, a growth stimulatory concentration of EGF is shown to lead to a moderate but persistent activation of p42 MAP kinase. Conversely, an early peak of MAP kinase activation, that rapidly falls below the basal level, is observed in the presence of a growth-inhibitory concentration of EGF. To assess the mechanism of the p42 MAP kinase inactivation under circumstances of negative growth regulation by EGF, we have investigated the role of the serine/threonine phosphatase 2A in this process. A constitutive phosphatase 2A activity was observed in untreated cells, that decreases rapidly in response to both high and low EGF concentrations. However, after this early inactivation, the phosphatase 2A activity was completely reversed concurrently with MAP kinase inactivation, after 40 min of treatment with 10 nM EGF. Conversely, in cells treated with 1 pM EGF, phosphatase 2A activity remained below the control level during all the time of the treatment, in association with a sustained MAP kinase activation. These results suggest that MAP kinase inactivation is closely related to phosphatase 2A activation. We then investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on the MAP kinase inactivation and observed that okadaic acid, at a concentration reported to specifically inhibit phosphatase 2A activity, totally reverses the MAP kinase inactivation induced by long-term treatment with 10 nM EGF. Additionally, we have shown that the protein synthesis inhibitor cycloheximide fails to affect the EGF-induced MAP kinase regulation, indicating that mitogen-induced protein phosphatases are not, or are only slightly, required in this regulation. In conclusion, our data demonstrate that the ambivalent action of EGF on the proliferation of A431 cells is associated with differential mechanisms of p42 MAP kinase regulation catalysed by the serine/threonine phosphatase 2A.

Amino Acid Sequence↗
PubMed1976

Histochemical studies on the effect of thyroid hormone on alkaline and acid phosphatase activities in liver of fish and amphibia.

The Lata fishes (Ophicephalus punctatus) showed increased alkaline and acid phosphatase activities in liver after immersion for 15-30 days in thyroxine-containing medium (0.025 mug/ml). A single injection of thyroxine (1-2 mug/g of body weight) caused increased acid phosphatase activity in liver of Lata fish in comparison to the controls on the 5th day after experiment but the alkaline phosphatase activity remained unchanged. Both alkaline and acid phosphatases showed increased activities in liver of Lata fishes treated with a single injection of 4 mug of thyroxine per g of body weight on the 5th day. Immersion of Lata fishes in thiourea solution (1 mg/ml) for 15 days did not show any alteration in alkaline or acid phosphatase activities but these enzyme activities decreased after 30 days' immersion in thiourea solution in comparison to the controls. A seasonal variation of alkaline and acid phosphatase activities was observed in liver of Lata fishes. More alkaline phosphatase activity was found in liver of summer fishes than in winter fishes. The winter fishes showed more acid phosphatase activity than the summer fishes. Three consecutive injections of thyroxine (0.1 mug/g of body weight) to toads (Bufo melanostictus) caused increased alkaline and acid phosphatase activities in liver on the 5th day of the experiment, in comparison to the controls.

Acid Phosphatase↗
PubMedDate not supplied

[Alkaline and acid phosphatase activity in the dynamics of ectopic osteogenesis].

Activities of marker enzymes of osteoblasts (alkaline phosphatase), osteoclasts (tartrate-resistant isoform of acid phosphatase), and macrophages (tartrate-sensitive isoform of acid phosphatase) in bone tissue forming after osteoinduction on the basis of demineralized bone matrix were under study. Early (1 week) stages of osteosynthesis were characterized by low activity of alkaline phosphatase, acid phosphatase being represented by the tartrate-sensitive isoform. Two-three weeks later alkaline phosphatase activity grew and the tartrate-resistant isoform of acid phosphatase appeared. By the fourth-sixth weeks of osteogenesis alkaline phosphatase activity stabilized at a certain level and acid phosphatase activity still grew at the expense of the tartrate-resistant isoform. Conformity of the enzymologic data to changes in cellular populations in ectopic osteogenesis is discussed.

Acid Phosphatase↗
PubMed1989

Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity.

Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.

Alkaline Phosphatase↗
PubMed1978

[Properties and isozymes of acid and alkaline phosphatase in cattle testes and endometrium].

The activity of acid phosphatase in the testicular parenchyma of cattle was found to be higher than that in the endometrium. The acid phosphatase of either tissue broke up p-nitrophenylphosphate at a rate twice as high as that reached for di-sodium phenylphosphate. Hydrolysis of phenolphthalein diphosphate was slower and that of beta-glycerophosphate very slow. Two optimum pH values and two isozymes were recordable from acid phosphatase in testicular supernatant and three optimum pH values as well as three isozymes from that in endometrial supernatant. Even concentrations pf 0.2 mM of copper and mercury ions were strongly inhibiting the acid phosphatase, whereas lead and cadmium ions proved less effective. The activity of acid phosphatase from testes dropped by 24 per cent and that from the endometrium by 16 per cent over eight weeks of storage at -20 degrees C. The average activity of alkaline phosphatase in the endometrium was much higher than that in testicular parenchyma. Alkaline phosphatase from testes exhibited the shortest break-up time for p-nitrophenylphosphate, whereas disodium-phenylphosphate was broken up at the highest rate by endometrial alkaline phosphatase. Two isozymes of alkaline phosphatase were recordable each from the testes and endometrium.

Acid Phosphatase↗
PubMed1985

The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin.

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.

Acid Phosphatase↗
PubMed1984

Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by F A/GSK-3 and casein kinase II (PC0.7).

The ATP-Mg-dependent phosphoprotein phosphatase is believed to consist of a catalytic subunit and a regulatory component identified as phosphatase inhibitor-2. It was found in this study that isolated inhibitor-2 was phosphorylated in serine residues by casein kinase II to at least 3 mol of phosphate per mol of inhibitor-2 while another protein kinase, F A/GSK-3, introduced no more than 0.3 mol of phosphate per mol exclusively in threonine residues. Analysis of tryptic digests by high performance liquid chromatography indicated that casein kinase II action resulted in two major (peaks 1 and 2) and two minor phosphopeptides whereas F A/GSK-3 action generated only peak 2. Combined action of the two protein kinases introduced an additional 0.4-0.6 mol of phosphate per mol over that predicted for simple additive behavior. This synergistic phosphorylation was associated with increased phosphate in peak 2 and correlated with unchanged phosphoserine but increased phosphothreonine, to a level approaching 1 mol/mol. ATP-Mg-dependent protein phosphatase was either reconstituted from purified inhibitor-2 and low molecular weight type 1 phosphatase or isolated as an inactive complex (Fc). Both phosphatase complexes were activated by F A/GSK-3 which caused a transient phosphorylation of the inhibitor-2 component. Casein kinase II alone phosphorylated the inhibitor-2 in both phosphatase complexes without affecting the enzyme activity. Exposure to the combination of F A/GSK-3 and casein kinase II resulted in a synergistic phosphorylation. Furthermore, the combined action of the two protein kinases caused a synergistic activation of the phosphatase at submaximal F A/GSK-3 levels. The results suggest that interactions between phosphorylation sites may play a role in the activation of the ATP-Mg-dependent phosphatase, in particular that phosphorylation by casein kinase II at serine can potentiate the phosphorylation of threonine by F A/GSK-3 with subsequent influence on phosphatase activation.

Animals↗
PubMed1981

Dephosphorylation of rabbit skeletal muscle glycogen synthase (phosphorylated by cyclic AMP-independent synthase kinase 1) by phosphatases.

Phosphorylation of rabbit skeletal muscle glycogen synthase by cyclic AMP-independent synthase kinase 1 results in the incorporation of 4 mol of PO4/subunit. Incubation of the phosphorylated synthase with rabbit muscle phosphoprotein phosphatase brings about the hydrolysis of phosphates from all four major tryptic peptides and an increase in the synthase activity ratio from 0.01 to 0.85. Incubation of the phosphorylated synthase with calf intestinal alkaline phosphatase brings about the preferential hydrolysis of phosphates from three of the four major tryptic peptides and a slight increase in the four major tryptic peptides and a slight increase in the synthase activity ratio from 0.01 to 0.1. The phosphorylation site which is resistant to hydrolysis by calf intestinal alkaline phosphatase can be dephosphorylated by subsequent incubation with rabbit muscle phosphoprotein phosphatase. This dephosphorylation is accompanied by an increase in the synthase activity ratio to approximately 0.9. Measurements of the changes in the kinetic properties of the synthase samples dephosphorylated by alkaline phosphatase reveal that the phosphorylation sites susceptible to hydrolysis by alkaline phosphatase mainly affect the binding of glucose-6-P to the synthase. Comparison of the kinetic properties of the synthase samples dephosphorylated by alkaline phosphatase and by phosphoprotein phosphatase we find that the phosphorylation site resistant to hydrolysis by alkaline phosphatase affects both the binding of UDP-glucose and glucose-6-P to the synthase.

Alkaline Phosphatase↗