RESUMEN
The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Trehalasa/metabolismo , Secuencia de Bases , Dominio Catalítico , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Glicerol/metabolismo , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2 , Proteínas Ribosómicas/efectos de los fármacos , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Trehalasa/antagonistas & inhibidores , Trehalasa/aislamiento & purificaciónRESUMEN
Type 2A protein phosphatase (PP2A) comprises a diverse family of phosphoserine- and phosphothreonine-specific enzymes ubiquitously expressed in eukaryotic cells. Common to all forms of PP2A is a catalytic subunit (PP2Ac) which can form two distinct complexes, one with a structural subunit termed PR65/A and another with an alpha4 protein. The PR65/A-PP2Ac dimer may further associate with a regulatory subunit and form a trimeric holoenzyme. To date, three distinct families of regulatory subunits, which control substrate selectivity and phosphatase activity and target PP2A holoenzymes to their substrates, have been identified. Other molecular mechanisms that regulate PP2Ac function include phosphorylation, carboxyl methylation, inhibition by intracellular protein inhibitors (I(1)(PP2A) and I(2)(PP2A)), and stimulation by ceramide. PP2A dephosphorylates many proteins in vitro, but in vivo protein kinases and transcription factors appear to represent two major sets of substrates. Several natural compounds can inhibit PP2A activity and are used to study its function. Mutations in genes encoding PR65/A subunits have been identified in several different human cancers and the PP2A inhibitor, termed fostriecin, is being tested as an anticancer drug. Thus, a more thorough understanding of PP2A structure and function may lead to the development of novel strategies against human diseases.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Transducción de Señal/fisiología , Ciclo Celular/fisiología , Activación Enzimática , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/efectos de los fármacos , Fosfoproteínas Fosfatasas/genética , Conformación Proteica , Proteínas Quinasas/metabolismo , Especificidad por Sustrato , Toxinas Biológicas/farmacología , Factores de Transcripción/metabolismoRESUMEN
Two holoenzymes of protein phosphatase 2A (PP2A), designated PP2AI and PP2AII, were purified from maize seedlings. The subunit composition of maize holoenzymes generally resembled those of animal PP2A. Using SDS/PAGE and Western blots with antibodies generated against peptides derived from animal PP2A, we established the subunit composition of plant protein phosphatase 2A. In both maize holoenzymes, a 38000 catalytic (PP2Ac) and a 66000 constant regulatory subunit (A) constituting the core dimer of PP2A were present. In addition, PP2AI (180000-200000) contained a protein of 57000 which reacted with antibodies generated against the peptide (EFDYLKSLEIEE) conserved in all eukaryotic Balpha regulatory subunits. In contrast, none of the proteins visualised in PP2AII (140000-160000) by double staining reacted with these antibodies. The activity of PP2AI measured with (32)P-labelled phosphorylase a in the presence of protamine and ammonium sulfate is about two times higher than that of PP2AII. PP2AI and PP2AII displayed different patterns of activation by protamine, polylysine and histone H1 and exhibit high sensitivity toward inhibition by okadaic acid. The data obtained provide direct biochemical evidence for the existence in plants of PP2A holoenzymes composed of a catalytic subunit complexed with one or two regulatory subunits.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Zea mays/enzimología , Secuencia de Aminoácidos , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/aislamiento & purificación , Proteína Fosfatasa 2 , Zea mays/crecimiento & desarrolloRESUMEN
Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N-termini, were introduced into the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriacetate/agarose. In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 microg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a [1.7 micromol.min-1.(mg protein)-1] and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state-specific antibodies, recombinant PP2Ac was carboxymethylated at the last C-terminal leucine residue. Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine-agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.
Asunto(s)
Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Pichia/enzimología , Animales , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Clonación Molecular , Cartilla de ADN , Escherichia coli , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoAsunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Proteínas/metabolismo , Animales , Bélgica , Sitios de Unión , Adhesión Celular , Ciclo Celular , División Celular , Transformación Celular Viral , Humanos , Inmunidad , Insulina/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfoproteínas Fosfatasas/química , Fosforilación , Proteínas Quinasas/metabolismo , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/enzimología , Transducción de Señal , Especificidad por SustratoRESUMEN
Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivo in response to phosphatase inhibitors and the dephosphorylation and deactivation of PDE3B in vitro by phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 microM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 microM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 microM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivo and can act as a PDE3B phosphatase in vitro. In comparison with insulin, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fosfoproteínas Fosfatasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Adipocitos/enzimología , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Activación Enzimática , Insulina/farmacología , Toxinas Marinas , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Mapeo Peptídico , Fosforilación , RatasRESUMEN
Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.
Asunto(s)
Proteínas de Ciclo Celular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Fosfatasa 1 de Especificidad Dual , Quinasa I-kappa B , Proteínas Inmediatas-Precoces/metabolismo , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-aktRESUMEN
Purification of type 2A protein phosphatase (PP2A) from rabbit skeletal muscle resulted in the isolation of a trimeric phosphatase which is composed of a catalytic (PP2Ac), a structural (PR65alpha/Aalpha), and a regulatory (PR55alpha/Balpha) subunit, together with translation termination factor 1 (eRF1) and another protein of 55 kD (EMBO J., 15, 101-112). Yeast two-hybrid system analysis demonstrated that the eRF1 interacted with PP2Acalpha but not with PR65alpha/Aalpha or PR55alpha/Balpha. The N-terminal region of PP2Acalpha, comprising 50 amino acid residues, and the C-terminal part of eRF1, corresponding to an internal region between amino acids 338-381, were found to be necessary for eRF1--PP2Acalpha interaction in yeast. Immunoprecipitations using 12CA5 antibodies and extracts from COS1 cells transiently transfected with eRF1 tagged with 9-amino acid epitope from influenza hemagglutinin (HA) demonstrated the presence of eRF1--PP2Acalpha--PR65alpha/Aalpha complex in these cells. In addition, polysomes obtained from COS1 cells overexpressing HA--eRF1 displayed several-fold higher PP2A activity than control polysomes. No effect of either PP2Ac or dimeric and trimeric PP2A holoenzymes on the rate of translation termination was detected using an in vitro reconstituted translation termination assay. In summary, eRF1 appears to represent a novel PP2A-targeting subunit that brings this phosphatase in contact with putative ribosomal substrate(s). It remains to be established whether termination of translation requires dephosphorylation of participating protein factor(s).
Asunto(s)
Factores de Terminación de Péptidos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Técnicas In Vitro , Músculo Esquelético/metabolismo , Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/genética , Fosfoproteínas Fosfatasas/genética , Biosíntesis de Proteínas , Proteína Fosfatasa 2 , Conejos , Ribosomas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
By a number of criteria, we have demonstrated that the translation termination factor eRF1 (eukaryotic release factor 1) associates with protein phosphatase 2A (PP2A). Trimeric PP2A1 was purified from rabbit skeletal muscle using an affinity purification step. In addition to the 36 kDa catalytic subunit (PP2Ac) and established regulatory subunits of 65 kDa (PR65) and 55 kDa (PR55), purified preparations contained two proteins with apparent Mrs of 54 and 55 kDa. Protein microsequencing revealed that the 55 kDa component is a novel protein, whereas the 54 kDa protein was identified as eRF1, a protein that functions in translational termination as a polypeptide chain release factor. Using the yeast two-hybrid system, human eRF1 was shown to interact specifically with PP2Ac, but not with the PR65 or PR55 subunits. By deletion analysis, the binding domains were found to be located within the 50 N-terminal amino acids of PP2Ac, and between amino acid residues 338 and 381 in the C-terminal part of human eRF1. This association also occurs in vivo, since PP2A can be co-immunoprecipitated with eRF1 from mammalian cells. We observed a significant increase in the amount of PP2A associated with the polysomes when eRF1 was transiently expressed in COS1 cells, and eRF1 immunoprecipitated from those fractions contained associated PP2A. Since we did not observe any dramatic effects of PP2A on the polypeptide chain release activity of eRF1 (or vice versa), we postulate that eRF1 also functions to recruit PP2A into polysomes, thus bringing the phosphatase into contact with putative targets among the components of the translational apparatus.
Asunto(s)
Factores de Terminación de Péptidos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Células COS , Catálisis , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Factores de Terminación de Péptidos/genética , Fosfoproteínas Fosfatasas/genética , Pruebas de Precipitina , Unión Proteica , Proteína Fosfatasa 2 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.
Asunto(s)
Isoenzimas/genética , Fosfoproteínas Fosfatasas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Músculo Esquelético/enzimología , Fragmentos de Péptidos , Fosfoproteínas Fosfatasas/metabolismo , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteína Fosfatasa 2 , Conejos , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Association of the catalytic subunit (C2) with a variety of regulatory subunits is believed to modulate the activity and specificity of protein phosphatase 2A (PP2A). In this study we report the cloning and expression of a new family of B-subunit, the B', associated with the PP2A0 form. Polymerase chain reactions and cDNA library screening have identified at least seven cDNA isotypes, designated alpha, beta 1, beta 2, beta 3, beta 4, gamma, and delta. The different beta subtypes appear to be generated by alternative splicing. The deduced amino acid sequences of the alpha, beta 2, beta 3, beta 4 and gamma isoforms predict molecular weights of 57,600, 56,500, 60,900, 52,500, and 68,000, respectively. The proteins are 60-80% identical and differ mostly at their termini. Two of the isoforms, B' beta 3 and B' gamma, contain a bipartite nuclear localization signal in their COOH terminus. No homology was found with other B- or B- related subunits. Northern analyses indicate a tissue-specific expression of the isoforms. Expression of B' alpha protein in Escherichia coli generated a polypeptide of approximately 53 kDa, similar to the size of the B' subunit present in the purified PP2A0. The recombinant protein was recognized by antibody raised against native B' and interacted with the dimeric PP2A (A.C2) to generate a trimeric phosphatase. The deduced amino acid sequences of the B' isoforms show significant homology to mammalian, fungal, and plant nucleotide sequences of unknown function present in the data bases. Notably, a high degree of homology (55-66%) was found with a yeast gene, RTS1, encoding a multicopy suppressor of a rox3 mutant. Our data indicate that at least seven B' subunit isoforms may participate in the generation of a large number of PP2A0 holoenzymes that may be spatially and/or functionally targeted to different cellular processes.
Asunto(s)
Isoenzimas/genética , Fosfoproteínas Fosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Encéfalo/enzimología , Bovinos , Clonación Molecular , Secuencia Conservada , ADN Complementario , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Reacción en Cadena de la Polimerasa , Proteína Fosfatasa 2 , Conejos , Homología de Secuencia de AminoácidoRESUMEN
The physiological role of type 2A protein phosphatases (PP2A) is dependent upon the association of the catalytic subunit with a variety of regulatory subunits. In order to understand the function of PP2A, we have undertaken purification of the holoenzymes and molecular cloning of the regulatory subunits. Two trimeric forms containing distinct B-subunits, PP2A0 and PP2A1, have been purified from rabbit skeletal muscle. The B-subunits associated with PP2A0 and PP2A1 migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with slightly different mobility, approximately 52.5 and approximately 51.5 kDa, respectively and showed distinct immunological properties. The B' form of B-subunit associated with PP2A0 was recognized by antibodies against the B-subunit present in bovine heart PP2A but not by antibodies specific to the B subunit isoforms of rabbit PP2A1. Cloning of cDNAs encoding the B subunit of PP2A1 resulted in the isolation of a cDNA highly homologous to, but distinct from, the B alpha subunit isoform. The deduced amino acid sequence of this novel isoform, which was designated B gamma, encoded a protein which was 81% and 87% identical to the B alpha and B beta isoforms, respectively. Northern blot analysis indicated that the B gamma isoform is highly expressed in rabbit brain as a transcript of 3.9 kb. Analysis of B-subunit expression by Western blot indicated a general parallel with the message levels. In conclusion, our data reveal even greater complexity of PP2A trimeric holoenzymes due to the identification of a novel B regulatory subunit isoform of PP2A1 and a distinct B' subunit associated with PP2A0.
Asunto(s)
Encéfalo/enzimología , Músculo Esquelético/enzimología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/aislamiento & purificación , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/inmunología , Conformación Proteica , Proteína Fosfatasa 2 , ARN Mensajero/análisis , Conejos , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The gene encoding the calcitonin receptor (CTR) was isolated from a porcine kidney epithelial cell line (LLC-PK1) genomic library and found to span approximately 70 kilobases. Analysis of the gene sequence revealed that the CTR mRNA encompasses 14 exons with 12 exons encoding the protein. Two splicing acceptor sites separated by 48 nucleotides were found in intron 7. The expression of two mRNA species in LLC-PK1 cells was subsequently confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In LLC-PK1 cells the mRNA encoding the shorter CTR (CTR-1a) is approximately 1,000 times more abundant than the longer variant (CTR-1b), as estimated by the competitive RT-PCR. The transcription initiation site of the CTR gene was mapped by primer extension, S1 nuclease, and RT-PCR analysis. The proximal promoter region of 500 base pair is GC-rich (66%) and CpG-rich (CpG/GpC ratio 0.71). Transient transfection of CTR gene promoter-luciferase chimeras in LLC-PK1 cells led to the expression of luciferase activity. The CTR gene was mapped to chromosome band 9q11-q12.
Asunto(s)
Mapeo Cromosómico , Receptores de Calcitonina/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Exones/genética , Variación Genética , Intrones/genética , Luciferasas/biosíntesis , Luciferasas/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Porcinos , Transcripción GenéticaRESUMEN
We have used polyclonal antibodies against an internal peptide (residues 169 to 182; Ab169/182) and a peptide corresponding to the carboxyl terminus (residues 299 to 309; Ab299/309) to look for in vivo modifications of protein phosphatase 2A catalytic (PP2Ac) subunit. Treatment of extracts from human breast cancer (MCF7) cells with either alkali or ethanol increased immunoreactivity of PP2Ac subunit severalfold on Western blots with Ab299/309, but did not apparently change molecular weight or isoelectric point of the protein. In contrast, immunoreactivity with Ab169/182 was unchanged by these treatments. Subsequently, we demonstrated that the increase in PP2Ac subunit recognition by Ab299/309 coincides with the demethylation of this protein at the carboxyl-terminal leucine (Leu309). Methylation of PP2Ac subunit, in vitro, increases its activity toward both phosphorylase a and a phosphopeptide. The carboxyl-terminal sequence (TPDYFL) of PP2Ac subunit is completely conserved between mammals, yeast, fruit fly, and plants which suggests that regulation of this enzyme activity by carboxyl-terminal methylation has been conserved during evolution.
Asunto(s)
Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Proteína Metiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Neoplasias de la Mama , Catálisis , Secuencia Conservada , Femenino , Humanos , Leucina/análogos & derivados , Metilación , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Fosfoproteínas Fosfatasas/inmunología , Fosforilasa a/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.
Asunto(s)
Isoenzimas/fisiología , Familia de Multigenes , Fosfoproteínas Fosfatasas/fisiología , Secuencia de Aminoácidos , Animales , Isoenzimas/genética , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Conejos , Transducción de Señal/fisiologíaRESUMEN
Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Genes Fúngicos , Fosfoproteínas Fosfatasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Fúngicos , Biblioteca de Genes , Genes Supresores , Genotipo , Sustancias Macromoleculares , Datos de Secuencia Molecular , Morfogénesis/genética , Músculos/enzimología , Oligonucleótidos , Fenotipo , Proteína Fosfatasa 2 , Conejos , Mapeo Restrictivo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Ácido NucleicoRESUMEN
The relationship of adenotin, a low-affinity adenosine-binding protein, to adenosine receptors was examined in two human tissues and two mammalian cultured cell lines. An adenosine A2 receptor exists in the membranes from platelets, PC-12 cells, and JAR cells as shown by a stimulation of adenylate cyclase related to 5'-N-ethylcarboxamidoadenosine (NECA) or a NECA-related increase in intracellular cAMP levels. In contrast, binding studies with tritiated NECA revealed typical adenotin-like low-affinity binding sites on the membranes from the sources studied with agonist potencies as follows: NECA greater than 2-chloroadenosine greater than R-PIA. No evidence was found of coupling to a guanine nucleotide regulatory protein. Solubilization of platelet and placental membranes and precipitation with polyethylene glycol separated adenotin or the adenotin-like protein from a second adenosine binding site in each tissue. The pharmacologic properties of the precipitated binding sites were compatible with an adenosine A2 receptor in platelets and an adenosine A1 receptor in placenta. Our observations indicate that adenotin-like proteins exist outside the placenta. In addition, adenotin and adenotin-like proteins coexist with the adenosine A1 or A2 receptor in a number of cells and tissues and do not couple to a guanine nucleotide regulatory protein and stimulate adenylate cyclase. Therefore, adenotin is pharmacologically distinct from adenosine receptors, and its function remains to be discovered.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/patología , Plaquetas/ultraestructura , Proteínas Portadoras/metabolismo , Coriocarcinoma/patología , Feocromocitoma/patología , Placenta/ultraestructura , Receptores Purinérgicos/metabolismo , Neoplasias Uterinas/patología , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida) , Adenilil Ciclasas/metabolismo , Neoplasias de las Glándulas Suprarrenales/química , Neoplasias de las Glándulas Suprarrenales/metabolismo , Neoplasias de las Glándulas Suprarrenales/ultraestructura , Animales , Plaquetas/química , Plaquetas/metabolismo , Proteínas Portadoras/análisis , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Coriocarcinoma/química , Coriocarcinoma/metabolismo , Coriocarcinoma/ultraestructura , Cromatografía Líquida de Alta Presión , AMP Cíclico/metabolismo , Femenino , Humanos , Feocromocitoma/química , Feocromocitoma/metabolismo , Feocromocitoma/ultraestructura , Placenta/química , Placenta/metabolismo , Polietilenglicoles , Embarazo , Radioinmunoensayo , Receptores Purinérgicos/análisis , Células Tumorales Cultivadas , Neoplasias Uterinas/química , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/ultraestructura , Vasodilatadores/análisis , Vasodilatadores/farmacologíaRESUMEN
The effects of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP) on respiration and oxidative phosphorylation in rat liver mitochondria were examined in vitro. Respiration rates of glutamate, malate and succinate were investigated in the presence of each herbicide (0.1-4.0 mM). At lower concentrations, all herbicides stimulated state 4 respiration, decreased the respiratory control ratio and the ADP/O ratio. The respiration rate in state 3 and uncoupled state was unaffected. At higher concentrations all bioenergetic parameters, respiration in state 4, 3 and uncoupled state, as well as respiratory control ratio and ADP/O, were inhibited in a concentration-dependent manner. These data indicate that these herbicides alter energy metabolism in rat liver mitochondria by uncoupling of oxidative phosphorylation. 2,4,5-TP possesses the strongest uncoupling properties followed by 2,4,5-T, MCPA and 2,4-D in that order.
Asunto(s)
Herbicidas/farmacología , Mitocondrias Hepáticas/metabolismo , Ácido 2,4,5-Triclorofenoxiacético/análogos & derivados , Ácido 2,4,5-Triclorofenoxiacético/farmacología , Ácido 2,4-Diclorofenoxiacético/farmacología , Ácido 2-Metil-4-clorofenoxiacético/farmacología , Adenosina Difosfato/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Masculino , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[3H]ethylcarboxamidoadenosine [( 3H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [3H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.