Effect of ligands on Drosophila phosphorylase a as monitored by its enzymic inactivation

The dephosphorylation of Drosophila phosphorylase a with the catalytic subunit of fruit-fly protein phosphatase-1 was inhibited by AMP, IMP, ADP, ATP, glucose-6-P, glucose-1-P and UDPG. Glucose, caffeine and glycogen did not influence the reaction. The inhibitory effect of AMP was reduced by glucose and caffeine. The above ligands acted through the modification of phosphorylase a conformation. This conclusion was drawn from the ligands' effect on the dephosphorylation of phosphohistone by Drosophila phosphatase-1 and on the tryptic digestion of fruit-fly phosphorylase a.     

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)     

Effects of acidic and basic macromolecules on the activity of protein phosphatase-1

The dephosphorylation of phosphorylase a by the catalytic subunit of protein phosphatase-1 obtained from rabbit skeletal muscle is inhibited by heparin in a noncompetitive manner with respect to phosphorylase a (Ki = 8 micrograms/ml). The inhibitory effect of heparin is also observed in the presence of effectors (e.g., glucose and AMP) modifying the dephosphorylation of phosphorylase a. Heat-stable protein inhibitors of protein phosphatase-1 can develop their inhibitory effect of the activity of protein phosphatase-1 even in the presence of heparin. The inhibitory effect of heparin and the heat-stable inhibitor-2 of phosphatase is additive. Polybrene, a heparin antagonist, prevented phosphatase-1 from the inhibition caused by heparin or the inhibitors. Proteins with basic character, histone fractions (H1, H3) and protamine sulfate, can counteract with the inhibitory effect of heparin, but they cannot intercept the actions of inhibitor-1 or -2.     

The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein phosphatase-1, 2A and 2C but not by protein phosphatase-2B

The activity of hormone-sensitive lipase, the rate-limiting enzyme in adipose tissue lipolysis, is controlled by cAMP-mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Str?lfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317-3321]. The capacity of protein phosphatase-1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase-4 and cAMP-dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (alpha subunit). Protein phosphatase-1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone-sensitive lipase, while protein phosphatase-2B had no measureable activity towards any of the sites. When the activities of protein phosphatase-1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4. Protein phosphatase-1 showed 20% higher and protein phosphatase-2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase-2A and 2C, but relatively poor substrates for protein phosphatase-1. Protein phosphatase-2C activity towards the lipase was completely dependent on Mg2+ with a half-maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase-1 3-5-fold with half-maximal effect at 0.6 microgram/ml, and by protein phosphatase-2A about 2-fold with half-maximal effect at 3-5 micrograms/ml, thus illustrating the potential for control of these lipase phosphatase activities.     

The protein phosphatases involved in cellular regulation. 1. Modulation of protein phosphatases-1 and 2A by histone H1, protamine, polylysine and heparin

The phosphorylase phosphatases in rat and rabbit liver cytosol that are markedly stimulated by histone H1, protamine and polylysine were identified as protein phosphatases-2A0, 2A1 and 2A2 by anion-exchange chromatography, gel-filtration and immunotitration experiments. Histone H1 and protamine also stimulated the dephosphorylation of phosphorylase kinase, glycogen synthase, fructose-1,6-bisphosphatase, pyruvate kinase, acetyl-CoA carboxylase and phenylalanine hydroxylase by phosphatases-2A1 and 2A2, and with several of these substrates activation was even more striking (20-100-fold) than that observed with phosphorylase (approximately 5-fold). Activation by basic polypeptides did not involve dissociation of these phosphatases to the free catalytic subunit. The dephosphorylation of phosphorylase by protein phosphatase-1 was suppressed by basic polypeptides, protamine and polylysine being the most potent inhibitors. However, the dephosphorylation of glycogen synthase, pyruvate kinase and acetyl-CoA carboxylase were markedly stimulated by histone H1 and protamine (2-13-fold). Consequently, with the appropriate substrates, protein phosphatase-1 can also be regarded as a basic-polypeptide-activated protein phosphatase. Heparin stimulated (1.5-2-fold) the dephosphorylation of phosphorylase by phosphatases-2A0 and 2A1, provided that Mn2+ was present, but phosphatase-2A2 and the free catalytic subunit of phosphatase-2A were unaffected. Heparin, in conjunction with Mn2+, also stimulated (1.5-fold) the dephosphorylation of glycogen synthase (labelled in sites 3 abc), phosphorylase kinase and phenylalanine hydroxylase by phosphatase-2A1, but not by phosphatase-2A2. By contrast, the dephosphorylation of phosphorylase and phosphorylase kinase by protein phosphatase-1 was inhibited by heparin. However, dephosphorylation of glycogen synthase and pyruvate kinase by phosphatase-1 was stimulated by this mucopolysaccharide. The studies demonstrate that basic proteins can be used to distinguish protein phosphatase-1 from protein phosphatase-2A, but only if phosphorylase is employed as substrate. Optimal differentiation of the two phosphatases is observed at 30 micrograms/ml protamine or at heparin concentrations greater than 150 microM.     

Glucose and caffeine regulation of liver glycogen phosphorylase activity in the freeze-tolerant wood frog Rana sylvatica

We have examined the effect of glucose and caffeine inhibition on the activity of liver glycogen phosphorylase a from the freeze-tolerant frog Rana sylvatica. Kinetic studies indicate that this enzyme exhibits similar sensitivity to glucose inhibition (glucose dissociation constant = 12.5 mM) as the mammalian enzyme. Little inhibition (less than 25%) was observed at normal glucose concentrations (1-5 mM), while significant inhibition (60-95%) occurred at glucose concentrations (50-500 mM) present in freezing-exposed animals. These results favour the hypothesis that in the normal state glucose regulates phosphorylase activity primarily through the promotion of dephosphorylation of phosphorylase a, whereas during freezing regulation is achieved through phosphorylase a inactivation. The caffeine dissociation constant (0.93 mM) and the degree of synergism between caffeine and glucose (interaction factor, alpha = 0.14) were also similar to that observed for the mammalian enzyme. Hence, if a caffeine-like ligand exists in vivo, it must be in low enough amounts during freezing to allow sufficient phosphorylase a activity for high glucose production.     

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.     

Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A

The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.     

Phosphorylase a is an allosteric inhibitor of the glycogen and microsomal forms of rat hepatic protein phosphatase-1

The dephosphorylation of glycogen synthase by protein phosphatase-1 in hepatic glycogen and microsomes was inhibited by nanomolar concentrations of phosphorylase a. The I50 for phosphorylase a was 1000-fold lower than its Km as a substrate, while tryptic digestion increased the I50 1000-fold without affecting Km. Protein phosphatase-1 from skeletal muscle and protein phosphatase-2A from liver were only inhibited at 1000-fold higher concentrations. Protein phosphatase-1 became desensitized to phosphorylase a when released from hepatic microsomes, but sensitivity was partially restored by readdition of the solubilized enzyme to the microsomes. The results demonstrate that phosphorylase a is a potent allosteric inhibitor of hepatic protein phosphatase-1 and suggest that inhibition may be conferred by a novel phosphorylase a-binding subunit.     

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.     

Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mm glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mm glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or alpha-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nm insulin, 30 mm potassium chloride, or 0.25 mm diazoxide, indicating that insulin secretion and/or depolarization of beta cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.     

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.     

The protein phosphatases of Drosophila melanogaster and their inhibitors

Protein phosphatases-1, 2A and 2B have been identified in membrane and soluble fractions of Drosophila melanogaster heads. Similarities between Drosophila and mammalian protein phosphatase-1 included specificity for the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition by protamine, retention by heparin-Sepharose and selective interaction with membranes. In addition, an inactive form of protein phosphatase-1, termed protein phosphatase-1I, was detected in the soluble fraction that could be activated by preincubation with MgATP and mammalian glycogen synthase kinase-3. Inhibitor-2 partially purified from Drosophila had an identical molecular mass to its mammalian counterpart, and recombined with mammalian protein phosphatase-1 to form a hybrid protein phosphatase-1I. Similarities between Drosophila and mammalian protein phosphatase-2A included preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and -2, activation by protamine, exclusion from heparin-Sepharose and apparent molecular mass. A Ca2+-dependent calmodulin-stimulated protein phosphatase (protein phosphatase-2B) that was inhibited by trifluoperazine was identified in the soluble fraction. The remarkable similarities between Drosophila protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation and demonstrate that the procedures used to classify mammalian protein phosphatases have a wider application. Characterisation of the Drosophila phosphatases will facilitate genetic analysis of dephosphorylation systems and their possible roles in neuronal and behavioural plasticity in Drosophila.     

Skeletal muscle glycogen phosphorylase a kinetics: effects of adenine nucleotides and caffeine.

This study aimed to determine physiologically relevant kinetic and allosteric effects of P(i), AMP, ADP, and caffeine on isolated skeletal muscle glycogen phosphorylase a (Phos a). In the absence of effectors, Phos a had Vmax = 221 +/- 2 U/mg and Km = 5.6 +/- 0.3 mM P(i) at 30 degrees C. AMP and ADP each increased Phos a Vmax and decreased Km in a dose-dependent manner. AMP was more effective than ADP (e.g., 1 microM AMP vs. ADP: Vmax = 354 +/- 2 vs. 209 +/- 8 U/mg, and Km = 2.3 +/- 0.1 vs. 4.1 +/- 0.3 mM). Both nucleotides were relatively more effective at lower P(i) levels. Experiments simulating a range of contraction (exercise) conditions in which P(i), AMP, and ADP were used at appropriate physiological concentrations demonstrated that each agent singly and in combination influences Phos a activity. Caffeine (50-100 microM) inhibited Phos a (Km approximately 8-14 mM, approximately 40-50% reduction in activity at 2-10 mM P(i)). The present in vitro data support a possible contribution of substrate (P(i)) and allosteric effects to Phos a regulation in many physiological states, independent of covalent modulation of the percentage of total Phos in the Phos a form and suggest that caffeine inhibition of Phos a activity may contribute to the glycogen-sparing effect of caffeine.     

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.     

Inhibition of rabbit muscle glycogen phosphorylase by D-gluconohydroximo-1,5-lactone-N-phenylurethane

The effect of the beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone-N-phenylurethane (PUG) on the kinetic and ultracentrifugation properties of glycogen phosphorylase has been studied. Recent crystallographic work at 2.4 A resolution [D. Barford et al. (1988) Biochemistry 27, 6733-6741] has shown that PUG binds in the catalytic site of phosphorylase b crystals with its gluconohydroximolactone moiety occupying a position similar to that observed for other glucosyl compounds and the N-phenylurethane side chain fitting into an adjacent cavity with little conformational change in the enzyme. In solution, PUG was shown to be a potent inhibitor of phosphorylase b, directly competitive with alpha-D-glucopyranose 1-phosphate (glucose-1-P) (Ki = 0.40 mM) and noncompetitive with respect to glycogen and AMP. When PUG was tested for synergistic inhibition in the presence of caffeine, the Dixon plots of reciprocal velocity versus PUG concentration at different fixed caffeine concentrations provided intersecting lines with interaction constant (alpha) values of 0.95-1.38, indicating that the binding of one inhibitor is not significantly affected by the binding of the other. For glycogen phosphorolysis, PUG was noncompetitive with respect to phosphate, suggesting that it can bind to the central enzyme-AMP-glycogen-phosphate complex. PUG was shown to inhibit phosphorylase alpha (without AMP) activity (Ki = 0.43 mM) in a manner similar to that of the b form. However, in the presence of AMP, PUG exhibited complex kinetics, acting as a noncompetitive inhibitor with respect to glucose-1-P, while a twofold decrease of PUG binding to the enzyme-AMP-glycogen complex was observed. Ultracentrifugation experiments demonstrated that PUG does not cause any significant dissociation of phosphorylase alpha tetramer. Furthermore the dimerization of phosphorylase alpha by glucose is completely prevented in the presence of PUG. These observations are consistent with PUG binding to both the R and the T conformations of phosphorylase.     

Activity of protein phosphatases against initiation factor-2 and elongation factor-2.

The protein phosphatases active against phosphorylase a, elongation factor-2 (EF-2) and the alpha-subunit of initiation factor-2 (eIF-2) [eIF-2(alpha P)] were studied in extracts of rabbit reticulocytes. Swiss-mouse 3T3 fibroblasts and rat hepatocytes, by use of the specific phosphatase inhibitors okadaic acid and inhibitor proteins-1 and -2. In all three extracts tested, both phosphatase-1 and phosphatase-2A contributed to overall phosphatase activity against phosphorylase and eIF-2(alpha P), but phosphatase-2B and -2C did not. In contrast, only protein phosphatase-2A was active against EF-2. Furthermore, in hepatocytes there was substantial type-2C phosphatase activity against EF-2, but not against phosphorylase or eIF-2 alpha. These findings in cell extracts were borne out by data obtained by studying the activities of purified protein phosphatase-1 and -2A against eIF-2(alpha P) and eIF-2(alpha P) was a moderately good substrate for both enzymes (relative to phosphorylase a). In contrast, EF-2 was a very poor substrate for protein phosphatase-1, but was dephosphorylated faster than phosphorylase a by protein phosphatase-2A. The implications of these findings for the control of translation and their relationships to previous work are discussed.     

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 1. Phosphorylation by cAMP-dependent protein kinase at site 2 releases catalytic subunit from the glycogen-bound holoenzyme

The glycogen-associated form of protein phosphatase-1 (PP-1G) is a heterodimer comprising a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit, the latter being phosphorylated by cAMP-dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C-terminal to site 1 in the primary structure. The sequence in this region is: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 4000-fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate-limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti-site-1 Fab fragments to glycogen-bound PP-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type-2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the alpha subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type-2A and type-2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10-fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP-1 activity towards the glycogen-metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen--protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin-stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP-1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.     

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.     

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A_0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a. Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction (A_0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 mm) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.