RESUMO
Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Vírus 40 dos Símios/imunologia , Proteína Supressora de Tumor p53/metabolismo , Mapeamento de Peptídeos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Testes de Precipitina , Proteína Fosfatase 2 , Especificidade por SubstratoRESUMO
A model for medulloblastoma-like primitive neuroectodermal tumors was established in rat using retrovirally transduced SV40 large T antigen (LT) as an inducing agent (O. D. Wiestler et al., Brain Pathol., 2: 47-59, 1992). A cell line isolated from such a tumor and clonal derivatives thereof were biologically and molecularly characterized. In the parental tumor cell line, TZ870, which had been selected for G418 resistance, virtually all cells expressed LT and wild-type p53, which were complexed to each other. When plated in soft agar, these cells grew relatively slowly and formed disperse colonies. However, when grown without selection pressure, these cells reproducibly gave rise to LT-negative and G418-sensitive derivatives, LT-0 cells. Surprisingly, these latter cells exhibited a higher degree of malignancy both in vitro, growing readily to large colonies in soft agar, and in vivo, where they gave rise to a rapidly growing malignant tumor. Clonal selection from TZ870 cells revealed two types of clones: in one type, LT expression was stably maintained, even without selection pressure, whereas the other type lost the LT coding sequences. All LT-negative clones exhibited the same phenotype as the LT-0 cells. Reexpression of LT had no effect. However, LT no longer formed complexes with p53, and p53 was metabolically stable, suggesting that it had been mutated. Sequence analyses and diagnostic restriction digests of the p53 gene revealed that (a) both the parental LT-transformed cells and their derivatives contained only one complete p53 allele and (b) all LT-positive clones expressed wild-type p53, whereas all LT-negative clones expressed a mutant allele with a common mutation at Cys-174-->Tyr, indicating their clonal origin. We assume that the loss of LT coding sequences is the consequence of the p53 mutation, perhaps by inducing genomic instability, and that both the p53 mutation and additional genetic alterations that accompany the loss of LT coding sequences might contribute to enhanced malignancy.
Assuntos
Antígenos Virais de Tumores/genética , Tumores Neuroectodérmicos Primitivos Periféricos/genética , Proteína Supressora de Tumor p53/genética , Animais , Antígenos Virais de Tumores/biossíntese , Neoplasias Encefálicas/patologia , Testes de Carcinogenicidade , Linhagem Celular , Transformação Celular Viral , Mutação , Tumores Neuroectodérmicos Primitivos Periféricos/induzido quimicamente , Ratos , Reprodutibilidade dos Testes , Retroviridae/genética , Vírus 40 dos Símios/genética , Células Tumorais Cultivadas , Integração ViralRESUMO
The tumor suppressor protein p53 is phosphorylated at multiple sites in the amino-terminal transactivation domain and at several sites in the carboxy-terminal region. Phosphorylation appears to modulate its DNA binding activity. Here we demonstrate that phosphorylation of p53 also modulates its transcriptional activity. Okadaic acid treatment of cells resulted in enhanced phosphorylation of p53 and concomitantly in enhanced transactivation of an mdm2 promoter-linked luciferase reporter gene. This effect was cell type specific, however, since transactivation was enhanced in rat and mouse fibroblasts but reduced in the human Saos-2 cell line. Moreover, the effect was dependent on the promoter. In rat cells transcription from the mdm2, waf1 (cip1) and bax gene promoters, and the artificial PG13 promoter was enhanced by okadaic acid treatment whereas that from the cyclin G promoter was reduced. When various phosphorylation site mutants of p53 were tested for transactivation of these promoters, they behaved differently. Amino-terminal mutants exhibited reduced transcriptional activities on mdm2, waf1 and cyclin G promoters but enhanced activities with bax and PG13 promoters. On the other hand, a mutant at the cdk phosphorylation site, A313, showed reduced activity with mdm2 and waf1 promoters but enhanced activity with the cyclin G promoter, and finally, mutant A390 exhibited enhanced activity on waf1 and bax promoters, but reduced activity on the cyclin G promoter. These results suggest that phosphorylation of p53 may have different effects on its transcriptional activity, depending on the cellular environment and the particular response element. Moreover, both, amino- and carboxy-terminal phosphorylation sites seem to be involved in modulating the DNA-binding and the transactivation activities.
Assuntos
Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Ácido Okadáico/farmacologia , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular/efeitos dos fármacos , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Ratos , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
In SV40-transformed or infected rat cells phosphorylation of the tumor suppressor protein p53 is enhanced due to activation of kinases. At least three different kinases can be co-precipitated with p53-large T (LT) immune complexes, casein kinase II representing the major activity, a cyclin dependent kinase (Cdk), and a kinase which appears to be specifically activated by LT (E Müller, B Boldyreff and KH Scheidtmann, Oncogene 8: 2193-2205, 1993). In this paper we describe the purification and identification of the LT-activated kinase that phosphorylates a site adjacent to the Cdk site in rat p53. To monitor the activity a synthetic peptide was used containing glutamic acid at the position of Ser-313, thus mimicking a phosphorylated Cdk site. With a combination of Mono Q chromatography and subsequent affinity chromatographies with p13suc1 and a p53-fragment as ligands a 42 kDa protein kinase was purified to near homogeneity from SV40-transformed rat cells. This kinase phosphorylated both the peptide substrate and the native rat p53. Interestingly, phosphorylation of the specific site seemed to depend on prior phosphorylation of the Cdk site. On the other hand, the kinase seemed to be activated by LT, as the activity towards the peptide substrate was significantly higher in extracts from wild-type LT-transformed cells than from normal or mutant LT-transformed cells. This activation was not restricted to rat cells but occurred in SV40-transformed mouse and infected monkey cells as well. Phosphorylation of the specific site by LT-activated kinase was not dependent on the presence of LT in vitro suggesting that activation of the LT-activated kinase is probably indirect rather than through direct interaction with LT. Cell cycle studies revealed that the LT-activated kinase is cell cycle regulated, since its activity was not detectable in M phase but increased during G1 phase after which it remained relatively constant.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Proteínas Quinases/isolamento & purificação , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular , Transformação Celular Viral , Células Cultivadas , Cromatografia por Troca Iônica , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ratos , Serina/metabolismo , Spodoptera , Especificidade por SubstratoRESUMO
Interaction of viral oncoproteins, such as SV40 large T, with cellular growth suppressor proteins Rb and p53 is presumed to inactive or modulate their growth suppression functions, thereby leading to transformation. An additional transformation-related activity of LT leads to hyperphosphorylation of p53. To search for kinases that might be responsible for this effect, p53-LT complexes were immunopurified from different SV40-transformed rat cell lines and assayed for associated kinase activities, in vitro. Protein kinase activity was readily observed in p53-LT immunecomplexes from wild-type transformed cells but was low or undetectable in p53 from mutant-transformed or normal cells. Optimal activity required the presence of Mn++. p53 was phosphorylated at all sites found in vivo. In contrast, LT was phosphorylated only at a subset of formerly identified sites and at additional sites not seen in vivo. The p53-LT-kinase complex was assayed for the presence of casein kinases, cdk like kinases, or DNA-activated kinase, using specific effectors, antibodies, or purified enzymes as tools. DNA-activated kinase or cdc2/cdk2 were not detectable, although the purified enzymes phosphorylated p53 in vitro. Casein kinase 2 represented the major activity, which on p53 phosphorylated not only the C-terminal Ser390 but also several sites in the N-terminal region. One additional activity, not identified so far, may represent an LT-induced or activated kinase. This kinase seems to enhance overall phosphorylation of p53 and, perhaps other substrates, and may thereby contribute to transformation.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Transformação Celular Viral , Proteínas de Ligação a DNA , Proteínas Quinases/metabolismo , Vírus 40 dos Símios/genética , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/imunologia , Proteína Quinase CDC2/metabolismo , Caseína Quinases , Linhagem Celular Transformada , Proteína Quinase Ativada por DNA , Ativação Enzimática , Camundongos , Fosforilação , Proteínas Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Vírus 40 dos Símios/imunologiaRESUMO
The tumor suppressor protein p53 exists in different phosphorylation states depending on the cellular environment and perhaps the stage of the cell cycle. These different phosphorylation states can be mimicked in the baculovirus expression system by employing the phosphatase inhibitor okadaic acid. Hyperphosphorylation of p53, particularly of Ser313 and/or Ser309, stimulated its DNA binding activity (Fuchs, Hecker and Scheidtmann, Eur. J. Biochem. 228, 625, 1995). Here we show that hyperphosphorylation of p53 has different effects on its DNA-binding activity, depending on the phosphorylation sites and the binding motif: (i) Phosphorylation of amino-terminal sites appeared to reduce binding to the RGC consensus motif, whereas additional phosphorylation of both, Ser313 and Ser309 led to enhanced binding. (ii) Upon hyperphosphorylation, binding to the RGC motif was enhanced whereas binding to the p53 response element of the bax1 gene promoter was diminished. (iii) DNA binding was also greatly enhanced by antibodies Pab 122 and 421 directed against the carboxyl terminus, but this latter effect was superimposed by the phosphorylation state of p53. Thus, the DNA binding activity of p53 appears to be regulated in a complex way in that (i) binding to a given sequence motif may be regulated by differential phosphorylation and/or by interaction with other factors; (ii) binding to different motifs may be modulated in opposite ways. Thus, the different genes that are regulated by p53 may be differently affected by these parameters.
Assuntos
DNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Anticorpos/farmacologia , Baculoviridae , Sequência de Bases , Linhagem Celular Transformada , Éteres Cíclicos/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Ratos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/imunologiaRESUMO
To investigate the effect of phosphorylation on the transcription activity of p53, ten phosphorylation mutants were constructed covering all the identified phosphorylation sites of rat p53. These included mutants of two casein kinase I sites (Ser6 and Ser9), two DNA-PK sites (Ser15 and Ser39), a p34cdc2 site (Ser313), the adjacent Ser312 and a casein kinase II site (Ser390). Two double phosphorylation mutants (Ser4, 6 and Ser15, 390) and one triple phosphorylation mutant (Ser4, 6 and 15) were also constructed. The transcription activity of all the p53 phosphorylation mutants was tested by transfection into two different types of cells, Saos-2 cells and p53(-/-) fibroblasts derived from p53 knock out mice, which both lack endogenouse p53. Surprisingly, all the p53 phosphorylation mutants retain transcription activity and the seven mutants tested can also suppress cell growth.
Assuntos
Proteína Supressora de Tumor p53/metabolismo , Animais , Divisão Celular , Células Cultivadas , Genes p53 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Ratos , Relação Estrutura-Atividade , Transcrição Gênica , Ativação Transcricional , Proteína Supressora de Tumor p53/químicaRESUMO
Dlk (also termed ZIP kinase) is a novel serine/threonine kinase with a unique C-terminal domain that is rich in arginine and contains three putative NLS motifs and a functional lecuine zipper. Dlk is indeed localized in the nucleus where it shows a speckled distribution. To elucidate the biological functions of Dlk, we wanted to identify the signals relevant for nuclear transport and further the nuclear structures which Dlk binds to. Expression of various deletion and point mutations of Dlk as GFP fusion proteins revealed that the leucine zipper is required for association with speckles and the most C-terminal NLS is necessary and sufficient for nuclear transport. Interestingly, a C-terminal deletion mutant defective for nuclear transport exhibited a pronounced colocalization with actin filaments and, even more strikingly, was a very potent inducer of apoptosis. This apoptotic activity was abrogated, however, when this mutant was retargeted to the nucleus via a heterologous NLS from large T, indicating that Dlk only exerts an apoptotic activity in the cytoplasm. To identify the speckle like structures to which Dlk binds we performed immunofluorescence analyses with antibodies directed against representative marker proteins of replication, transcription, or splicing centers. None of these marker proteins revealed a colocalization with Dlk. Instead, we found a partial colocalization with PML bodies which seem to play a key role in regulation of apoptosis. Taken together, these data strongly suggest a functional role for Dlk in control of cell survival which is dependent on its subcellular localization.
Assuntos
Apoptose/genética , Núcleo Celular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Transporte Biológico/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Linhagem Celular , Proteínas Quinases Associadas com Morte Celular , Zíper de Leucina , MutaçãoRESUMO
Dlk/ZIP kinase is a newly discovered serine/threonine kinase which, due to its homology to DAP kinase, was named DAP like kinase, Dlk. This kinase is tightly associated with nuclear structures, it undergoes extensive autophosphorylation and phosphorylates myosin light chain and core histones H3, H2A and H4 in vitro. Moreover, it possesses a leucine zipper which mediates interaction with transcription factor ATF-4, therefore it was called ZIP kinase. We employed the yeast two-hybrid system to identify interaction partners of Dlk that might serve as regulators or targets. Besides ATF-4 and others we found Par-4, a modulator of transcription factor WT1 and mediator of apoptosis. Complex formation between Dlk and Par-4 was confirmed by GST pull-down experiments and kinase reactions in vitro and coexpression experiments in vivo. The interaction domain within Dlk was mapped to an arginine-rich region between residues 338 - 417, rather than to the leucine zipper. Strikingly, coexpression of Dlk and Par-4 lead to relocation of Dlk from the nucleus to the cytoplasm, particularly to actin filaments. These interactions provoked a dramatic reorganization of the cytoskeleton and morphological symptoms of apoptosis, thus suggesting a functional relationship between Dlk and Par-4 in the control of apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Proteínas de Transporte/genética , Linhagem Celular Transformada , Citoplasma/fisiologia , Proteínas Quinases Associadas com Morte Celular , Escherichia coli , Zíper de Leucina , MAP Quinase Quinase Quinases/genética , Fosforilação , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
We cloned a cDNA coding for a novel serine/threonine kinase, Dlk, a protein of 448 amino acids with a predicted molecular weight of 51.3 kDa. The kinase domain shows 81% amino acid sequence identity to the recently identified DAP kinase (death associated protein kinase) (Deiss et al., Genes & Dev., 9, 15-30, 1995), therefore, the new kinase was called Dlk, for DAP like kinase. Northern analyses revealed a single mRNA species of 1.7 kb which was ubiquitously expressed. However, expression levels varied considerably in different cell lines and tissues. Moreover, expression was downregulated upon UV irradiation. Dlk exhibited autophosphorylation activity, predominantly towards threonine residues and phosphorylated the regulatory subunit of myosin light chain, but in this case exclusively at serine residues. Dlk seems to be tightly associated with insoluble nuclear structures, presumably chromatin, since it was resistant to various rigorous extraction procedures but it was partially released upon DNase I digestion of nuclei. Consistent with this, purified Dlk phosphorylated core histones H3, H2A and H4 as exogenous substrates and endogenous histone H3 in kinase assays with nuclear extracts. Expression as GFP-fusion protein revealed a diffuse as well as a speckled nuclear staining suggesting an association with replication or transcription centers.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Linhagem Celular Transformada , Células Cultivadas , DNA Complementar/genética , Proteínas Quinases Associadas com Morte Celular , Indução Enzimática/efeitos da radiação , Fibroblastos/enzimologia , Zíper de Leucina , MAP Quinase Quinase Quinases , Dados de Sequência Molecular , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Raios UltravioletaRESUMO
Primitive neuroectodermal tumors (PNETs) such as human medulloblastomas are genetically heterogeneous and therefore poorly understood. In a rat model the SV40 large T antigen was used to induce neoplasms with characteristic features of PNETs. Tumor development requires a latency period of 8-11 months implicating secondary genetic alterations. To identify such secondary alterations we performed comparative analyses of two phenotypically identical PNET-derived cell lines. Indeed, these cell lines displayed distinct high-level amplification sites. Using a combination of subtractive cDNA analysis and radiation hybrid mapping we have now identified genes in the amplicon regions of the two cell lines. Interestingly, one of these genes encodes the rat homolog of a cytosolic branched chain aminotransferase (BCAT(C)) previously shown to be amplified in a mouse teratocarcinoma cell line. We propose that this simple cloning strategy may serve as a powerful tool for the isolation of genes implicated in known chromosomal aberrations in primary tumors and tumor cell lines.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Neoplasias Encefálicas/genética , Amplificação de Genes , Tumores Neuroectodérmicos Primitivos/genética , Sequência de Aminoácidos , Animais , Neoplasias Encefálicas/imunologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Camundongos , Dados de Sequência Molecular , Tumores Neuroectodérmicos Primitivos/imunologia , Hibridização de Ácido Nucleico , Ratos , Homologia de Sequência de Aminoácidos , Transaminases/genética , Células Tumorais CultivadasRESUMO
A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.
Assuntos
Arabidopsis/metabolismo , Histonas/metabolismo , Hordeum/metabolismo , Fosfotreonina/metabolismo , Secale/metabolismo , Treonina , Vicia faba/metabolismo , Arabidopsis/genética , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Hordeum/genética , Imuno-Histoquímica , Meiose , Microscopia Eletrônica , Mitose , Fosforilação , Fosfosserina/metabolismo , Proteínas de Plantas/metabolismo , Secale/genética , Vicia faba/genéticaRESUMO
Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half-maximal inactivation U0 = 44 +/- 7 mV. The corresponding values of connexin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 40 +/- 2 pS as well as z = 3.7 +/- 0.8 and U0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants. In order to characterize cell and tissue distribution as well as phosphorylation of connexin40 and -43 proteins, antibodies to C-terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitation, and immunofluorescence analysis of transfected cells where they exhibited the punctate pattern characteristic of gap junctions on contacting membranes. Phosphorylation of connexin40 was shown by immunoprecipitation from 32P-labeled, transfected SkHep1 cells. Analyses of protein distribution in tissues revealed that the amount of connexin40 detected in heart was higher than in lung which is the inverse of the level of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and skin is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phosphorylated than in embryonic heart or in transfected human cells.
Assuntos
Conexina 43/fisiologia , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Adenocarcinoma/patologia , Animais , Comunicação Celular , Conexina 43/imunologia , Conexinas/imunologia , Coração Fetal/química , Junções Comunicantes/química , Junções Comunicantes/ultraestrutura , Células HeLa , Humanos , Camundongos , Miocárdio/química , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/fisiologia , Especificidade de Órgãos , Técnicas de Patch-Clamp , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína alfa-5 de Junções ComunicantesRESUMO
We recently reported intriguing properties of neural tumors generated by retrovirus-mediated transfer of the SV40 large T antigen into fetal rat brain transplants. Histopathologically, these neoplasms displayed characteristic features of primitive neuroectodermal tumors (PNET) and exhibited a striking potential for migration into the host brain. In the present study, four cell lines were derived from these PNETs and characterized. Two lines with an immature phenotype expressed the embryonal form of the neural cell adhesion molecule and nestin. They showed spheroid formation and delicate cell processes. The remaining cell lines had a flat, epitheloid appearance and were immunoreactive for synaptophysin, neurofilament proteins and glial fibrillary acidic protein. These cells constitute valuable tools to study the cellular origin(s) and molecular basis of PNETs, differentiation of neural progenitors and tumor cell migration in the brain.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Tumores Neuroectodérmicos Primitivos/imunologia , Tumores Neuroectodérmicos Primitivos/patologia , Neurônios/fisiologia , Animais , Antígenos Transformantes de Poliomavirus/análise , Encéfalo/embriologia , Transplante de Tecido Fetal , Técnicas de Transferência de Genes , Imuno-Histoquímica , Testes de Precipitina , Ratos/embriologia , Ratos Endogâmicos F344 , Retroviridae/genética , Células Tumorais CultivadasRESUMO
We transiently exposed SV 40 infected TC 7 cell cultures to a reduced O2 tension (4-8 h, about 200 ppm relative to 10(5) Pa total pressure). Under the hypoxic conditions, 'working' viral replication forks were greatly retarded or stopped, and initiation of daughter strand synthesis in further SV 40 DNA molecules was suppressed. Reoxygenation released an immediate burst of SV 40 replication which mainly consisted of a synchronous viral replication round. This synchronous in vivo replication began at the known origin of replication and proceeded at normal rates to the known termination region. Viral replicons seemed to accumulate under hypoxia in a state fully prepared to begin replication immediately after recovery of a normal pO2. The shut-down and sudden reactivation of DNA synthesis under hypoxia and reoxygenation, respectively, were not accompanied by changes of the phosphorylation state of large T antigen. The described synchronization procedure can be applied to optionally large SV 40 infected cell cultures.
Assuntos
Replicação do DNA , DNA Viral/biossíntese , Vírus 40 dos Símios/genética , Animais , Antígenos Transformantes de Poliomavirus/biossíntese , Antígenos Transformantes de Poliomavirus/metabolismo , Hipóxia Celular , Linhagem Celular , Chlorocebus aethiops , Células Clonais , DNA Viral/isolamento & purificação , Cinética , Fosfatos/metabolismo , Fosfopeptídeos/análise , Timidina/metabolismo , Fatores de Tempo , TrítioRESUMO
Dlk, also known as ZIP kinase, is a serine/threonine kinase that is tightly associated with nuclear structures. Under certain conditions, which require cytoplasmic localization, Dlk can induce apoptosis. In search for interaction partners that might serve as regulators or targets of this kinase we identified apoptosis antagonizing transcription factor (AATF), a nuclear phosphoprotein of 523 amino acids. The 1.8 kb mRNA seems to be ubiquitously expressed. AATF contains an extremely acidic domain and a putative leucine zipper characteristic of transcription factors. Indeed, a Gal4-BD-AATF fusion protein exhibited strong transactivation activity. Interestingly, AATF interfered with Dlk-induced apoptosis.
Assuntos
Apoptose , MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Células Cultivadas , DNA Complementar/análise , Proteínas Quinases Associadas com Morte Celular , Dados de Sequência Molecular , Proteínas Nucleares , Ratos , Fatores de Transcrição/metabolismoRESUMO
The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating beta-subunits and two catalytic alpha- or alpha'-subunits and the interaction with p53 is mediated by the regulatory beta-subunit of CK2. Recently we showed that the beta-subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the beta-subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the beta-subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the beta-subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21(WAF1/CIP1) and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down-regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 beta-subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 beta-subunit in vivo, which does not affect p53 mediated transrepression.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Células COS , Caseína Quinase II , Linhagem Celular , DNA/genética , DNA/metabolismo , Expressão Gênica , Genes fos , Genes p53 , Humanos , Camundongos , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
Dlk/ZIP kinase is one of five members of the death associated protein (DAP) kinase family. DAP kinase is able to induce apoptosis in a p19ARF/p53-dependent manner. We elucidated the potential role of the p19ARF/p53 pathway in Dlk/ZIP kinase-triggered cell death. Overexpression of a constitutively pro-apoptotic form of Dlk/ZIP kinase induced apoptosis in rat fibroblast cells which express wild-type p19ARF and p53. Cell death was characterised by apoptotic membrane blebbing, mitochondrial depolarisation, cytochrome c release and activation of caspase-3. However, Dlk/ZIP kinase-triggered cell death was also observed in p19ARF-deficient and p53-deficient mouse fibroblast cells. Quantitative analysis revealed that the status of p53 had no major influence on cellular susceptibility to Dlk/ZIP kinase-triggered cell death. Loss of p53 did not prevent Dlk/ZIP kinase-induced mitochondrial membrane depolarisation and release of cytochrome c. Furthermore, overexpression of Dlk/ZIP kinase did not lead to an increased expression of pro-apoptotic p53 target genes in either cell line. These data suggest that Dlk/ZIP kinase is able to trigger the mitochondrial apoptosis pathway independent of the p19ARF/p53 signalling pathway.
Assuntos
Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Genes p53/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor p14ARF/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Proteínas Quinases Associadas com Morte Celular , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Thirteen synthetic peptides corresponding to different regions of the SV40 large T antigen were used as immunogens after coupling to a carrier protein. All peptide conjugates elicited sera that recognized the inducing peptide. In 10 cases the corresponding sites in the native large T antigen also were recognized, as determined by immunoprecipitation. The degree of recognition of the native protein varied between 0.5 and 80%, the most reactive sera being those induced by the terminal peptides. The ability of internal peptides to induce antibodies reactive with native large T antigen appeared to be correlated with peptide hydrophilicity and possibly atomic mobility. No such correlation was apparent with predicted features of secondary structure. The influence of peptide length on induction of protein-recognizing antisera will also be discussed.
Assuntos
Antígenos Virais de Tumores/análise , Soros Imunes/isolamento & purificação , Proteínas Oncogênicas Virais/análise , Peptídeos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores/imunologia , Antígenos Virais de Tumores/fisiologia , Proteínas de Transporte , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Imunização/métodos , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/fisiologia , Peptídeos/síntese química , Peptídeos/metabolismo , Testes de Precipitina/métodos , Coelhos/imunologia , Relação Estrutura-AtividadeRESUMO
The SV40 large T antigen is a viral oncoprotein which performs multiple interactions with cellular factors to achieve a proliferative state required for viral replication as well as for transformation. The major targets in this scenario are members of the Rb family, pRb, p107, and p130, and tumor suppressor protein p53. These interactions of large T with Rb proteins and p53 are required but not sufficient for transformation. To search for unknown interaction partners of large T that might participate in its transforming activity we employed the yeast two-hybrid system. Screening a cDNA library from a large T-induced brain tumor cell line revealed a total of 86 positive clones representing 37 individual clones. Of these, four clones were selected for further analyses. Interestingly, the cDNA inserts of these clones coded for different components of the cytoskeleton, lamin C, laminin gamma1, thymosin beta4, and gelsolin. Complex formation between large T and these proteins was confirmed in vitro. Interaction of large T with these components might influence activities such as intracellular transport, signal transduction, adhesion, or migration.