RESUMEN

BACKGROUND: Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B', and PR72. Specific functions for each variable subunit are not well understood. RESULTS: Two fission yeast genes pbp1+ and pbp2+ homologous to the regulatory subunit B' were isolated. Physical in vivo interaction of the gene products with the catalytic subunit was demonstrated. A double disruption haploid mutant (Deltapbp1Deltapbp2) showed growth defect, cell shape and size abnormality, multiseptation and anucleated cell formation due to abnormality in septum positioning. These phenotypes were suppressed by human B' cDNA, indicating the striking conservation of the B' function from yeast to human. Over-expression of fission yeast B' led to growth defects, a loss of cell shape polarity, septal abnormality and anucleated cell formation. Deltapbp1Deltapbp2 and pbp1 null haploids were hypersensitive to calcineurin inhibitors, cyclosporin A and FK506, with which the mutants underwent arrest at post-anaphase and cell lysis. Double disruption of calcineurin and pbp1+, but not pbp2+, genes led to synthetic lethality. CONCLUSION: The fission yeast B' subunit of PP2A plays critical roles in cell shape control and septum formation, and shares essential functions with calcineurin for viability, possibly through their roles in cytokinesis and cell wall integrity.

Asunto(s)

Proteínas Bacterianas , Calcineurina/metabolismo , Proteínas Portadoras/genética , Hexosiltransferasas , Mitosis/fisiología , Muramoilpentapéptido Carboxipeptidasa/genética , Peptidil Transferasas , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Calcineurina/genética , Calcineurina/fisiología , Proteínas Portadoras/fisiología , Dominio Catalítico/genética , Secuencia Conservada , Ciclosporina/metabolismo , Humanos , Immunoblotting , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación , Proteínas de Unión a las Penicilinas , Fosfoproteínas Fosfatasas/genética , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Proteína Fosfatasa 2 , Subunidades de Proteína , Mapeo Restrictivo , Schizosaccharomyces/citología , Schizosaccharomyces/fisiología , Especificidad por Sustrato , Temperatura

RESUMEN

A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.

Asunto(s)

Eritrocitos/enzimología , Fragmentos de Péptidos/fisiología , Fosfoproteínas Fosfatasas/química , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Humanos , Manganeso/metabolismo , Peso Molecular , Conformación Proteica , Proteína Fosfatasa 2

RESUMEN

Human erythrocyte Mn(2+)-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn(2+)-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'.

Asunto(s)

Eritrocitos/enzimología , Hierro/metabolismo , Manganeso/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Zinc/metabolismo , Animales , Dominio Catalítico , Inhibidores Enzimáticos/farmacología , Humanos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Proteína Fosfatasa 2 , Fluoruro de Sodio/farmacología , Vanadatos/farmacología

RESUMEN

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.

Asunto(s)

Eritrocitos/enzimología , Hierro/análisis , Manganeso/farmacología , Fosfoproteínas Fosfatasas/química , Zinc/análisis , Apoenzimas/química , Apoenzimas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Magnesio/farmacología , Metaloproteínas/química , Fosfoproteínas Fosfatasas/efectos de los fármacos , Proteína Fosfatasa 2

RESUMEN

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Eritrocitos/enzimología , Humanos , Cinética , Ácido Ocadaico/farmacología , Fragmentos de Péptidos/análisis , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteína Fosfatasa 2 , Análisis de Secuencia , Serina/metabolismo , Especificidad por Sustrato

Asunto(s)

Fosfoproteínas Fosfatasas/química , Alquenos/farmacología , Animales , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Chaperonas de Histonas , Humanos , Metilación , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Poliaminas/farmacología , Polielectrolitos , Polienos , Proteína Fosfatasa 2 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/farmacología , Pironas , Factores de Transcripción

RESUMEN

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Asunto(s)

Encéfalo/metabolismo , Fosfoproteínas Fosfatasas/inmunología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citosol/metabolismo , Eritrocitos/inmunología , Eritrocitos/metabolismo , Humanos , Sueros Inmunes , Mucosa Intestinal/metabolismo , Cinética , Masculino , Ratones , Microsomas/metabolismo , Mitocondrias/metabolismo , Peso Molecular , Músculo Esquelético/metabolismo , Fosfoproteínas Fosfatasas/química , Fosforilación , Proteína Fosfatasa 2 , Ratas , Ratas Wistar , Fracciones Subcelulares , Distribución Tisular

RESUMEN

Two cDNAs for possible splicing variants of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A, were isolated. These variants were identified from human cerebral cortex by library screening and PCR, and designated delta1 and delta3 isoforms, while the previously reported isoform [Tanabe et al. (1996) FEBS Lett. 379, 107-1111 was designated delta2. Compared with the delta2 isoform, the delta1 isoform contained a 32-residue insertion beginning at residue 84, and consisted of 602 amino acids in all. The delta3 isoform lacked a 74-residue sequence corresponding to residues 1083 of the delta2 isoform, and consisted of 496 amino acids. Using isoform-specific antipeptide antisera, the 74-kDa subunit (B" or delta) originally purified from human erythrocytes was identified as the delta1 isoform.

Asunto(s)

Corteza Cerebral/enzimología , Isoenzimas/genética , Fosfoproteínas Fosfatasas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Escherichia coli/genética , Expresión Génica , Humanos , Isoenzimas/química , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Reacción en Cadena de la Polimerasa , Proteína Fosfatasa 2 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análisis de Secuencia , Eliminación de Secuencia

RESUMEN

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.

Asunto(s)

Membrana Celular/química , Hígado/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Familia-src Quinasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Membrana Celular/genética , Electroforesis , Regulación Neoplásica de la Expresión Génica , Hígado/citología , Masculino , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas c-yes , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido , Tirosina/metabolismo

RESUMEN

Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Asunto(s)

Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Corteza Cerebral/enzimología , Clonación Molecular , ADN Complementario/genética , Escherichia coli/genética , Humanos , Masculino , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Proteína Fosfatasa 2 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Secuencias Repetitivas de Ácidos Nucleicos , Distribución Tisular