Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1(143-217), were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1(191-210) in the far-Western assay. NIPP-1(191-210) competed for PP-1 inhibition by full-length NIPP-1(1-351), inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1(143-217)-Sepharose but not from full-length NIPP-1(1-351)-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.     

Separation and identification of type 1 and type 2 protein phosphatases from rabbit reticulocyte lysates

Protein phosphatase type 1 and type 2 activities (designated PP-1 and PP-2, respectively) from rabbit reticulocyte lysates have been identified and characterized based on criteria previously established for similar activities in rabbit skeletal muscle and rabbit liver. These include (a) chromatographic separation on DEAE-cellulose, (b) substrate specificity toward glycogen phosphorylase a and the alpha- and beta-subunits of phosphorylase kinase, (c) differential sensitivity to the heat-stable protein phosphatase inhibitors-1 and -2, and (d) sensitivity to MgATP. When total lysate phosphatases are assayed in the presence of 1 mM MnCl2, protein phosphatase type 2 represents 84% of lysate phosphorylase phosphatase activity. However, when phosphatase assays are carried out with MgATP concentrations similar to those in the lysate, type 2 activity is diminished, and the levels of type 1 (41%) and type 2 (59%) phosphatase activities are comparable. A small proportion (6%) of total lysate phosphatase is tightly bound to the ribosomes, where type 1 phosphatase predominates. At least five species of protein phosphatases can be identified in lysates. These constitute two forms of protein phosphatase type 1, one of which (designated FC) is dependent on MgATP and a lysate activator protein FA; both FC and FA have been identified previously in skeletal muscle. Three species of protein phosphatase type 2 have been identified and designated PP-2B, PP-2A1, and PP-2A2 based on criteria recently established for rabbit skeletal muscle and rabbit liver phosphatases, which display similar phosphatase profiles. Lysate protein phosphatases types 1, FC, 2A1, and 2A2 can all act on phosphorylase a and the alpha- (type 2) or beta-(type 1) subunit of phosphorylase kinase. PP-2B, a Ca2+/calmodulin-dependent phosphatase, specifically dephosphorylates the alpha-subunit of phosphorylase kinase, but does not act on phosphorylase alpha. The heat-stable protein phosphatase inhibitor-2 from skeletal muscle completely blocks the activity of the two type 1 phosphatases (PP-1, FC), but has no effect on the three species of type 2 protein phosphatase. A preliminary assay of the two heat-stable phosphatase inhibitors in lysates indicates significant levels of inhibitor-2, but little or no detectable inhibitor-1.     

Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.     

Protein phosphatases of the guinea-pig parotid gland

The nature of protein phosphatases of the guinea-pig parotid gland was investigated. The protein phosphatases were characterized by (a) the use of five different 32P-labelled substrate proteins (phosphorylase a, histone H2B, casein, and the alpha and beta subunits of phosphorylase kinase), (b) their behaviour during ion-exchange chromatography, (c) their relative molecular mass distribution during gel filtration, (d) their sensitivity towards inhibition by inhibitor 2, (e) their ability to be stimulated by protamine and (f) by their behaviour during freezing and thawing in the presence of 2-mercaptoethanol. The following results were obtained. 1. The 'cytosol' (100,000 X g supernatant) contains protein phosphatases of the types 1, 2A and 2B. 2. On the basis of inhibition with inhibitor 2 (1.2 micrograms/ml) the 'cytosolic' phosphorylase phosphatase activity consists to about 40% of protein phosphatase 1 and to about 60% of protein phosphatase 2A. 3. In the cytosol about 80-90% of the protein phosphatases 1 and 2A exist in an inactive state. 4. A 5-10-fold activation can be achieved by ethanol precipitation, which results in the generation of a mixture of forms of low apparent molecular mass of about 30 kDa. 5. Microsome-associated phosphorylase phosphatase activities can be extracted in a highly active state by detergent (1% Triton X-100) or by 0.8 M NaCl. 6. Activity measurements in the presence of inhibitor 2 (1.2 micrograms/ml) indicate that the microsomal activities consist to about 75% of protein phosphatase 1 and to about 25% of protein phosphatase 2A. Activities corresponding to protein phosphatases 2B and 2C could not be detected. 7. The 'microsomal' protein phosphatase activities exhibit lower apparent molecular masses (70 kDa and 30 kDa) than the 'cytosolic' protein phosphatases (about 260 kDa). 8. After ethanol treatment of the microsomal protein phosphatases only activities with apparent molecular masses of about 30 kDa can be detected. These share several similarities with the ethanol-treated cytosolic protein phosphatases. 9. Both cytosolic and microsomal protein phosphatases display activity towards histone H2B and casein.     

Okadaic Acid-Induced Inhibition of B-50 Dephosphorylation by Presynaptic Membrane-Associated Protein Phosphatases

The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.     

Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle

Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.     

Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.     

Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP-dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase-1 (PP-1). Conversely, Thr34-phosphorylated DARPP-32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase-2A (PP-2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP-32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 microM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP-32 by 17+/-2-fold. Okadaic acid (1 microM), an inhibitor of PP-1 and PP-2A, had a smaller effect, increasing phospho-DARPP-32 by 5.1+/-1.3-fold. The effect of okadaic acid on DARPP-32 phosphorylation was shown to be due to inhibition of PP-2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP-1/PP-2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP-32. The use of Ca2(+)-free/EGTA medium mimicked the effects of cyclosporin A on DARPP-32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP-32 phosphorylation was attributable to blockade of the Ca2(+)-dependent activation of calcineurin. The results indicate that calcineurin and PP-2A, but not PP-1, act synergistically to maintain a low level of phosphorylated DARPP-32 in neostriatal slices.     

Activation/dephosphorylation of muscle glycogen synthase phosphorylated by phosphorylase kinase

1. Glycogen synthase from rabbit skeletal muscle was phosphorylated by phosphorylase kinase to yield synthase b2. 2. Dephosphorylation and activation of synthase b2 by the catalytic subunits of protein phosphatase-1 (PP-1c) and protein phosphatase-2A (PP-2Ac) was studied. The apparent Km of PP-1c and PP-2Ac were 3.3 microM and 6.2 microM, respectively. The apparent Vmax of PP-1c was about two times larger than that of PP-2Ac. 3. Ligands with phosphate moiety (AMP, glucose-6-P at high concentration) caused an inhibition in dephosphorylation by both phosphatases. Spermine inhibited the dephosphorylation by PP-1c and stimulated the action of PP-2Ac. Therefore it can be employed to distinguish the phosphatases using synthase b2 as substrate.     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver.

The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.     

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using β-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.     

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle.

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.     

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.     

The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.     

The structure, role, and regulation of type 1 protein phosphatases.

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.     

Protein Phosphatases 1 and 2A Dephosphorylate B-50 in Presynaptic Plasma Membranes from Rat Brain

The protein B-50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phosphatase type 1 and 2A (PP-1 and PP-2A)-like activities. The present studies further demonstrate that B-50 is dephosphorylated not only by a spontaneously active PP-1-like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 micrograms of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor-2 and by high concentrations (microM) of okadaic acid, identifying it as a latent form of PP-1. In the presence of inhibitor-2 to block PP-1, histone H1 (16-64 micrograms/ml) and spermine (2 mM) increased B-50 dephosphorylation. This sensitivity to polycations and the reversal of their effects on B-50 dephosphorylation by 2 nM okadaic acid are indicative of PP-2A-like activity. PP-1- and PP-2A-like activities from SPM were further displayed by using exogenous phosphorylase alpha and histone H1 as substrates. Both PP-1 and PP-2A in rat SPM were immunologically identified with monospecific antibodies against the C-termini of catalytic subunits of rabbit skeletal muscle PP-1 and PP-2A. Okadaic acid-induced alteration of B-50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity-purified anti-B-50 immunoglobulin G. These results provide further evidence that SPM-bound PP-1 and PP-2A-like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B-50.