-
[show abstract]
[hide abstract]
ABSTRACT: The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1.
Molecular and Biochemical Parasitology 10/2000; 110(1):161-6. · 2.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cells use phosphorylation/dephosphorylation mechanisms to regulate the activity of several proteins required to transmit information from the cell surface to the nucleus. Recent studies have significantly increased our knowledge regarding the structure/function of one major regulator of cell phosphorylation: protein phosphatase 2A (PP2A). This review will discuss the role of PP2A in virology and parasitology.
Microbes and Infection 05/2000; 2(4):401-7. · 3.10 Impact Factor
-
Parasitology Today 04/1999; 15(3):90-2.
-
[show abstract]
[hide abstract]
ABSTRACT: The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. We have developed a cell-free system prepared from prophase-arrested Xenopus oocytes to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase, in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. We propose that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity, the second one requires a new kinase counteracted by phosphatase 2A. Furthermore we demonstrate that, under our conditions, Cdc2 amplification and MAP kinase activation are two independent events.
Experimental Cell Research 12/1998; 244(2):491-500. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of HIV-1 encoded proteins NCp7, vpr and NCp7:vpr complex on the activity of protein phosphatase-2A0 have been tested. We report that NCp7 is an activator of protein phosphatase-2A0 and that vpr activated protein phosphatase-2A0 only slightly. We also report that NCp7 and vpr form a tight complex which becomes a more potent activator of protein phosphatase-2A0 than NCp7 alone. The ability of NCp7 to activate protein phosphatase-2A0 is regulated by vpr. The C-terminal portion of vpr prevents NCp7 from activating protein phosphatase-2A0 while the N-terminal portion of vpr potentiates the effect of NCp7 on the activity of protein phosphatase-2A0. Our findings indicate that vpr may be acting as a targeting subunit which directs NCp7 to activate protein phosphatase-2A0. In view of the fact that protein phosphatase-2A functions as an inhibitor of G0 to M transition of the cell cycle and is involved in other key cellular processes such as the control of RNA transcription, the results presented in this report may explain how HIV-1 causes cell cycle arrest which may lead to CD4+ T cell depletion and also how it disturbs normal cellular processes of its host cell.
FEBS Letters 02/1997; 401(2-3):197-201. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two types of PP2A catalytic subunit cDNA clones were isolated from a Xenopus oocyte library. One type corresponds to the Xenopus C36 alpha (XC36 alpha) isoform as published by Cormier et al. (1991) and another type encodes for a novel isoform of PP2AC36 (XC36 beta), more homologous to the C36 beta isoform cloned from Mammalia. Northern blot analysis with isoform specific probes showed that the XC36 alpha mRNA transcript of 2 kb is more abundant than the XC36 beta mRNA of 1.9 kb during embryogenesis and in adult tissues. Both transcripts are highly expressed in ovary and heart relative to the other adult tissues examined. The high expression levels of both XC36 alpha and XC36 beta transcripts in the ovary decrease during the first mitotic embryonic divisions. Resumption of zygotic expression starts at about the same time for both mRNAs, during the tailbud stage, but the XC36 beta transcript shows only a moderate and transient increase and declines again during the tadpole stage whereas expression of XC36 alpha increases further during the same period until the onset of metamorphosis.
Biochemical and Biophysical Research Communications 11/1995; 215(2):666-73. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5' flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-kappa B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5' of a luciferase reporter gene revealed that the 5' flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis.
Genomics 08/1995; 28(2):261-72. · 3.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: cDNA clones encoding the 65-kDa (PR65) and 55-kDa (PR55) regulatory subunits of protein phosphatase 2A from Xenopus laevis were isolated by homology screening with the corresponding human cDNAs, and used to analyze the developmental expression patterns of these genes. The PR65 subunit was found to be encoded by two genes, termed XPR65 alpha and XPR65 beta. The open reading frames of the alpha and beta cDNAs both span 1767 bp, and predict proteins of 64.4 kDa and 65.3 kDa, respectively, that are 87% identical. The predicted amino acid sequence of XPR65 alpha showed 95% and 84% identity with human PR65 alpha and PR65 beta proteins, respectively, whereas the identity of XPR65 beta with the same proteins was 87% and 86.5%, respectively. Only one type of Xenopus PR55 (XPR55) was isolated that showed 93% and 84% similarity to human PR55 alpha and PR55 beta, respectively. Analysis of the N-terminal region of XPR55 with the same regions of human PR55 alpha and PR55 beta, indicates that the XPR55 is the Xenopus homolog of the human PR55 alpha isoform. Despite the overall similarity with PR55 from other species, XPR55 has an N-terminal extention of at least 24 amino acids. In the ovary, a transcript of 2.8 kb, encoding the XPR65 beta, was predominantly expressed and these XPR65 beta mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4-kb XPR65 alpha was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4-kb alpha-specific mRNA was more abundant than the 2.8-kb beta transcript. Two transcripts of 2.4 kb and 2.5 kb, encoding the XPR55 subunit, were detected at a constant level throughout Xenopus oogenesis and during embryogenesis. Both transcripts were also expressed at similar levels in all adult tissues, but in a tissue-specific manner. Analysis of the XPR55 and XPR65 proteins using antibodies to recombinant proteins revealed that the overall levels of the two proteins were constant, in good agreement with mRNA data.
European Journal of Biochemistry 07/1995; 230(3):1037-45. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A simple, improved procedure for the isolation of the phosphotyrosyl phosphatase activator (PTPA) from rabbit skeletal muscle has been developed. The majority of the protein phosphatase 2A (PP2A) was separated from PTPA at an early stage in the procedure. The procedure yields approximately 1 mg essentially pure PTPA/kg rabbit skeletal muscle; it was also applied to porcine brain and the yeast Saccharomyces cerevisiae. The physico-chemical properties of PTPA obtained from all sources are very similar. The pure rabbit skeletal muscle protein was used to raise polyclonal goat antibodies and to affinity purify these antibodies. Immunological studies revealed the presence of PTPA in all mammalian tissues and cell lines examined with differences in tissue distribution, brain showing the highest concentration. PTPA could only be detected in cytosolic fractions. Using a semi-quantitative immunological assay (Western blot), the in vivo concentration could be estimated to be micromolar, which is in the same range as the PP2A target. The purified Xenopus oocyte PTPA showed only a weak cross reactivity, whereas yeast PTPA was not recognised by the antibody indicating some evolutionary diversity of the protein. In a PTPA-affinity column chromatography, the weak interaction with PP2A was independent of the presence of ATP.Mg, a necessary cofactor in the activation process. Interaction of PTPA with PP2A in a 1:1 ratio induces a low (kcat = 3 min-1) ATPase activity that is inhibited by okadaic acid, ADP and non-hydrolysable ATP analogues.
European Journal of Biochemistry 01/1995; 226(3):899-907. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: PTPA, or phosphotyrosyl phosphatase activator, is a protein that stimulates the tyrosyl phosphatase activity of protein phosphatase 2A in an ATP, Mg(2+)-requiring reaction (Cayla, X., Goris, J., Hermann, J., Hendrix, P., Ozon, R., and Merevede, W. (1990) Biochemistry 29, 658-667). We constructed oligonucleotide probes based on the amino acid sequences of peptides isolated from purified PTPA and used them to probe rabbit muscle and human heart cDNA libraries. A putative full-length clone was isolated from the rabbit skeletal muscle as well as from the human heart library. The nucleotide sequence of both clones contains an open reading frame of 969 nucleotides starting from an assigned initial ATG codon and encodes for a protein of 323 amino acids. The predicted rabbit and human PTPA protein sequences show an identity of 96.6%. The predicted protein matched all the peptide sequences obtained from the rabbit skeletal muscle protein. Bacterially expressed protein, as well as the in vitro reticulocyte lysate translation product, comigrated with the purified 37-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Both proteins reacted with immunopurified, anti-PTPA polyclonal antiserum. The recombinant protein was a soluble and active protein. Northern blot analysis revealed two transcripts of 2.8 and 4 kilobases, respectively, in human placenta but only one 2.8-kilobase transcript in rabbit and rat tissues. High levels of PTPA mRNA were detected in testis, which contrasted with the low levels present in skeletal muscle.
Journal of Biological Chemistry 07/1994; 269(22):15668-75. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Papovavirus tumor antigens have been shown to associate with the cellular phosphoserine/threonine-specific protein phosphatase 2A (PP2A). We were interested in the consequences that T-antigen association might have on PP2A activity and so studies of the phosphatase activity in immunoprecipitates, prepared from polyoma virus-transformed or polyoma virus-infected mouse 3T3 fibroblasts, were performed. The phosphoserine/threonine phosphatase activity, measured with phosphorylase a as the substrate, showed all the characteristics of PP2A. It was stimulated by polycations, inhibited by fluoride or p-nitrophenyl phosphate, sensitive to okadaic acid and microcystin and insensitive to inhibitor-1 and inhibitor-2. Phosphotyrosyl phosphatase (PTPase) activity was associated with the middle-T/small-T-associated complex when reduced, carboxamidomethylated and maleylated lysozyme, phosphorylated exclusively on tyrosyl residues, was used as the substrate. This PTPase activity was as sensitive to okadaic acid as was the phosphorylase phosphatase activity; it could be inhibited by phosphorylase a and did not dephosphorylate poly(Glu80Tyr20). The level of middle-T/small-T-associated PTPase activity relative to the phosphorylase phosphatase activity was tenfold higher than that of the purified dimeric PP2A. A similar activity ratio was observed with the purified phosphatase after stimulation with a cellular protein, designated phosphotyrosyl phosphatase activator. These results suggest that the same enzyme may possess dual specificity. In contrast to the cellular trimeric PP2A, containing the 55-kDa putative regulatory subunit, the middle-T/small-T-associated enzyme had low activity towards a retinoblastoma peptide phosphorylated by p34cdc2. These results indicate how middle-T/small-T might effect the activity of PP2A in polyoma virus-transformed cells.
European Journal of Biochemistry 06/1993; 214(1):281-6. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.
European Journal of Biochemistry 10/1990; 192(3):633-42. · 3.58 Impact Factor
-
Advances in Enzyme Regulation 02/1990; 30:265-85.
-
[show abstract]
[hide abstract]
ABSTRACT: PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].
Biochemistry 01/1990; 29(3):658-67. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemical Journal 06/1989; 260(1):45-51. · 4.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tubulin can stimulate specifically the aryl phosphatase activity of the low-Mr polycation-stimulated (PCSL) phosphatase, measured as p-nitrophenyl phosphatase activity, or using reduced carboxamidomethylated and maleylated (RCM) lysozyme, phosphorylated on tyrosyl residues, as a substrate. This stimulation is independent of the degree of polymerization of tubulin (A50 = 60 nM) and is due to an increase in Vmax. It is mechanistically different from the ATP-induced activation and resistant to heat and trypsin treatment. Chymotrypsin destroys the stimulatory effect of tubulin. The polycation-stimulated phosphorylase phosphatase activity is inhibited by tubulin, probably by a polycation/polyanion interaction. The microtubule-associated protein, MAP2, is inhibitory to the p-nitrophenyl phosphatase activity and tubulin can eliminate this inhibitory effect. MAP2 also inhibits the polycation-stimulated phosphorylase phosphatase activity.
European Journal of Biochemistry 04/1989; 180(1):15-22. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
European Journal of Biochemistry 05/1988; 173(1):17-25. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.
European Journal of Biochemistry 02/1988; 171(1-2):107-17. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5′ flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains up-stream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-κB, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5′ of a luciferase reporter gene revealed that the 5′ flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis.
Genomics.
