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The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and is regulated by the opposing actions of protein kinases and protein phosphatases. Protein kinases in the lens system have been well studied, but very little is known about lens protein phosphatases. Protein phosphatases can be divided several families, such as protein phosphatase types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C) and protein tyrosine phosphatases (PTP). In this study we evaluated what kinds of protein phosphatases are present in the lens by using various specific substrates and inhibitors. Samples were prepared from lenses of 17-day-old chick embryos, and fractionated by high-resolution gel permeation column chromatography, then the fractions were assayed for phosphatase activities. The results with 32P-labeled glycogen phosphorylase A, okadaic acid and inhibitor-1, which are a specific substrate and inhibitors of PP1 and/or PP2A, showed that PP1activities were present in the 500-, 115- and 45-kDa fractions of the lens protein. The 115-kDa fraction also contained PP2A activity. By using a phosphothreonine-containing peptide as a substrate, three peaks of phosphatase activities were found at around 115, 55 and 35 kDa. Based on their response to various phosphatase inhibitors and their metal dependency, the fractions of 115 and 35 kDa were concluded to contain PP2A, while the 55-kDa fraction contained PP2C. Immunoblot using specific antibodies against PP1, PP2A and PP2C confirmed that each fraction above contained corresponding protein phosphatases as proteins. When a phosphotyrosine-containing peptide substrate was examined at pH 7.4, we observed a major peak at 500 kDa, which was presumed to contain receptor-like PTP(s). On the other hand, at pH 5.5, we observed a peak of 18 kDa, which was confirmed to contain a low-molecular-weight PTP. These protein phosphatases have recently been suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are of much interest.
The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.
Inositol polyphosphate 4-phosphatase (4-phosphatase) is a Mg2+-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 3,4-bisphosphate. We have isolated cDNA encoding a 105,257-Da protein that is 37% identical to the previously cloned 4-phosphatase. Recombinant protein was expressed in Escherichia coli and shown to hydrolyze all three 4-phosphatase substrates with enzymatic properties similar to the original enzyme. We designate the original 4-phosphatase and the new isozyme as inositol polyphosphate 4-phosphatase types I and II, respectively. 4-Phosphatase II is highly conserved with the human and rat enzymes having 90% amino acid identity. A conserved motif between 4-phosphatase I and II is the sequence CKSAKDRT that contains the Cys-Xaa5-Arg active site consensus sequence identified for other Mg2+-independent phosphatases. Northern blot analysis indicated that 4-phosphatase II is widely expressed with the highest levels occurring in the skeletal muscle and heart. In addition, cDNA encoding alternatively spliced forms of human 4-phosphatase I (107, 309 Da) and rat 4-phosphatase II (106,497 Da) were also isolated that encode proteins with a putative transmembrane domain near their C termini. These alternatively spliced forms were expressed as recombinant proteins in E. coli and SF9 insect cells and found to possess no detectable enzymatic activity suggesting that additional factors and/or processing may be required for these alternatively spliced isozymes.
Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.
The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and is regulated by the opposing actions of protein kinases and protein phosphatases. Protein kinases in the lens system have been well studied, but very little is known about lens protein phosphatases. Protein phosphatases can be divided several families, such as protein phosphatase types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C) and protein tyrosine phosphatases (PTP). In this study we evaluated what kinds of protein phosphatases are present in the lens by using various specific substrates and inhibitors. Samples were prepared from lenses of 17-day-old chick embryos, and fractionated by high-resolution gel permeation column chromatography, then the fractions were assayed for phosphatase activities. The results with 32P-labeled glycogen phosphorylase A, okadaic acid and inhibitor-1, which are a specific substrate and inhibitors of PP1 and/or PP2A, showed that PP1 activities were present in the 500-, 115- and 45-kDa fractions of the lens protein. The 115-kDa fraction also contained PP2A activity. By using a phosphothreonine-containing peptide as a substrate, three peaks of phosphatase activities were found at around 115, 55 and 35 kDa. Based on their response to various phosphatase inhibitors and their metal dependency, the fractions of 115 and 35 kDa were concluded to contain PP2A, while the 55-kDa fraction contained PP2C. Immunoblot using specific antibodies against PP1, PP2A and PP2C confirmed that each fraction above contained corresponding protein phosphatases as proteins. When a phosphotyrosine-containing peptide substrate was examined at pH 7.4, we observed a major peak at 500 kDa, which was presumed to contain receptor-like PTP(s). On the other hand, at pH 5.5, we observed a peak of 18 kDa, which was confirmed to contain a low-molecular-weight PTP. These protein phosphatases have recently been suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are of much interest.
Chromatography of cardiac muscle and brain extracts on DEAE-cellulose resolved phosphotyrosyl-protein phosphatase activity into three fractions, termed Y-1, Y-2, and Y-3. These were eluted at 0.05, 0.15, and 0.3 M KCl, representing about 33, 55, and 12%, respectively, of the enzymatic activity recovered from the resin. Comparative studies demonstrated that the properties of phosphatases Y-1, Y-2, and Y-3 were distinctly different from those of previously identified phosphoseryl-protein phosphatases-1, -2, -3, and -4. Phosphatases Y-1, Y-2, and Y-3 were stimulated by EDTA, and exhibited optimal activity at neutral pH. These properties were different from those of the two minor phosphotyrosyl-protein phosphatase activities associated with phosphoseryl-protein phosphatases-3, and -4, which were divalent cation dependent, and exhibited optimal activity at alkaline pH. Further purification of phosphatase Y-2 from bovine heart has been carried out. The enzyme had a Mr = 65,000 (Stokes radius = 3.8 nm; S020,W = 4.1). Its activity was stimulated by 5- to 10-fold in the presence of EDTA (Ka = 15 microM) and was strongly inhibited by micromolar concentrations of vanadate. Phosphatase Y-2 was highly specific for phosphotyrosyl-IgG and -casein, and showed little activity toward phosphoseryl-casein, -phosphorylase a, phosphothreonyl-inhibitor-1, and p-nitrophenyl phosphate. The present studies indicate that phosphotyrosyl-protein phosphatase activity in animal tissues exists in multiple forms. The major active species are specific for phosphotyrosyl proteins, and represent enzymes different from the known phosphoseryl-protein phosphatases and p-nitrophenyl phosphatases.
The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.
Three forms of protein phosphatase-1 were isolated from rabbit skeletal muscle that had Mr values of 37 000, 34 000 and 33 000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the beta-subunit of phosphorylase kinase very much faster than the alpha-subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase-3 and Mg-ATP for activity by incubation with inhibitor-2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr-37 000 and Mr-34 000 species were converted to the Mr-33 000 form by incubation with chymotrypsin. It is concluded that the Mr-33 000 and Mr-34 000 forms are derived from the Mr-37 000 component by limited proteolysis. Conversion of the Mr-37 000 to the Mr-33 000 form was accompanied by a two-fold increase in activity, indicating that an Mr-4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36 000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr-37 000 (15-20 kU/mg) or Mr-33/34 000 (40-50 kU/mg) forms of protein phosphatase-1. It dephosphorylated the alpha-subunit of phosphorylase kinase 4-5-fold faster than the beta-subunit, was unaffected by inhibitor-1 or inhibitor-2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase-3 and Mg-ATP-dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr-37 000 form of protein phosphatase-1 to the Mr-33 000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observed with protein phosphatase 1. Antibody to protein phosphatase-2A raised in sheep did not cross-react with any of the forms of protein phosphatase-1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase-1 and protein phosphatase-2A are distinct gene products.
An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.
Fluoride is a potent therapeutic agent that increases spinal bone density in osteoporotic subjects. Based on work with animal cells previously, we proposed fluoride acts by inhibiting phosphotyrosyl protein phosphatase (EC 3.1.3.48) activity in bone cells. The presence of fluoride sensitive acid phosphatase (EC 3.1.3.2) activity was characterized in extracts of cultured human bone cells. Crude extracts contained acid phosphatase activity that was inhibited by fluoride with an apparent Ki of 12 mumol/L. The activity was investigated further by separating the acid phosphatase isoenzymes using CM Sepharose chromatography and a gradient of acetate pH 4.8-6.5. The major peak of activity recovered from CM Sepharose chromatography was characterized for stability, Km and inhibition by fluoride. The enzyme was sensitive to inhibition by tartrate, had a high affinity for paranitrophenylphosphate (apparent Km = 0.158 mupmol/L) and an apparent pH optimum of 4.8. Fluoride was a strong competitive inhibitor with an apparent Ki of 12.4 mumol/L. The column fractions containing the acid phosphatase were tested further for phosphotyrosyl protein phosphatase activity using [32P]labeled phosphotyrosyl histone as the substrate. Release of [32P]phosphate from this substrate at pH 7.0 was proportional to enzyme concentration and incubation time, demonstrating the presence of phosphotyrosyl protein phosphatase activity. The phosphotyrosyl protein phosphatase activity was inhibited by fluoride and had a pH optimum of approximately 7. These observations indicate that human osteoblasts contain a fluoride-sensitive phosphotyrosyl protein phosphatase. Thus, these results are consistent with the hypothesis that fluoride stimulates human bone cell proliferation by inhibiting the action of phosphotyrosyl protein phosphatase, thereby increasing the level of phosphorylated tyrosine residues which are known to play a role in increasing cell proliferation.
The phosphorylation by casein kinase TS (II) of the modulator protein of the ATP, Mg-dependent phosphatase increases after preincubation with the PCSH1 phosphatase or with the catalytic subunit of the ATP, Mg-dependent phosphatase. Dephosphorylation by the two phosphatases combined leads to the incorporation of 2 mol phosphate per mol modulator (at Ser residues). Occupancy of the ATP, Mg-dependent phosphatase phosphorylation site(s) is a negative determinant in the phosphorylation of the modulator by kinase TS. Among the PCS phosphatases PCSH1 shows the highest activity toward the 32P-Ser residues labeled by kinase TS in untreated or previously dephosphorylated modulator, while the ATP, Mg-dependent phosphatase is totally ineffective. Protamine stimulates all phosphatase activities, so that the catalytic subunit of the ATP, Mg-dependent phosphatase becomes almost as effective as the PCSC phosphatase in dephosphorylating the kinase TS sites.
Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.