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1.
J Biol Chem ; 276(39): 36320-6, 2001 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-11470787

RESUMO

The IkappaB kinase (IKK) complex, composed of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma), is the key enzyme in activation of nuclear factor kappaB (NF-kappaB). To study the mechanism and structure of the complex, we wanted to recombinantly express IKK in a model organism that lacks IKK. For this purpose, we have recombinantly reconstituted all three subunits together in yeast and have found that it is biochemically similar to IKK isolated from human cells. We show that there is one regulatory subunit per kinase subunit. Thus, the core subunit composition of IKKalpha.beta.gamma complex is alpha(1)beta(1)gamma(2), and the core subunit composition of IKKbeta.gamma is beta(2)gamma(2). The activity of the IKK complex (alpha+beta+gamma or beta+gamma) expressed in yeast (which lack NF-kappaB and IKK) is 4-5-fold higher than an equivalent amount of IKK from nonstimulated HeLa cells. In the absence of IKKgamma, IKKbeta shows a level of activity similar to that of IKK from nonstimulated HeLa cells. Thus, IKKgamma activates IKK complex in the absence of upstream stimuli. Deleting the gamma binding domain of IKKbeta or IKKalpha prevented IKKgamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKKgamma into IKK and large complex formation. The possibility of IKK complex being under negative control in mammalian cells is discussed.


Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Western Blotting , Catálise , Cromatografia em Gel , Clonagem Molecular , Ativação Enzimática , Deleção de Genes , Células HeLa , Humanos , Quinase I-kappa B , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína
2.
J Biol Chem ; 276(32): 30188-98, 2001 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-11402028

RESUMO

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.


Assuntos
Etídio/análogos & derivados , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Oxirredução , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Catalase/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Espectroscopia de Ressonância de Spin Eletrônica , Ativação Enzimática , Etídio/farmacologia , Regulação da Expressão Gênica , Genes Dominantes , Glutationa Transferase/metabolismo , Humanos , Quinase I-kappa B , Receptores de Lipopolissacarídeos/metabolismo , Luciferases/metabolismo , Camundongos , Modelos Biológicos , Mutação , Ligação Proteica , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Superóxidos/metabolismo , Fatores de Tempo , Regulação para Cima
4.
J Biol Chem ; 273(46): 30544-9, 1998 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-9804824

RESUMO

Vascular endothelial cells (ECs), forming a boundary between the circulating blood and the vessel wall, are constantly subjected to fluid shear stress due to blood flow. The aim of this study was to determine the role of the recently identified IkappaB kinases (IKKs) in shear stress activation of NF-kappaB and to elucidate the upstream signaling mechanism that mediates IKK activation. Our results demonstrate that IKKs in ECs are activated by shear stress in a rapid and transient manner. This IKK activation is followed by IkappaB degradation and NF-kappaB translocation into the nucleus. Transfection of plasmids encoding catalytic inactive mutants of IKKs, i.e. hemagglutinin (HA)-IKKalpha(K44M) and HA-IKKbeta(K44A), inhibits shear stress-induced NF-kappaB translocation. In addition, constructs encoding antisense IKKs, i.e. HA-IKKalpha(AS) and HA-IKKbeta(AS), attenuate shear stress induction of a promoter driven by the kappaB enhancer element. Preincubation of the EC monolayer with a monoclonal anti-alphavbeta3 integrin antibody (clone LM609) attenuates shear stress induction of IKK. Inhibition of tyrosine kinases by genistein causes a similar down-regulating effect. These results suggest that the integrin-mediated signaling pathway regulates NF-kappaB through IKKs in ECs in response to shear stress.


Assuntos
Hemorreologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Vitronectina/metabolismo , Animais , Catálise , Bovinos , Adesão Celular , Células Cultivadas , Endotélio Vascular/enzimologia , Ativação Enzimática , Fibronectinas/metabolismo , Quinase I-kappa B , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estresse Mecânico
5.
Nature ; 395(6699): 297-300, 1998 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-9751060

RESUMO

Pro-inflammatory cytokines activate the transcription factor NF-kappaB by stimulating the activity of a protein kinase that phosphorylates IkappaB, an inhibitor of NF-kappaB, at sites that trigger its ubiquitination and degradation. This results in the nuclear translocation of freed NF-kappaB dimers and the activation of transcription of target genes. Many of these target genes code for immunoregulatory proteins. A large, cytokine-responsive IkappaB kinase (IKK) complex has been purified and the genes encoding two of its subunits have been cloned. These subunits, IKK-alpha and IKK-beta, are protein kinases whose function is needed for NF-kappaB activation by pro-inflammatory stimuli. Here, by using a monoclonal antibody against IKK-alpha, we purify the IKK complex to homogeneity from human cell lines. We find that IKK is composed of similar amounts of IKK-alpha, IKK-beta and two other polypeptides, for which we obtained partial sequences. These polypeptides are differentially processed forms of a third subunit, IKK-gamma. Molecular cloning and sequencing indicate that IKK-gamma is composed of several potential coiled-coil motifs. IKK-gamma interacts preferentially with IKK-beta and is required for the activation of the IKK complex. An IKK-gamma carboxy-terminal truncation mutant that still binds IKK-beta blocks the activation of IKK and NF-kappaB.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Catálise , Clonagem Molecular , Ativação Enzimática , Células HeLa , Humanos , Quinase I-kappa B , Células Jurkat , Zíper de Leucina , Dados de Sequência Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Estrutura Secundária de Proteína , Deleção de Sequência
6.
J Biol Chem ; 273(33): 21132-6, 1998 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-9694868

RESUMO

The tax gene product of human T-cell leukemia virus I induces aberrant expression of various cellular genes, which contributes to transformation of host cells. Induction of many Tax target genes is mediated through transcription factor NF-kappaB. Here we show that Tax triggers activation of cellular protein kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, which phosphorylate the NF-kappaB inhibitory protein IkappaB alpha, resulting in its degradation and NF-kappaB activation. Constitutive IKK activation occurs in both Tax-transfected and human T-cell leukemia virus I-infected T cells. We further demonstrate that Tax-mediated NF-kappaB signaling also requires the NF-kappaB-inducing kinase (NIK). Consistently, inactive forms of either IKKs or NIK attenuate Tax-mediated NF-kappaB activation. Therefore, Tax activates NF-kappaB by targeting cellular signaling molecules, including both IKKs and NIK.


Assuntos
Produtos do Gene tax/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Catálise , Humanos , Quinase I-kappa B , Células Jurkat , Fosforilação , Quinase Induzida por NF-kappaB
7.
Science ; 281(5381): 1360-3, 1998 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-9721103

RESUMO

A large protein complex mediates the phosphorylation of the inhibitor of kappaB (IkappaB), which results in the activation of nuclear factor kappaB (NF-kappaB). Two subunits of this complex, IkappaB kinase alpha (IKKalpha) and IkappaB kinase beta (IKKbeta), are required for NF-kappaB activation. Purified recombinant IKKalpha and IKKbeta expressed in insect cells were used to demonstrate that each protein can directly phosphorylate IkappaB proteins. IKKalpha and IKKbeta were found to form both homodimers and heterodimers. Both IKKalpha and IKKbeta phosphorylated IkappaB bound to NF-kappaB more efficiently than they phosphorylated free IkappaB. This result explains how free IkappaB can accumulate in cells in which IKK is still active and thus can contribute to the termination of NF-kappaB activation.


Assuntos
NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Animais , Linhagem Celular , Dimerização , Ativação Enzimática , Células HeLa , Sequências Hélice-Alça-Hélice , Humanos , Quinase I-kappa B , Zíper de Leucina , Mutação , NF-kappa B/antagonistas & inibidores , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Spodoptera , Fator de Transcrição RelB
8.
Cell ; 91(2): 243-52, 1997 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-9346241

RESUMO

Recently we purified a 900 kDa cytokine-responsive IkappaB kinase complex (IKK) and molecularly cloned one of its subunits, IKKalpha, a serine kinase. We now describe the molecular cloning and characterization of IKKbeta, a second subunit of the IKK complex. IKKbeta is 50% identical to IKKalpha and like it contains a kinase domain, a leucine zipper, and a helix-loop-helix. Although IKKalpha and IKKbeta can undergo homotypic interaction, they also interact with each other and the functional IKK complex contains both subunits. The catalytic activities of both IKKalpha and IKKbeta make essential contributions to IkappaB phosphorylation and NF-kappaB activation. While the interactions between IKKalpha and IKKbeta may be mediated through their leucine zipper motifs, their helix-loop-helix motifs may be involved in interactions with essential regulatory subunits.


Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Clonagem Molecular , Ativação Enzimática , Células HeLa , Sequências Hélice-Volta-Hélice , Humanos , Quinase I-kappa B , Zíper de Leucina , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição RelA
9.
Mol Cell Biol ; 17(8): 4687-95, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9234725

RESUMO

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.


Assuntos
Mutação , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas de Saccharomyces cerevisiae , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/genética , Tri-Iodotironina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Sequência Conservada/genética , Proteínas de Ligação a DNA , Células HeLa , Humanos , Ligantes , Modelos Moleculares , Coativador 2 de Receptor Nuclear , Conformação Proteica , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/genética , Proteínas Recombinantes de Fusão , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Nature ; 388(6642): 548-54, 1997 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-9252186

RESUMO

Nuclear transcription factors of the NF-kappaB/Rel family are inhibited by IkappaB proteins, which inactivate NF-kappaB by trapping it in the cell cytoplasm. Phosphorylation of IkappaBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kappaB. A cytokine-activated protein kinase complex, IKK (for IkappaB kinase), has now been purified that phosphorylates IkappaBs on the sites that trigger their degradation. A component of IKK was molecularly cloned and identified as a serine kinase. IKK turns out to be the long-sought-after protein kinase that mediates the critical regulatory step in NF-kappaB activation.


Assuntos
NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Ativação Enzimática , Células HeLa , Humanos , Quinase I-kappa B , Mediadores da Inflamação/metabolismo , Cinética , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Especificidade por Substrato , Fator de Necrose Tumoral alfa/metabolismo
11.
Mol Cell Biol ; 17(6): 3094-102, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9154808

RESUMO

Transcription factor AP-1 transduces environmental signals to the transcriptional machinery. To ensure a quick response yet maintain tight control over AP-1 target genes, AP-1 activity is likely to be negatively regulated in nonstimulated cells. To identify proteins that interact with the Jun subunits of AP-1 and repress its activity, we developed a novel screen for detecting protein-protein interactions that is not based on a transcriptional readout. In this system, the mammalian guanyl nucleotide exchange factor (GEF) Sos is recruited to the Saccharomyces cerevisiae plasma membrane harboring a temperature-sensitive Ras GEF, Cdc25-2, allowing growth at the nonpermissive temperature. Using the Sos recruitment system, we identified new c-Jun-interacting proteins. One of these, JDP2, heterodimerizes with c-Jun in nonstimulated cells and represses AP-1-mediated activation.


Assuntos
Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Repressoras/isolamento & purificação , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , Dimerização , Eletroforese em Gel de Poliacrilamida , Técnicas Genéticas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Fosfoproteínas Fosfatases , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Repressoras/metabolismo , ras-GRF1
12.
Genes Dev ; 11(10): 1299-314, 1997 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-9171374

RESUMO

In normally growing Drosophila cultured cells the Drosophila heat shock transcription factor (dHSF) is localized in the cytosol and translocates into the nucleus after heat shock. In the cytosol of nonshocked cells, the dHSF is present as a monomer that cannot bind DNA. Upon stress, the dHSF enters the nucleus where it is observed to be a trimer. A novel nuclear localization sequence (NLS) in the dHSF was found to be responsible for stress-dependent nuclear entry. Deletion of the NLS prevents nuclear entry, as expected, yet surprisingly also allows constitutive oligomerization and DNA binding in the cytosol. Further analysis of the NLS by mutagenesis suggests that the two functions of nuclear entry and oligomerization are separable in that distinct residues present in the NLS are responsible for each. Mutations in certain basic residues completely block nuclear entry, as expected for a constitutive NLS. In addition, two residues were found in the NLS that, when altered, allowed constitutive nuclear entry of dHSF independent of stress. These residues may interact with a putative cellular component or possibly other domains of the HSF to prevent nuclear entry in normally growing cells. The NLS can also function autonomously to target a beta-galactosidase fusion protein into the nucleus in a heat shock-dependent fashion.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Biopolímeros , Linhagem Celular , Drosophila melanogaster , Fatores de Transcrição de Choque Térmico , Dados de Sequência Molecular , Estresse Oxidativo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , beta-Galactosidase/metabolismo
13.
Curr Opin Cell Biol ; 9(2): 240-6, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9069263

RESUMO

AP-1 (activating protein-1) is a collective term referring to dimeric transcription factors composed of Jun, Fos or ATF (activating transcription factor) subunits that bind to a common DNA site, the AP-1-binding site. As the complexity of our knowledge of AP-1 factors has increased, our understanding of their physiological function has decreased. This trend, however, is beginning to be reversed due to the recent studies of gene-knockout mice and cell lines deficient in specific AP-1 components. Such studies suggest that different AP-1 factors may regulate different target genes and thus execute distinct biological functions. Also, the involvement of AP-1 factors in functions such as cell proliferation and survival has been made somewhat clearer as a result of such studies. In addition, there has been considerable progress in understanding some of the mechanisms and signaling pathways involved in the regulation of AP-1 activity. In addition to regulation by heterodimerization between Jun, Fos and ATF proteins, AP-1 activity is regulated through interactions with specific protein kinases and a variety of transcriptional coactivators.


Assuntos
Fator de Transcrição AP-1/fisiologia , Animais , Apoptose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação da Expressão Gênica , Genes fos , Genes jun , Humanos
14.
Mol Cell Biol ; 14(7): 4929-37, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8007989

RESUMO

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.


Assuntos
AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Choque Térmico/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Proteínas ras , Células 3T3 , Animais , Sequência de Bases , Linhagem Celular Transformada , Núcleo Celular/metabolismo , AMP Cíclico/farmacologia , Primers do DNA , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Genes ras , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Mamíferos , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Ratos , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Transfecção
15.
J Mol Biol ; 230(1): 124-36, 1993 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-8450530

RESUMO

The DNA binding domain of steroid receptors coincides with the cysteine-rich region encompassing the two conserved zinc fingers. In the case of the glucocorticoid receptor (GR), a weak transactivation function has been described to be adjacent or partly overlapping to the DNA binding domain, whereas stronger trans-acting functions are encoded by the amino and the carboxy domain. In this report we describe the phenotype produced by stochastic mutations of the zinc finger region. The mutants were obtained either by selected rearrangements of the rat GR cDNA, or by semi-random nucleotide substitutions. All the identified permissive rearrangements were confined to a region downstream from the first zinc finger (duplications starting between residue 474 and 492). In general, the phenotype of point mutations is compatible with established structural data. Nevertheless, we found two unexpected phenotypes. First, we noticed that the double mutant His451 Asn/Ser459Gly is stronger than the wild-type sequence in DNA binding. Secondly, substitution of the conserved Lys461 results in an abnormal behavior of the mutated GR. In particular, the mutant Lys461Tyr (61Y) displays about the same transactivation when tested in form of a minimal GR fragment (amino acids 407/556) as when tested in the amino-prolongued GR fragment (amino acids 3-556, which contains the major transactivation domain of the GR). This is in contrast with the behavior of the other mutants in which the residue 461 is intact. In these cases, transactivation capacity is normally increased more than 30-fold from GR407-556 to GR3-556. These results are discussed in terms of possible cross-talk among the DNA binding domain and other functions residing in the amino domain of the GR.


Assuntos
Proteínas de Ligação a DNA/química , Receptores de Glucocorticoides/química , Fatores de Transcrição/química , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Chlorocebus aethiops , Análise Mutacional de DNA , Elementos Facilitadores Genéticos , Rearranjo Gênico , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Relação Estrutura-Atividade , Transcrição Gênica
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