The regulation of glycogen metabolism. Phosphorylation of inhibitor-1 from rabbit skeletal muscle, and its interaction with protein phosphatases-III and -II.

Inhibitor-1 from rabbit skeletal muscle was phosphorylated by protein kinase dependent on adenosine 3' :5'-monophosphate (cyclic AMP), but not by phosphorylase kinase or by glycogen synthetase kinase-2. Protein phosphatase-III, isolated and stored in the presence of manganese ions to keep it stable, was in a form which catalysed a rapid dephosphorylation and inactivation of inhibitor-1. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 0.7 micron, V(rel) = 40] were comparable to those for the dephosphorylation of phosphorylase kinase [Km =1.1 micron, V (rel) = 62] and phosphorylase [Km = 5.0 micron, V (rel) = 100]. The dephosphorylation of inhibitor -1 was inhibited by inhibitor-2, indicating that it was catalysed by protein phosphatase-III, and not by another enzyme that might be contaminating the preparation. When protein phosphatase-III was diluted into buffers containing excess EDTA, it lost activity initially, but after 90 min, the activity reached a plateau that remained stable for at least 20h. The initial loss in activity varied with the substrate that was tested; it was 20-30% with phosphorylase a, 50-60% with phosphorylase kinase and greater than or equal to 95% with inhibitor-1. This form of protein phosphatase-III was inhibited by inhibitor-1 in a noncompetitive manner, and the Ki for inhibitor-1 was 1.6 +/- 0.3 nM. The phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthetase phosphatase activities of protein phosphatase-III were inhibited in an identical manner by inhibitor-1. This result emphasizes the potential importance of inhibitor-1 in the regulation of glycogen metabolism, since it can influence the state of phosphorylation of three different enzymes. The formation of the inactive complex between inhibitor-1 and protein phosphatase-III was reversed by incubation with trypsin (which destroyed inhibitor-1, but not protein phosphatase-III) or by dilution of the inactive complex. Kinetic studies, using the form of protein phosphatase-III which dephosphorylated inhibitor-1 very rapidly, demonstrated three unusual features of the system: (a) inhibitor-1 was still as powerful and inhibitor of the dephosphorylation of phosphorylase a and phosphorylase kinase a even under conditions where it was being rapidly dephosphorylated; (b) inhibitor-1 was not an inhibitor of its own dephosphorylation; (c) phosphorylase a did not effect the rate of dephosphorylation of inhibitor-1 even when it was present in a 50-fold molar excess over inhibitor-1. The result of these three properties is that inhibitor-1 is preferentially dephosphorylated by protein phosphatase-III even in the presence of a large excess of other phosphoprotein substrates. Inhibitor-1 was also dephosphorylated by protein phosphatase-II. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 2.8 micron, V (rel) = 200] and the alpha-subunit of phosphorylase kinase [Km = 3.7 micron, V (rel) = 100]were comparable...     

Separation and characterization of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle.

Two heat-stable and trypsin-labile inhibitors of phosphorylase phosphatase, designated inhibitor-1 and inhibitor-2, were partially purified from extracts of rabbit skeletal muscle by heating and coloumn chromatography using DEAE-dellulose and Bio-gel P-60. Inhibitor-1 exists in an active phosphorylated form and an inactive dephosphorylated form. The interconversion of phosphorylated inhibitor-1 and dephosphorylated inhibitor-1 is mediated by protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and a Mn2+-stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor-2 is not influenced by treatment with either the kinase or the Mn2+-stimulated phosphatase. The molecular weights of inhibitor-1 and inhibitor-2 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor-1 and inhibitor-2 inhibit phosphorylase phosphatase by a mechanism which appears to be non-competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic-AMP-dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor-1 and inhibitor-2.     

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 regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle.

Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorylated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann, W. H. (1976) Eur. J. Biochem. 70, 419--426]. Inhibitor-1 was purified by a heat treatment at 90 degrees C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally rechromatography of the phosphorylated protein on DEAE-cellulose, The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seven days corresponding to an overall yield of 15-20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 micron, which is at least as high as the concentration of phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibrium centrifugation was 19200 and by amino acid analysis was 20800. These values were lower than the mol. wt 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weight of 60000 estimated by gel filtration on Sephadex G-100. The gel filtration behaviour, stability to heating at 100 degrees C and amino acid composition suggest that inhibitor-1 may possess little ordered structure. The phosphorylated from of inhibitor-1 contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with the previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Thr- [Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEBS Lett. 76, 182-186].the phosphorylated form of inhibitor-1 inhibited phosphorylase phosphatase activity (0.02U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000-2000 times less effective as an inhibitor.     

Effects of phosphorylation of protein phosphatase 1 by pp60v-src on the interaction of the enzyme with substrates and inhibitor proteins

Phosphorylation of protein phosphatase 1 by pp60v-src decreased its activity towards phosphorylase kinase and glycogen synthase as well as towards phosphorylase a. Kinetic experiments indicated that the primary effect of phosphorylation was to increase the Km for each of the substrate proteins. There was little or no change in the Vmax for the reactions. The possibility that phosphorylation of protein phosphatase 1 altered its regulation by inhibitors-1 and -2 was also examined. Phosphorylation of protein phosphatase 1 did not prevent the reversible inhibition of the enzyme by inhibitor-1 or inhibitor-2 nor did it prevent the association of inhibitor-2 with protein phosphatase 1 to form the MgATP-dependent protein phosphatase. Protein phosphatase 1 is not a substrate for pp60v-src when it is complexed with inhibitor-2 to form the inactive MgATP-dependent protein phosphatase. Here we have shown that protein phosphatase 1 is also not phosphorylated by pp60v-src following activation of the MgATP-dependent protein phosphatase with glycogen synthase kinase-3 and MgATP. This indicates that the inability of pp60v-src to phosphorylate protein phosphatase 1 is not due to the change in protein phosphatase 1 conformation which accompanies the inactivation of the MgATP-dependent protein phosphatase. Rather, it appears to be the result of steric hindrance by inhibitor-2. This suggests that the pp60v-src phosphorylation site is closely associated with the inhibitor-2 binding site involved in the formation of the MgATP dependent protein phosphatase. The pp60v-src phosphorylation site was previously localized to a small (Mr less than or equal to 4000) domain which can be selectively degraded by chymotrypsin. Here we have shown that chymotryptic digestion increased the Km of unphosphorylated protein phosphatase 1 for each of the three phosphoprotein substrates used in this study. This effect was similar to that observed after phosphorylation of protein phosphatase 1. These results indicate that the pp60v-src phosphorylation site is in a region of protein phosphatase 1 which influences substrate binding and which may be near the active site.     

Kinetic analysis of the separate phosphorylation events in the phosphorylase kinase reaction

Glycogen phosphorylase, a dimer of identical subunits, is activated by phosphorylase kinase-catalyzed phosphorylation of one serine residue in each subunit. In this paper, the effect of the phosphorylation of one subunit on the phosphorylation of the other subunit was examined. The three forms of phosphorylase, phosphorylase b (nonphosphorylated), phosphorylase ab (one subunit phosphorylated), and phosphorylase a (both subunits phosphorylated), were separated by anion-exchange high-performance liquid chromatography (HPLC). Purified phosphorylase ab was found to be stable under the conditions of the phosphorylase kinase assay. Initial rate kinetics showed that phosphorylase kinase had a lower KM for phosphorylase ab (3.9 +/- 0.24 microM) than for phosphorylase b (14.9 +/- 2.6 microM). Using the HPLC separation as a simultaneous assay for the three forms of phosphorylase during the phosphorylase kinase reaction, it was found that the pseudo-first-order rate constant for the second phosphorylation step (k2) was 3.7 times greater than that for the first step (k1). The activator AMP reduced the ratio k2/k1 from 3.7 without AMP to 1.4. When the monomeric gamma delta complex of phosphorylase kinase subunits was used as the enzyme, the ratio k2/k1 was 2.1, compared to 3.7 with the multimeric holophosphorylase kinase. One explanation for these data is that phosphorylation of one subunit of phosphorylase b causes conformational changes that make the other subunit a better substrate for the kinase. In this context, the effect of AMP is to reduce the conformational differences between phosphorylases b and ab, and the gamma delta complex is less sensitive to the conformational differences between the two forms of phosphorylase.     

The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 microM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo.     

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.     

Larvicidal toxin from Bacillus sphaericus spores: Isolation of toxic components

Meiotic maturation of amphibian oocytes induced by progesterone is known to be regulated by protein phosphorylation. To investigate a possible role for protein phosphatase-1 in this process, the effect of phosphatase inhibitor-2 was determined on the in vivo rate of dephosphorylation of phosphorylase a and on the rate of oocyte maturation. Dephosphorylation of microinjected phosphorylase a was inhibited up to 40% in the presence of inhibitor-2, with half-maximal inhibition at an intracellular concentration of 0.6 μM. Inhibitor-2 also caused over a 3-fold increase in the half-time for maturation, suggesting a possible role for protein phosphatase-1 in the regulation of meiosis.     

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.     

Autonomic regulation of type 1 protein phosphatase in cardiac muscle

Muscarinic cholinergic agonists such as acetylcholine attenuate phosphorylation of phospholamban induced by agents that activate cAMP-dependent protein kinase. However, cAMP accumulation is variably affected or only slightly reduced; thus, the choline ester might produce effects in addition to inhibition of adenylate cyclase. We hypothesized that acetylcholine might regulate a phosphatase in mammalina myocardium. Exposure of Langendoff-perfused guinea pig ventricles to isoproterenol (10 nM) for 45 s increased phosphatase inhibitor-1 activity 2-fold. Co-administration of acetylcholine (100 nM) antagonized the effect of isoproterenol, and atropine (1 microM) blocked the effect of acetylcholine. Forskolin (1 microM) caused a 3-fold increase in inhibitor-1 activity, and acetylcholine markedly attenuated the effect of forskolin. However, acetylcholine did not lower cAMP levels in the same tissues. Both isoproterenol and forskolin reduced the type 1 phosphatase activity intrinsic to sarcoplasmic reticulum by 25-50%, using [32P]phosphorylase a or 32P-labeled membrane vesicles as a substrate for the phosphatase. Co-administration of acetylcholine markedly attenuated these effects of isoproterenol and forskolin. Acetylcholine alone caused a 50% increase in type 1 phosphatase activity. We concluded that inhibitor-1 and type 1 phosphatase can be regulated in intact cardiac muscle by agents that increase intracellular cAMP and by acetylcholine.     

A kinetic analysis of the effects of inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-1

The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase alpha as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitor-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn2+, and its dephosphorylation was inhibited competitively by inhibitor-2 (Kis = 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as a substrate (190 nM) was very much higher than its Ki as an inhibitor (1.5-7.5 nM). The results are consistent with a model in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase alpha. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exclusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase alpha and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results.     

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.     

Hormonal regulation of protein dephosphorylation. Identification and hormonal regulation of protein phosphatase inhibitor-1 in rat adipose tissue

An inhibitor (inhibitor-1) of phosphorylase a phosphatase has been identified in rat epididymal fat pads. This heat-stable, acid-soluble protein only exhibits phosphatase inhibitory activity when it itself is phosphorylated. Inhibitor-1 in rat adipose tissue migrates at 32,000 Da on sodium dodecyl sulfate-polyacrylamide gels, and at 64,000 Da on gel filtration. Exposure of fat pads to insulin (1 milliunit/ml) resulted in a 50% decrease in inhibitor-1 activity, compared to control (p less than 0.001). Isoproterenol (10(-6) M) caused a 25% increase in inhibitor-1 activity (p less than 0.05). Electrophoresis of heat-stable proteins prepared from hormone-treated 32P-labeled fat cells showed that insulin caused a dephosphorylation of the 32,000 Da phosphoprotein by 30% (p less than 0.01), whereas isoproterenol stimulated 32P incorporation in this protein by 35% compared to control (p less than 0.05). Thus, insulin appears to dephosphorylate and inactivate inhibitor-1, and might thereby result in an increase of protein phosphatase activity. Insulin regulation of inhibitor-1 is a mechanism which may underlie other of insulin's effects in adipose tissue, such as the activation of glycogen synthase.     

Reduced inhibitor 1 and 2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats

In failing hearts, although protein phosphatase type 1 (PP1) activity has increased, information about the regulation and status of PP1 inhibitor-1 (INH-1) and inhibitor-2 (INH-2) is limited. In this study, we examined activity and protein expression of PP1, INH-1 and INH-2 and phosphorylation of sarcoplasmic reticulum (SR) phospholamban (PLB), a substrate of PP1 and modulator of SR Ca2+-ATPase activity, in failing and non-failing hearts. These studies were performed in LV myocardium of seven rats with chronic renal hypertension produced by Goldblatt's one-kidney, one-clip procedure and seven age-matched sham-operated normal controls (CTR). Eight weeks after surgery, LV ejection fraction, LV hypertrophy, and pulmonary congestion were determined in all rats. PP1 activity (nmol 32P/min/mg non-collagen protein) was assessed in LV homogenates using 32P-labeled phosphorylase a as substrate. INH-1 and INH-2 activity was determined in the immunoprecipitate of LV homogenates and expressed as percentage inhibitory activity. Using a specific antibody, LV tissue levels of PP1C and calsequestrin (CSQ), a SR calcium binding protein, which is not altered in failing hearts, were also determined. Further, total and phosphorylated PLB, INH-1 and INH-2 protein levels were determined in the LV homogenate and phosphoprotein-enriched fraction, respectively. The band density of each protein was quantified in densitometric units and normalized to CSQ. Results: rats with chronic renal hypertension exhibited significantly reduced LV ejection fraction and increased LV hypertrophy and pulmonary congestion, characteristics of chronic heart failure (CHF). We found that compared to CTR, (1) both INH-1 (10.2+/-2 versus 57.5+/-1; p < 0.05) and INH-2 activity (3.8+/-0.4 versus 36.2+/-4; p < 0.05) were reduced, (2) total and phosphorylated PLB amount reduced, (3) protein level of phosphorylated INH-1 was reduced (2.32+/-0.1 versus 0.73+/-0.04; p < 0.05) whereas that of phosphorylated INH-2 increased (3.05+/-0.3 versus 1.42+/-0.1; p < 0.05), and (4) PP1 activity was increased approximately 2.6-fold in rats with CHF (1.59+/-0.05 versus 0.61+/-0.01; p < 0.05) while protein level of the catalytic subunit of PP1 (PP1C) increased 3.85-fold (0.77+/-0.05 versus 0.20+/-0.02; p < 0.05). These results suggest that reduced inhibitory INH-1 and INH-2 activity, increased PP1C protein level, and reduced PLB phosphorylation are associated with increased PP1 activity in failing hearts.     

Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells. The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase-1 from mammalian tissues, including preferential dephosphorylation of the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin-Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase-2A, including preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and 2, activation by protamine and heparin, and exclusion from heparin-Sepharose. An acid-stable and heat-stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor-2 from mammalian tissues. It inhibited protein phosphatase-1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase-1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase-3. The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1-methyladenine.     

The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...     

Features of glycogen phosphorylase from the body wall musculature of the lugworm Arenicola marina and the mode of activation during anoxia

The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 +/- 1.2 nmol . min-1 . (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 +/- 0.5 mumol . min-1 . (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5'-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex G-200 (Mr = 98000 +/- 4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The alpha-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the beta-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-1. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 microM Ca2+ in the absence, and at 0.5 microM Ca2+ in the presence, of 0.03 microM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half-maximal inhibition occurred at 45 microM in the absence and 35 microM in the presence of 0.03 microM calmodulin. The metabolic role of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin-binding protein of neural tissue termed calcineurin or CaM-BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84].     

Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.