RESUMEN
ATM is a member of the phosphatidylinositol 3-kinase (PIK)-like kinases, some of which are active in regulating DNA damage-induced mitotic cell-cycle checkpoints. ATM also plays a role in meiosis. Spermatogenesis in Atm-/- male mice is disrupted, with chromosome fragmentation leading to meiotic arrest; in human patients with ataxia-telangiectasia (A-T), gonadal atrophy is common. Immuno-localization studies indicate that ATM is associated with sites along the synaptonemal complex (SC), the specialized structure along which meiotic recombination occurs. Recombination, preceded by pairing of homologous chromosomes, is thought to require heteroduplex formation between homologous DNA, followed by strand exchange. These early meiotic steps (entailing the formation and processing of meiotic recombination intermediates with DNA-strand interruptions) require ssDNA-binding proteins such as replication protein A (RPA; refs 5-7). In somatic cells, DNA damage induces ATM-dependent phosphorylation of RPA. We demonstrate here that ATM and RPA co-localize along synapsed meiotic chromosomes and at sites where interactions between ectopic homologous chromosome regions appear to initiate. In Atm-/- meiotic prophase spermatocytes, immuno-localization shows that RPA is present along synapsing chromosomes and at sites of fragmentation of the SC. These results suggest that RPA and ATM co-localize at sites where interhomologous-DNA interactions occur during meiotic prophase and where breaks associated with meiotic recombination take place after synapsis, implying a possible functional interaction between these two proteins.
Asunto(s)
Ataxia Telangiectasia/genética , Proteínas de Unión al ADN/genética , Meiosis/genética , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Recombinación Genética , Animales , Ataxia Telangiectasia/enzimología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Fragmentación del ADN/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Profase/genética , Proteína de Replicación A , Espermatocitos/citología , Espermatocitos/enzimología , Espermatocitos/metabolismo , Complejo Sinaptonémico/genética , Proteínas Supresoras de TumorRESUMEN
A cell can be thought of as a well-coached sports team. To win, it needs superstar players with specialized tasks, but it also needs team players who can be relied on to maintain constant performance. Growth factor receptors or transcriptional activators might be considered to be the cell's superstars, whereas ribosomes could be considered team players that faithfully carry out directions from mRNA. The team also needs a head coach for overall direction and assistant coaches to direct the basic skills. The assistant coaches should ensure that basic cellular functions proceed correctly and that the cell responds to specific stimuli. Since almost every phosphorylatable protein is modified by several protein kinases, protein kinases like casein kinases I and II might be the assistant coaches of cellular regulation.
RESUMEN
The ability to survive spontaneous and induced DNA damage, and to minimize the number of heritable mutations that this causes, is essential to the maintenance of genome integrity for all organisms. Early studies on model eukaryotes focused on genes acting in defined DNA repair pathways. More recent work with the budding and fission yeasts and mammalian cells has started to integrate the DNA damage response with cell physiology and the cell cycle.
RESUMEN
In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH2-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.
Asunto(s)
Quinasa de la Caseína I , Daño del ADN , Reparación del ADN , Proteínas Fúngicas/genética , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/metabolismo , Biblioteca de Genes , Genes Fúngicos , Meiosis , Metilmetanosulfonato/farmacología , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Fenotipo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Homología de Secuencia de Ácido NucleicoRESUMEN
In mammalian cells, four protein kinases form the PI3-kinase-related protein kinase (PIK) superfamily. These four enzymes-FRAP, DNA-PK, ATM, and ATR-are distinguished by their large size (all are >2500 amino acids), their common primary sequence relatedness through the carboxy-terminal protein kinase domain, and their sequence similarity to the p110 lipid kinase subunit of PI3-kinase. FRAP (FKBP12 and rapamycin-binding protein kinase) participates in mitogenic and growth factor responses in G1 and may regulate specific mRNA translation signals. DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad 3 related) are thought to participate in responses to nuclear cues that activate DNA rearrangements or cell cycle arrests. Recent studies in this protein kinase family indicate an important role for ATM and ATR in a meiotic surveillance mechanism that may regulate proper chromosome transmission.
Asunto(s)
Ciclo Celular , Daño del ADN , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas/metabolismo , Transducción de Señal , Adenosina Trifosfatasas/metabolismo , Animales , Ataxia Telangiectasia , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , ADN Helicasas/metabolismo , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Meiosis , Proteínas Nucleares , Fosfatidilinositol 3-Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión a Tacrolimus , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essential part in the signal transduction pathways that regulate cell growth in response to extracellular signals. A great number of protein kinases have been discovered, and the identification of their biological targets is still a very active research area. Protein kinases must have the appropriate substrate specificity to ensure that signals are transmitted correctly. Previous studies have demonstrated the importance of primary sequences within substrate proteins in determining protein kinase specificity, but efficient ways of identifying these sequences are lacking. RESULTS: We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates. In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. The optimal substrate predicted for cAMP-dependent protein kinase (PKA) by this technique is consistent with the sequences of known PKA substrates. The optimal sequences predicted for cyclin-dependent kinases (CDKs) cyclin B-Cdc2 and cyclin A-CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases. In addition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specificity. We also discuss a model incorporating the optimal cyclin B-Cdc2 substrate into the known crystal structure of this kinase. CONCLUSIONS: Using the new technique we have developed, the sequence specificity of protein kinases can rapidly be predicted and, from this information, potential targets of the kinases can be identified.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Péptidos/química , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Quinasa CDC2/metabolismo , Secuencia de Consenso , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fosforilación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.
Asunto(s)
Meiosis/fisiología , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas/fisiología , Recombinación Genética/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteínas de Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromosomas/metabolismo , ADN Complementario , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Mamíferos , Meiosis/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Quinasas/genética , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Testículo/metabolismo , Proteínas Supresoras de TumorRESUMEN
Genetic analysis of protein kinases in Saccharomyces cerevisiae has revealed protein phosphorylation as a key regulatory mechanism both in the mitotic cell cycle and in meiosis. This article reviews genetically identified protein kinases that are associated with DNA metabolism and the meiotic pathway.
Asunto(s)
ADN de Hongos/metabolismo , Meiosis/fisiología , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Studies from a wide array of different fields using Saccharomyces cerevisiae as an experimental organism have uncovered protein phosphorylation as a recurrent theme in the regulation of diverse cellular activities. Protein kinases in yeast regulate a variety of processes; this article discusses several genetically identified protein kinases and the roles that these kinases play in cell growth and development.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Aminoácidos/metabolismo , Transporte Biológico , Fusión Celular , Proteínas Fúngicas/metabolismo , Factor de Apareamiento , Péptidos/fisiología , Fosforilación , Biosíntesis de Proteínas , Proteínas Quinasas/fisiología , Reproducción , Saccharomyces cerevisiae/fisiología , Transducción de Señal , Sacarosa/metabolismo , Transcripción GenéticaRESUMEN
Chromosome translocations involving antigen receptor loci are a genetic hallmark of non-Hodgkin's lymphomas in humans. Most commonly, these translocations result in juxtaposition of the immunoglobulin heavy-chain (IgH) locus with one of several cellular proto-oncogenes, leading to deregulated oncogene expression. The V(D)J recombinase, which mediates physiologic rearrangements of antigen receptor genes, may play a mechanistic role in some lymphoma translocations, although evidence is indirect. A high incidence of B-lineage lymphomas has been observed in mice with severe combined immunodeficiency (SCID) and p53-null mutations. We show that these tumors are characteristic of the pro-B-cell stage of development and that they harbor recurrent translocations involving chromosomes 12 and 15. Fluorescence in situ hybridization (FISH) shows retention of IgH sequences on the derivative chromosome 12, implying that breakpoints involve the IgH locus. Pro-B-cell lymphomas were suppressed in SCID p53(-/-) mice by a Rag-2-null mutation, demonstrating that DNA breaks generated during V(D)J recombination are required for oncogenic transformation, and suggesting that t(12;15) arise during attempted IgH rearrangement in pro-B cells. These studies indicate that the oncogenic potential inherent in antigen receptor diversification is controlled in vivo by efficient rejoining of DNA ends generated during V(D)J recombination and an intact cellular response to DNA damage.
Asunto(s)
ADN Nucleotidiltransferasas/genética , Linfoma de Células B/enzimología , Linfoma de Células B/genética , Translocación Genética , Animales , Linfocitos B/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas Pesadas de Inmunoglobulina/genética , Inmunofenotipificación , Linfoma de Células B/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Ratones SCID , Receptores de Antígenos de Linfocitos B/genética , Células Madre/inmunología , Translocación Genética/inmunología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , VDJ RecombinasasRESUMEN
The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E. coli shuttle vector. Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences. Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation. The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure. The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined. A small but definite general increase was found in the frequency of mitotic recombination. A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency. The effects on mutation appear to be locus (or allele) specific. Recombination in meiotic cells was measured and was not detectably altered by the presence of 6-methyladenine in GATC sequences.
Asunto(s)
ADN de Hongos/genética , Metilación , Metiltransferasas/genética , Mutación , Recombinación Genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Reparación del ADN , Escherichia coli/enzimología , Escherichia coli/genética , Meiosis , Mitosis , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)RESUMEN
Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.
Asunto(s)
Reparación del ADN , Escherichia coli/genética , Genes Bacterianos , Saccharomyces cerevisiae/genética , Adenina , Enzimas de Restricción del ADN , Genotipo , Metilación , PlásmidosRESUMEN
To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.
Asunto(s)
Proteínas de Ciclo Celular/genética , Reparación del ADN , Proteínas Fúngicas/genética , Genes Fúngicos , Recombinación Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , FN-kappa B , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-rel , Proto-Oncogenes , Proteínas Recombinantes de Fusión/fisiología , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.
Asunto(s)
Quinasa de la Caseína I , Ciclo Celular , Isoenzimas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , Caseína Quinasas , Bovinos , División Celular , Genes Fúngicos , Genotipo , Isoenzimas/biosíntesis , Isoenzimas/química , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis , Proteína Quinasa C/metabolismo , Proteínas Quinasas/biosíntesis , Prenilación de Proteína , Mapeo Restrictivo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Supresión GenéticaRESUMEN
We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Proteínas de Caenorhabditis elegans , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/metabolismo , Quinasa de la Caseína II , Caseína Quinasas , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Bases de Datos Factuales , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Modelos Moleculares , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Quinasa 1 Relacionada con NIMA , Quinasas Relacionadas con NIMA , Oligopéptidos/química , Oligopéptidos/metabolismo , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Fosforilasa Quinasa/metabolismo , Conformación Proteica , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Especificidad por SustratoRESUMEN
We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.
Asunto(s)
Isoenzimas/metabolismo , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Caseína Quinasas , Genes Fúngicos , Isoenzimas/genética , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Especificidad por Sustrato , TirosinaRESUMEN
The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.
Asunto(s)
Isoenzimas/metabolismo , Proteínas Quinasas/metabolismo , Inhibidores de Topoisomerasa II , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células COS , Camptotecina/farmacología , Caseína Quinasas , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ratones , Fosfopéptidos/análisis , Fosforilación , ARN Mensajero/genética , RatasRESUMEN
To test the double-strand break repair model, we used HO nuclease to introduce double-strand breaks at several sites along a yeast chromosome containing duplicated DNA. Depending on the configuration of the double-strand break and recombining markers, different spectra of recombinant products were observed. Different repair kinetics and recombinant products were observed when a double-strand break was introduced in unique or duplicated DNA. The results of this study suggest that double-strand breaks in yeast stimulate recombination by several mechanisms, and we propose an alternative mechanism for double-strand break-induced gene conversion that does not depend on direct participation of the broken ends.
Asunto(s)
Reparación del ADN , ADN de Hongos/genética , ADN/genética , Alelos , Conversión Génica , Genes Fúngicos , Cinética , Modelos Genéticos , Familia de Multigenes , Recombinación Genética , Saccharomyces cerevisiaeRESUMEN
Mutations in the REM1 gene of Saccharomyces cerevisiae confer a semidominant hyper-recombination and hypermutable phenotype upon mitotic cells ( GOLIN and ESPOSITO 1977). These effects have not been observed in meiosis. We have examined the interactions of rem1 mutations with rad6-1, rad50 -1, rad52-1 or spo11 -1 mutations in order to understand the basis of the rem1 hyper-rec phenotype. The rad mutations have pleiotropic phenotypes; spo11 is only defective in sporulation and meiosis. The RAD6, RAD50 and SPO11 genes are not required for spontaneous mitotic recombination; mutations in the RAD52 gene cause a general spontaneous mitotic Rec- phenotype. Mutations in RAD50 , RAD52 or SPO11 eliminate meiotic recombination, and mutations in RAD6 prevent spore formation. Evidence for the involvement of RAD6 in meiotic recombination is less clear. Mutations in all three RAD genes confer sensitivity to X rays; the RAD6 gene is also required for UV damage repair. To test whether any of these functions might be involved in the hyper-rec phenotype conferred by rem1 mutations, double mutants were constructed. Double mutants of rem1 spo11 were viable and demonstrated rem1 levels of mitotic recombination, suggesting that the normal meiotic recombination system is not involved in producing the rem1 phenotype. The rem1 rad6 double mutant was also viable and had rem1 levels of mitotic recombination. Neither rem1 rad50 nor rem1 rad52 double mutants were viable. This suggests that rem1 causes its hyper-rec phenotype because it creates lesions in the DNA that are repaired using a recombination-repair system involving RAD50 and RAD52.