Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.     

Latent forms of type-1 protein phosphatase in rabbit skeletal muscle

We have examined the characteristics of partially purified forms of rabbit skeletal muscle type-1 protein phosphatase (PP-1). Over 90% of PP-1 in unfractionated extracts and in glycogen particles was inactive, but could be activated by the divalent cations, Mn2+ or Co2+ (Me2+) plus trypsin. Gel filtration of muscle extracts revealed two inactive forms of PP-1; one activated by Me2+ alone or Me2+ plus trypsin, and a second containing inhibitor-2 and activated only by Me2+ plus trypsin. The kinetics of Me2+ plus trypsin activation revealed that after DEAE-chromatography, PP-1 was altered from the forms in crude extracts, gel filtered extracts or glycogen particles. The results indicate that the purified form of PP-1 catalytic subunit has properties which distinguish it from the forms of the enzyme in muscle extracts.     

Purification of a latent protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.     

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.     

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.     

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.     

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.     

Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase

The small molecular weight (± 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase F_A. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.     

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine

Summary The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (K_i value of 0.3mm) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylasea.     

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeletal muscle

Summary A heat- and acid-stable proten inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does not occur when phosphorylated histone or phosphorylated protamine are used as substrates.     

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.     

Identification of the sites on rabbit skeletal muscle protein phosphatase inhibitor-2 phosphorylated by casein kinase-II

Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C-terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2.     

Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver

Protein phosphatase 2C was isolated from rabbit skeletal muscle by a procedure that involved chromatography on DEAE-cellulose, precipitation with ammonium sulphate, gel-filtration on Sephadex G-100, affinity chromatography on thiophosphorylated myosin-P-light-chain--Sepharose and chromatography on Mono Q. The enzyme was purified about 35,000-fold and 0.3-0.4 mg was isolated from 2500 g skeletal muscle within 5 days. The final step resolved the activity into two peaks, termed protein phosphatases 2C1 and 2C2, that possessed identical substrate specificities and enzymatic properties. About 2.5-fold more protein phosphatase 2C2 was isolated than protein phosphatase 2C1. Protein phosphatases 2C1 and 2C2 migrated as single bands on SDS/polyacrylamide gels yielding apparent molecular masses of 44 kDa and 42 kDa, respectively, and the native proteins were both monomeric at pH 7.5 as judged by their elution from Sephadex G-100 and Sephacryl S200. Peptide maps of protein phosphatases 2C1 and 2C2, obtained after separate digestions with four different proteinases, were different, indicating that they are isoenzymes. Protein phosphatases 2C1 and 2C2 were purified from rabbit liver by the same procedure, and 0.2 mg (2C1 + 2C2) was isolated from 120 g hepatic tissue. Hepatic protein phosphatases 2C1 and 2C2 were also isolated in a molar ratio of about 1:2.5, and their enzymatic properties and apparent molecular masses in the presence and absence of SDS were identical to the skeletal muscle enzymes. Protein phosphatases 2C1 from muscle and liver displayed identical peptide maps, as did protein phosphatases 2C2 from these two tissues. It is concluded that the same two isoenzymes of protein phosphatase 2C are present in skeletal muscle and liver.     

Histone H1-stimulated phosphorylase phosphatase from rabbit skeletal muscle

A major rabbit skeletal muscle phosphorylase phosphatase activity which is markedly stimulated by histone H1 has been resolved from inhibitor-sensitive phosphorylase phosphatase (type-1 phosphatase), glycogen synthase kinase 3-activated phosphatase, phosphatase heat-stable inhibitor proteins, and alkaline phosphatase activity by various purification techniques. Evidence is presented that this phosphatase is a high-molecular weight form of a type-2 phosphatase. Our data suggest that this phosphatase may be regulated by histone H1, protamine or analogous polycationic compounds.     

Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone