Sequence homology between purple acid phosphatases and phosphoprotein phosphatases. Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers?

The amino acid sequences of mammalian purple acid phosphatases and phosphoprotein phosphatases are shown to possess regions of significant homology. The conserved residues contain a high percentage of possible metal-binding residues. The phosphoprotein phosphatases 1, 2A and 2B are proposed to be iron-zinc metalloenzymes with active sites isostructural (or nearly so) with those of the purple phosphatases.     

Molecular evolution of phosphoprotein phosphatases in Drosophila

Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18-22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G-C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae.     

Characterization of nuclear phosphoprotein phosphatases from goat testis

Two nuclear phosphoprotein phosphatases (PPases I and II) that cause dephosphorylation of [32P]histone, have been partially purified from goat testis. The enzymic activity is associated with nucleoplasm and chromatin. PPase I is markedly stimulated (approx. 200-600%) by Mg2+ or Mn2+ (1 mM) whereas Ca2+ (1 mM) causes slight stimulation (approx. 35%) of the enzyme. On the contrary, PPase II is only slightly activated (20-40%) by these metal ions (5 mM). Both the phosphoprotein phosphatase isoenzymes are maximally active at pH 6-7. PPases I and II are strongly inhibited (approx. 60-100%) by ZnCl2 (1 mM), P1 (5 mM) and thiol reagents. NaF (5 mM) inhibits (approx. 40%) specifically the activity of PPase I rather than PPase II. PPases are strongly inhibited by relatively high concentration of NaCl (0.4 M), isoenzyme II being more sensitive (approx. 80%) than isoenzyme I (approx. 50%). In addition to histones, both the isoenzymes can as well cause dephosphorylation of protamine, casein, and testicular nuclear proteins. Enzymic characteristics of the testicular nuclear PPases are clearly different from those of the cytosolic enzyme previously characterized.     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

Some properties of purified phosphoprotein phosphatases from rabbit liver.

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.     

Expression of multiple type 1 phosphoprotein phosphatases in Arabidopsis thaliana

Type 1 phosphoprotein Ser/Thr phosphatases (PP1) are highly conserved enzymes found in all eukaryotes. These enzymes have multiple functions in fungal and animal cells but little is known of their function and regulation in plants. Previous studies in our laboratory indicated that maize and Arabidopsis contain a family of PP1 genes and/or pseudogenes. In this study, we report the isolation of five distinct Arabidopsis cDNA clones (TOPP1, TOPP2, TOPP3, TOPP4 and TOPP5) which encode the catalytic subunit (PP1c) of type 1 protein phosphatases. Genomic Southern blot analyses indicate that these clones are the products of five distinct genes and that an additional 2–3 PP1c genes and/or pseudogenes may be present in the Arabidopsis genome. The derived amino acid sequences of the TOPP clones are very similar to published sequences of PP1c from animals, fungi and plants. Four of the TOPP amino acid sequences show unique structural features not observed in other PP1c sequences from fungi or animals. All of the TOPP genes are expressed in Arabidopsis roots, rosettes and flowers, although TOPP1, TOPP2 and TOPP3 appear to be expressed at higher levels in these tissues than TOPP4 and TOPP5.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.     

Stimulation of mammalian and yeast phosphoprotein phosphatases by megamodulins from various sources

Phosphoprotein phosphatases (PPase) were isolated from either the rabbit cerebral cortex or Baker's yeast by excluding endogenous megamodulin with histone, and then by desalting cations with Bio-Gel P-6DG filtration. The stimulation of PPase in the presence of Mn2+ was greatly enhanced by megamodulins prepared from various sources including rabbit brain, Baker's yeast, wheat germ, and Escherichia coli (E. coli). Moreover, the augmented activity of PPase was also demonstrated in the presence of [megamodulin-Mn2+] complex.     

Somatostatin is a potent activator of phosphoprotein phosphatases in the digestisve tract

This study demonstrates that somatostatin is a potent (K_d = 0.5 · 10^?11 M) and apparently selective activator of phosphoprotein phosphatase activity in homogenates and cytosolic extracts prepared from liver and pancreas, as well as from gastric and intestinal epithelial cells. It is suggested that protein dephosphorylation could account for at least some of the physiological effects of the tetradecapeptide in the digestive tract.     

The effect of calmodulin and troponin-C on activities of homogeneous liver phosphoprotein phosphatases

The effect of calmodulin was determined on activities of two homogeneous liver phosphoprotein phosphatases with phosphorylase a and phosphorylated histones as substrates. Calmodulin in the absence or presence of calcium had no effect on the dephosphorylation of phosphorylase a by either phosphatases. However, calmodulin inhibited the dephosphorylation of histones catalyzed by both phosphatases. No difference was found whether the reactions were carried out in the absence or presence of calcium. The effect of calmodulin on histone dephosphorylation was variable depending on (i) the absence or presence of KCl and Mg²⁺, and (ii) the concentration of histone in the reaction mixture. In the presence of KCl and Mg²⁺ at a histone concentration of 0.1 mg/ml, calmodulin inhibited the enzyme activity. At 1 mg/ml histone, lower concentrations of calmodulin activated whereas higher concentrations of calmodulin inhibited the enzyme activity. Similar, but relatively less, effect was observed with troponin-C. In the absence of KCl and Mg²⁺, calmodulin as well as troponin-C activated the enzyme activity. The optimal concentration of calmodulin (or troponin-C) was approximately 30–50% of histone concentration in the reaction mixture. Calcium alone or with calmodulin (or troponin-C) had no additional effect on enzyme activities. DNA and RNA, two negatively charged nucleic acids, also showed similar effects on histone dephosphorylation. Depending on whether KCl and Mg²⁺ were absent or present in the reaction mixtures, both nucleic acids either activated or inhibited the dephosphorylation of histones.     

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.     

Modulation of the rhythmic patterns of expression of phosphoprotein phosphatases in human leukaemia cells

Temporal variations in the expression of phosphoprotein phosphatase 1 (PP1), phosphoprotein phosphatase 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B) were monitored in the human acute, promyelocytic leukaemia cell line, HL60. Granulocytic differentiation was induced using all-trans retinoic acid (ATRA) and monocytic differentiation by phorbol-12-myristate-13-acetate (PMA). Expression of the enzyme proteins in cell extracts was determined by SDS-PAGE and Western immunoblotting using specific antibodies. For PP1, a single immunospecific band of molecular mass 38 kDa was detected corresponding to the catalytic subunit; induction of differentiation with either ATRA or PMA showed differences in the patterns of expression and, in the case of the latter, the mean value. Two immunospecific bands, of mass 34 and 37 kDa, possibly corresponding to dephosphorylated and phosphorylated forms, respectively, were detected for PP2A, as well as a minor band of mass 46 kDa; dynamic variations in the expression of all 3 forms were observed and there were differences between the control and treated cells. The catalytic domain of PTP1B was detected as a 46 kDa band. A 42 kDa form of the protein was also seen, which may represent a change in phosphorylation state, or be the result of proteolytic cleavage; usually the 46 kDa band was the major form, but on occasion there was a change to predominance of the 42 kDa band.     

Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.     

Probable occurrence of brain basic protein factor I, an activator of phosphoprotein phosphatases

Basic protein factor I (BPFI was purified to homogeneity from bovine brain by boiling and trichloroacetic acid-precipitation of tissue homogenate, followed by DEAE-cellulose, Sephadex G-150, Affi-Gel-phenothiazine, and Bio-Gel P-6DG chromatographic procedures. The preparation appeared as a single protein band in the SDS-polyacrylamide gel electrophoresis with a minimal Mr of 13,200. The factor was a basic protein as indicated by an estimated isoelectric point of pH 8.3 and a high content of amino acids including arginine, histidine, lysine and others. In the absence of Mn2+, the factor stimulated the phosphoprotein phosphatases (PPase) from rabbit brains. Unlike histones or protamine, the factor was a poor substrate for megamodulin-dependent protein kinase. In addition, the factor did not interact significantly with E. coli megamodulin.