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. Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle

A type-1 protein phosphatase (protein phosphatase-1G) was purified to homogeneity from the glycogen-protein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1C) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels. Protein phosphatase-1G was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-1C, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-1G was inactivated very rapidly by inhibitor-1. Protein phosphatase-1G interacted with inhibitor-2 (I-2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3. The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-1G by inhibitor-1. The relationship of the G subunit to the 'deinhibitor protein' is discussed.     

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].     

The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle

Protein phosphatases-2A0, 2A1 and 2A2 have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase-2A0 and 2A1, and 0.5 mg of phosphatase-2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases-2A0 and 2A1 each comprised three subunits, termed A, B' and C (2A0) or A, B and C (2A1), while phosphatase-2A2 contained only two subunits, A and C. The A and C components of phosphatases-2A0, 2A1 and 2A2 had indistinguishable mobilities on sodium dodecyl sulphate/polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase-2A purified from ethanol-treated muscle extracts. The electrophoretic mobilities of the B and B' subunits were slightly different, and their peptide maps were distinct. The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 +/- 6 kDa (2A0), 202 +/- 6 kDa (2A1) and 107 +/- 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B') and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2 (2A0), ABC2 (2A1) and AC (2A2). Protein phosphatase-2A2 appears to be derived from 2A0 and/or 2A1 during purification through degradation or dissociation of the B' and/or B subunits. Protein phosphatases-2A0, 2A1 and 2A2 were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5 = 0.25 microM), histone H1 (A0.5 = 0.3 microM) and polylysine (A0.5 = 0.04 microM). Activation by protamine varied over 5-20-fold for phosphatase-2A0 and 5-7-fold for phosphatases-2A1 and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5-10-fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2-fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B' subunits, or substrates phosphorylase and glycogen synthase.     

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...     

Complete primary structure of protein phosphatase inhibitor-1 from rabbit skeletal muscle

The complete primary structure of protein phosphatase inhibitor-1 has been determined. The protein consists of a single polypeptide chain of 165 residues, molecular weight 18640. The threonine residue that must be phosphorylated for activation is at position 35 and the active cyanogen bromide peptide, CB-1, comprises residues 2-66. The N-terminal methionine is acetylated and 40% of the inhibitor-1 molecules lack the C-terminal dipeptide Ala-Val. Serine-67 is substantially phosphorylated in vivo, but this phosphoserine residue does not appear to influence the activity of inhibitor-1.     

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.     

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

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

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 protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

PCR cloning of the cDNA of rabbit skeletal muscle protein phosphatase inhibitor-2.

The coding region of the cDNA of protein phosphatase inhibitor-2 was determined by polymerase chain reaction amplification. The cDNA clone consisted of 621 nucleotides, and encoded 204 amino acids. The deduced amino-acid sequence was identical with that of the sequence reported by chemical sequencing methods.     

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.     

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta subunit of phosphorylase kinase [Cohen, P., Watson, D.C. and Dixon, G.H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.     

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...     

The heat labile phosphatase modulator (inhibitor-2) complex from rabbit skeletal muscle

The heat stable phosphatase modulator protein (inhibitor-2) has been shown to play a crucial role in the reversible ATP, Mg-dependent activation of a multisubstrate protein phosphatase. The modulator activity is acid and heat stable and resides in a small asymmetrical protein which, after boiling migrates in sucrose density gradient centrifugation with a molecular weight of 17K. The present report shows that in unboiled rabbit skeletal muscle preparations all the modulator activity is found associated with a heat labile protein component, which imposes an important regulatory feature on the heat stable activity. The heat labile complex migrates in sucrose density gradient centrifugation as a M_r = 70K protein.     

The protein phosphatases involved in cellular regulation. Influence of polyamines on the activities of protein phosphatase-1 and protein phosphatase-2A

The effects of polyamines on the oligomeric forms of protein phosphatase-1 (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2AC) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1-2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8-15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6-7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0, 2A1 and 2A2 was greater than that observed with 2AC. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3-3-fold with protein phosphatase-2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pronounced with protein phosphatase-2AC than with 2A0, 2A1 and 2A2. Spermine (I50 = 0.1 mM with protein phosphatase-2AC) was a more potent inhibitor than spermidine (I50 = 0.9 mM) or putrescine (I50 = 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar manner to the homogeneous enzymes from rabbit skeletal muscle. Spermine did not activate protein phosphatase-1 to the same extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1-2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1C. With four other substrates maximal activation by spermine was less than 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50 = 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50 = 0.9 mM) or putrescine (I50 = 9 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Rabbit skeletal muscle protein phosphatases C-I and C-II have been previously isolated as two proteins of Mr = approximately 35,000. Both enzymes display broad substrate specificities but have distinct enzymatic properties in regard to their susceptibility to heat-stable protein inhibitor-2 and their response to divalent cations. Monoclonal antibodies against both protein phosphatase C-I and C-II were produced by fusion of spleen cells of immunized BALB/c mice with SP2/0-Ag14 mouse myeloma cells. The products of the hybrid cells were screened by solid phase radioimmunoassay for the production of antibodies to protein phosphatase C-I and C-II. Positive cells were cloned and injected into mice to produce ascitic fluids. Ten monoclonal antibodies against phosphatase C-I and eight monoclonal antibodies against phosphatase C-II were obtained. These antibodies were characterized with regard to their relative binding affinities to the two protein phosphatases and their abilities to inhibit the phosphorylase phosphatase activities of the two enzymes. All ten of the phosphatase C-I monoclonal antibodies inhibited the phosphorylase phosphatase activity of phosphatase C-I, and three of these also inhibited phosphatase C-II. Only one of the eight antibodies to phosphatase C-II was inhibitory and inhibited the activities of both phosphatase C-I and C-II. Examination of the binding of these monoclonal antibodies by a solid phase radioimmunoassay showed that eight of the ten phosphatase C-I antibodies cross-reacted with phosphatase C-II, while all eight of the phosphatase C-II antibodies cross-reacted with phosphatase C-I. These findings show that phosphatases C-I and C-II possess common antigenic determinant(s) and may, therefore, be structurally related proteins.     

Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.