Polycation-stimulated Protein Phosphatase Research Articles

Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing

The trimeric form of protein phosphatase 2A consisting of 36-, 65-, and 72-kDa subunits (previously termed polycation-stimulated protein phosphatase M) was purified from rabbit skeletal muscle. Amino acid sequence data of the 72-kDa regulatory subunit (termed PR72) were used to isolate cDNAs from human heart and fetal brain libraries and libraries derived from WI-38 and MCF-7 cells. The clones isolated from the heart cDNA library revealed an open reading frame encoding a protein with a predicted molecular mass of 62 kDa. All the peptides sequenced from the protein matched with the sequence predicted from the cDNA. However, in vitro transcription and translation from this cDNA yielded a protein with an apparent molecular mass of 72 kDa on sodium dodecyl sulfate-polyacrylamide gels. From brain we isolated cDNA clones spanning an open reading frame encoding a 130-kDa protein (termed PR130). The apparent molecular mass of the protein produced by in vitro transcription and translation was 130 kDa. This protein has exactly the same deduced C-terminal protein sequence as the PR72 subunit from amino acids 45 to 527 but has an N-terminal extension of 665 amino acids. It is likely, therefore, that these two proteins arise from the same gene by alternative splicing. In human tissues several transcripts were detected by Northern analysis generated probably by the use of different polyadenylation signals and alternative splicing. High levels of the PR72 mRNAs were detected in heart and muscle, while lower levels of PR130 transcripts were found in heart, brain, placenta, lung, muscle, and kidney.

Specificity of the polycation-stimulated (type-2A) and ATP,Mg-dependent (type-1) protein phosphatases toward substrates phosphorylated by P34cdc2 kinase.

p34cdc2 kinase, a critical regulator of the cell cycle, has been shown to recognize the consensus sequence S/TP in proteins such as histone H1, the retinoblastoma gene product RB and the carboxyl-terminal domain of eukaryotic RNA polymerase II. Using phosphorylated synthetic peptides, representing the p34cdc2 phosphorylation sites in these proteins and histone H1 protein as substrates, we investigated the substrate specificity of the different oligomeric forms of the polycation-stimulated (PCS/type-2A) protein phosphatase and the active catalytic subunit of the ATP,Mg-dependent (AMDc/type 1) protein phosphatase. The results show that the oligomeric structure of the PCS phosphatases is an important determinant for efficient dephosphorylation. The trimeric PCSH1 and PCSM phosphatases are about 10-20-fold-better histone H1 phosphatases than the dimeric PCSH2 and PCSL phosphatases and about 100-fold better than the catalytic subunit (PCSC), suggesting a regulatory role for the 72-kDa, 65-kDa and 55-kDa subunits. The RB peptide = INGS(P)PRT(P)PRRGQNR, is preferred over phosphorylase a (8-fold) by the PCSH1 phosphatase and is about a 40-fold and 95-fold-better substrate for the PCSH1 phosphatase than for the PCSM and PCSL phosphatases, respectively. The primary structure surrounding the S/T(P)P motif, by itself a strong negative determinant for dephosphorylation, can harbour positive features which relieve the constraint imposed by the carboxyl-terminal proline. Thus, the RB peptide INGS(P)PRT(P)PRRGQNR, in which the T(P)P configuration is preferred over the S(P)P sequence, is an extremely good and specific substrate for the PCSH1 phosphatase (Km = 10 microM, Vmax = 3882 nmol.min-1.mg-1). The AMDC phosphatase is a poor phosphatase for all the phosphopeptides tested, unless Mn2+ is added. Its histone H1 phosphatase activity is much less sensitive than its phosphorylase a and phosphopeptide phosphatase activity to inhibition by the modulator or inhibitor-1. The results strongly suggest a role for the trimeric PCSH1 phosphatase in reversing the p34cdc2 phosphorylations.

Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform

The trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.

.alpha.- And .beta.-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure

Protein phosphatase 2A (polycation-stimulated protein phosphatase L) was purified from porcine kidney and skeletal muscle. The 36-kDa catalytic and the 65-kDa putative regulatory (hereafter termed PR65) subunits of protein phosphatase 2A2 were separated by reverse-phase HPLC. Partial amino acid sequence data (300 residues) was obtained for PR65. Molecular cloning showed that two distinct mRNAs (termed alpha and beta) encoded the PR65 subunit. The cDNA encoding the alpha-isotype spanned 2.2 kilobases (kb) and contained an open reading frame of 1767 bases predicting a protein of 65 kDa, which was in good agreement with the size of the purified protein. The cDNAs encoding the beta-isotype contained an open reading frame of size similar to that of alpha-form but lacked an initiator ATG. Northern analysis, using RNA isolated from several human cell lines, indicated that the alpha-isotype was encoded by a mRNA of 2.4 kb that was much more abundant than the beta mRNA of 4.0 kb. Comparison of the predicted amino acid sequences of the two isotypes revealed 87% identity. The deduced protein sequences of the alpha- and beta-isotypes were found to be made up of 15 imperfect repeating units consisting of 39 amino acids. This repeating structure was conserved between species.

Synthetic peptides as model substrates for the study of the specificity of the polycation‐stimulated protein phosphatases

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.

Phosphotyrosyl phosphatase activity of the polycation-stimulated protein phosphatases and involvement of dephosphorylation in cell cycle regulation

Phosphotyrosyl phosphatase activity of the polycation-stimulated protein phosphatases and involvement of dephosphorylation in cell cycle regulation

Phosphorylation/dephosphorylation of the β light chain of clathrin from rat liver coated vesicles

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.

Identification of the ATP + Mg-dependent and polycation-stimulated protein phosphatases in the germinal vesicle of the Xenopus oocyte

Two protein phosphatase activities were characterized in the germinal vesicle of Xenopus laevis oocytes after manual dissection of the nucleus. One enzyme can be classified as an active form of the ATP + Mg-dependent (AMD) phosphatase, the other as a polycation-stimulated (PCS) phosphatase. The activity of the PCS phosphatase is localized exclusively in the soluble compartment of the nucleus (nucleoplasm). The catalytic subunit of the AMD phosphatase activity is associated either with the nuclear particulate fraction or with an inhibitory subunit in the nucleoplasm.

Stimulation of the ATP, Mg-dependent protein phosphatase by p-nitrophenyl phosphate

The phosphorylase phosphatase activity of the ATP, Mg-dependent protein phosphatase is stimulated by p-nitrophenyl phosphate ( pNPP). All the active forms of this type of enzyme show this property, which seems to be unrelated to any pNPP-hydrolyzing activity. The increase in activity is due to an increase in V m, the K m being unchanged. The possibility that pNPP acts as a deinhibitor is excluded. pNPP acts as a competitive inhibitor on the phosphorylase phosphatase activity of the different polycation-stimulated protein phosphatases. Stimulation by pNPP can be used as a differential criterion in a specific assay of the active forms of the ATP,Mg-dependent phosphatase.

Modulation of the substrate specificity of the polycation-stimulated protein phosphatase from Xenopus laevis oocytes

A polycation-stimulated (PCS) protein phosphatase was isolated in high yield (280 micrograms/100 g ovaries) from Xenopus laevis oocytes through a procedure involving a tyrosine-agarose hydrophobic chromatography. The 220-kDa enzyme contains a 35-kDa and a 62-kDa subunit. It was identified as the low-Mr polycation-stimulated (PCSL) protein phosphatase. The labile p-nitrophenyl phosphatase activity, copurifying with the phosphorylase phosphatase activity, can be increased severalfold by preincubating the purified enzyme with ATP, its analogues or PPi. This activation is time-dependent and accompanied by a parallel decrease of the phosphorylase phosphatase activity. Although the stimulation was antagonized by metal ions during the preincubation, the basal and ATP-stimulated p-nitrophenyl phosphatase requires Mg2+ or Mn2+ in the assay, with pH optima of 8.5-9 and 7.5 respectively.

Identification of pseudo ‘phosphothreonyl-specific’ protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [ 32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565–8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255–261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the α-subunit of phosphorylase kinase. The M r of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2A o of protein phosphatase 2A.

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.

The polycation-stimulated protein phosphatases: regulation and specificity

Four classes of protein phosphatases are presumed to play an important role in dephosphorylating the major proteins involved in the control of general metabolism. Based on the enzyme-directed regulation of activity they have been classified as ATP,Mg-dependent-, polycation-stimulated-, Mg 2+-dependent protein phosphatases and calcineurin. We have recently purified from rabbit skeletal muscle four distinct PCS protein phosphatases, classified according to the apparent molecular weight of the native enzymes in gel filtration at an early stage of the purification as: PCS H (390 kDa), PCS M (250 kDa) and PCS L (200 kDa) phosphatases. The PCS H phosphatase could be resolved into a 3(65 : 55 35 kDa)-subunit PCS H1 phosphatase and a 2(65 : 35 kDa)-subunit PCS H2 enzyme probably derived from the PCS H1 phosphatase, both characterized as specific deinhibitor phosphatases. PCS M phosphatase, a 3(72 : 65 35 kDa)-subunit enzyme, shows a high degree of stimulation with a low concentration optimum of polycations and is sensitive to a Ca 2+-dependent protease, which brings about a five- to ten-fold increase in inhibitor-1 phosphatase activity. PCS L phosphatase is characterized by a 2(65 : 35 kDa)-subunit structure, a low intrinsic deinhibitor phosphatase activity and a low degree of stimulation of phosphorylase phosphatase activity requiring high concentrations of polycations. At low concentrations of polycations the stimulation of phosphorylase phosphatase activity of the PCS enzymes is enzyme-directed, since it occurs at concentrations far below the substrate concentration. The degree of stimulation is also typical for each type of enzyme (PCS M > PCS H1 > PCS H2 > PCS L > PCS C) and dependent on the polycation used; at the optimum concentration the most effective polycations (polylysine, protamine, histone H1) stimulate the phosphorylase phosphatase activity to about the same extent. Polycation concentrations above the optimum are less effective on phosphorylase phosphatase activity and can even become inhibitory to the basal activity. Whether this effect is enzyme- or substrate-directed (or both) is not known. The stimulation by polycations could be completely lost following preincubation of the PCS phosphatase with polycations. This deactivation is time-, temperature- and concentration-dependent. However the polycations did not affect the basal phosphorylase phosphatase activity. In addition to phosphorylase a and inhibitor-1, casein, myosin light chains and phosphorylase b kinase (α-subunit) are choice substrates for these enzymes. Protamine, histone H1 and polylysine also stimulate the dephosphorylation of phosphocasein, phosphorylated myosin light chains and the α-subunit of phosphorylase kinase by the PCS phosphatases. The polycation stimulation of inhibitor-1 dephosphorylation was always less prominent (up to two-fold) and absent with the deinhibitor or synthetic peptides as substrates. Paranitrophenylphosphate phosphatase activity of the PCS phosphatases is extremely low and cannot be stimulated by polycations. Unlike the catalytic subunit of the ATP,Mg-dependent protein phosphatase, the PCS phosphatases exhibit a remarkable preference for the phosphothreonyl- over the phosphoseryl peptides. The catalytic subunits isolated from the three basic types of PCS phosphatases are apparently identical. The migration of the catalytic subunits obtained from the different types of PCS phosphatases in sucrose density gradient centrifugation was identical (37–41 kDa). In contrast with the holoenzymes they are less sensitive to polycation stimulation, showing an identical concentration optimum and the stimulation by metal ions is lost.

Catalytic subunit of the polycation-stimulated protein phosphatase: Effect of proteolysis on polycation stimulation

The phosphorylase phosphatase activity of the holoenzyme form of phosphatase 2A isolated from extracts of porcine renal cortex or bovine heart was stimulated 600% and 500%, respectively, by the addition of histone H1. After conversion of the phosphatase to the catalytic subunit form by treatment with ethanol at room temperature, histone H1 stimulated activity by about 150% only. Purification of the catalytic subunit from porcine renal cortex yielded two forms of the enzyme which were separated by heparin-Sepharose chromatography. These forms were designated peak 1 and peak 2 according to their order of elution from the column. Peak 1 catalytic subunit was stimulated by more than 400% by histone H1, whereas peak 2 was stimulated by about 50% only. Based on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, peak 2 had a slightly higher M r value than peak 1 (35 500 vs. 35 000). Incubation of the peak 2 phosphatase with trypsin converted it to a form that was similar to peak 1 with respect to M r and stimulation by histone H1. When the catalytic subunit of phosphatase 2A was purified from bovine heart only one form was obtained. Bovine heart enzyme was similar to renal peak 2 in that it had an apparent M r of 35 500 and was only slightly stimulated by histone H1. Treatment of the bovine heart catalytic subunit with trypsin, chymotrypsin or type 2 Ca 2+-dependent proteinase decreased the apparent M r by about 500 and increased histone H1 stimulation to about 500%. Thus, when a small peptide was removed by proteolysis, histone H1 stimulation of the ‘nicked’ catalytic subunit was similar to that obtained with the holoenzyme.

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.

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35 000 both before and after 4 M urea treatment. Its activity was specific for the β-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 ( A 0.5 = 0.6 μM). This enzyme had a molecular weight of approx. 70 000 which was reduced to approx. 35 000 after treatment with 4 M urea. It dephosphorylated both the α- and β-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

The predominant of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of β-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent M r of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly( d-lysine), poly( l-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the α-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.

Polycation-stimulated protein phosphatases from rabbit skeletal muscle

Polycation-stimulated protein phosphatases from rabbit skeletal muscle

Activation of the PCS M-protein phosphatase by a Ca 2+-dependent protease

The inhibitor-1 phosphatase but not the phosphorylase phosphatase activity of a newly discovered 250 kDa polycation-stimulated (PCS M) protein phosphatase in rabbit skeletal muscle is increased up to 10-fold by a Ca 2+-dependent protease. The enzyme-directed protease effect to which the PCS H and PCS L. phosphatases are insensitive was progressively lost during purification of the enzyme. This could be explained by either a slow conversion of the enzyme to an active form of the enzyme with a change in specificity, or the loss of a protease-sensitive inhibitor of the inhibitor-1 phosphatase activity, resulting in a PCS phosphatase characterized by its high inhibitor-1/phosphorylase a activity ratio. The Ca 2+-dependent protease is completely inhibited by EGTA or leupeptin.

Dephosphorylation of the deinhibitor protein by the PCS H protein phosphatase

The deinhibitor protein, responsible for the decreased sensitivity of the ATP, Mg-dependent protein phosphatase to inhibitor-1 and the modulator protein, is inactivated by cyclic AMP-dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP, Mg-dependent phosphatase in its activated or spontaneously active form, four different forms of polycation-stimulated phosphatases (PCS H, PCS M, PCS L and PCS C) and calcineurin. Only the high - M r, polycation-stimulated protein phosphatase (PCS H), but not its catalytic subunit (PCS C), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCS H is affected neither by polycations nor by Mn ions.