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1.
Proc Natl Acad Sci U S A ; 106(20): 8198-203, 2009 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-19416843

RESUMO

Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen." Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small ( approximately 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.


Assuntos
Antígenos CD/metabolismo , Epitopos , Lipocalinas/metabolismo , Engenharia de Proteínas , Proteínas de Fase Aguda/genética , Anticorpos/química , Antígenos CD/química , Sítios de Ligação , Antígeno CTLA-4 , Cristalografia por Raios X , Humanos , Indicadores e Reagentes/síntese química , Indicadores e Reagentes/química , Lipocalina-2 , Lipocalinas/química , Lipocalinas/genética , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas/genética
2.
Br J Cancer ; 101(8): 1456-60, 2009 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-19707196

RESUMO

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93-1.10, P(trend)=0.77; MDM2: HR=0.96, 95%CI: 0.84-1.09, P(trend)=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87-1.12, P(trend)=0.83; MDM2: HR=0.98, 95%CI: 0.80-1.21, P(trend)=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.


Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Genes p53 , Predisposição Genética para Doença , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/genética , Neoplasias da Mama/etiologia , Feminino , Heterozigoto , Humanos , Fatores de Risco
3.
Curr Biol ; 5(10): 1191-200, 1995 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-8548291

RESUMO

BACKGROUND: The mammalian response to stress results in the activation of stress-activated protein kinases (also known as cJun N-terminal kinases; SAPKs or JNKs), which are a sub-group of the mitogen-activated protein (MAP) kinase family. The SAPKs are involved in the upregulation of activity of the transcription factor AP-1 by post-translational modification of two of its components, cJun and ATF2. AP-1 activity can also be elevated by increased expression of the Fos protein, a further AP-1 component. Elk-1 (also called p62TCF), a transcription factor involved in the induction of the expression from the c-fos promoter through the promoter's serum response element, is known to be activated as a result of phosphorylation by the MAP kinases ERK1 and ERK2. However, induction of c-fos expression in response to noxious agents takes place in the absence of ERK activation. We therefore investigated whether SAPKs similarly upregulate c-fos expression by phosphorylating Elk-1. RESULTS: Elk-1 is activated in response to stimuli other than mitogenic signals. Both p46SAPK and p54SAPK interact physically with, and phosphorylate, Elk-1. The capacity of Elk-1 to form a ternary complex with serum response factor in vitro is thereby elevated. In vivo, selective activation of SAPKs stimulates formation of the ternary complex containing Elk-1, serum response factor and the serum response element, and enhances Elk-1-dependent transcription. Expression of the SAPK upstream-activator kinase, MEKK1, induces SAPK activation and c-fos transcription in the absence of ERK activity. Phosphopeptide mapping of Elk-1 phosphorylated with p46SAPK or p54SAPK reveals Ser383, a residue critical for ternary complex formation and transcriptional activation, to be the major phosphorylation site. CONCLUSION: Elk-1 responds to stress-induced, as well as mitogenic, signals by stimulating c-fos transcription through the serum response element. Phosphorylation of Elk-1 by SAPKs and the ensuing expression of Fos protein thus constitutes an additional mechanism by which cells can upregulate AP-1 activity in response to stress.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Camundongos , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Resposta Sérica , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Proteínas Elk-1 do Domínio ets
4.
Mol Cell Biol ; 16(3): 1094-102, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8622654

RESUMO

The rapid and transient induction of the human proto-oncogene c-fos in response to a variety of stimuli depends on the serum responses element (SRE). In vivo footprinting experiments show that this promoter element is bound by a multicomponent complex including the serum response factor (SRF) and a ternary complex factor such as Elk-1. SRF is thought to recruit a ternary complex factor monomer into an asymmetric complex. In this report, we describe a quaternary complex over the SRE which, in addition to an SRF dimer, contains two Elk-1 molecules. Its formation at the SRE is strictly dependent on phosphorylation of S-383 in the Elk-1 regulatory domain and appears to involve a weak intermolecular association between the two Elk-1 molecules. The influence of mutations in Elk-1 on quaternary complex formation in vitro correlates with their effect on the induction of c-fos reporter expression in response to mitogenic stimuli in vivo.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Técnicas de Transferência de Genes , Camundongos , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Fator de Resposta Sérica , Proteínas Elk-1 do Domínio ets
5.
Mol Cell Biol ; 13(1): 123-32, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8417320

RESUMO

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Clonagem Molecular , Sequência Consenso , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Fator de Resposta Sérica
6.
Res Microbiol ; 142(2-3): 119-25, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1925008

RESUMO

We describe several experimental approaches relating to the early steps in the initiation of DNA replication at oriC. 1) A matrix is given which enables calculatation of the relative affinity of DnaA boxes for DnaA protein; 2) base changes within single Dna A boxes in oriC have little effect on oriC function; 3) mutations which change the distance between DnaA boxes inactivate oriC, but changes by one helical turn (+ and -) result in near wild-type oriC activity; 4) a Fis binding site was located at oriC coordinates 206-220; 5) KMnO4 probing demonstrates Dna-A-dependent unwinding in the left part of oriC in vivo and in vitro.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Replicação do DNA/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Dados de Sequência Molecular , Mutação
7.
J Clin Pathol ; 56(1): 26-30, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12499428

RESUMO

BACKGROUND: Retinoblastoma is the most common intraocular malignancy in childhood and is responsible for approximately 1% of all deaths caused by childhood cancer. AIMS/METHODS: Comparative genomic hybridisation was performed on 13 consecutive, histologically confirmed retinoblastomas to analyse patterns of chromosomal changes and correlate these to clinicopathological variables. Six cases were hereditary and seven cases were sporadic. RESULTS: In 11 of the 13 tumours chromosomal abnormalities were detected, most frequently gains. Frequent chromosomal gains concerned 6p (46%), 1q (38%), 2p, 9q (30%), 5p, 7q, 10q, 17q, and 20q (23%). Frequent losses occurred at Xq (46%), 13q14, 16q, and 4q (23%). High level copy number gains were found at 5p15 and 6p11-12. A loss at 13q14 occurred in three cases only. Relatively few events occurred in the hereditary cases (27) compared with the non-hereditary cases (70 events). The number of chromosomal aberrations in these 13 retinoblastomas showed a bimodal distribution. Seven tumours showed less than four chromosomal aberrations, falling into a low level chromosomal instability (CIN) group, and six tumours showed at least eight aberrations, falling into a high level CIN group. In the low level CIN group the mean age was half that seen in the high level CIN group, there were less male patients, and there were more hereditary and bilateral cases. Microsatellite instability was not detected in either of the two groups. CONCLUSION: Despite the complex pattern of genetic changes in retinoblastomas, certain chromosomal regions appear to be affected preferentially. On the basis of the number of genetic events, retinoblastomas can be divided in low and a high level chromosomal instability groups, which have striking differences in clinical presentation.


Assuntos
Aberrações Cromossômicas , Neoplasias da Retina/genética , Retinoblastoma/genética , Fatores Etários , Pré-Escolar , DNA de Neoplasias/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Repetições de Microssatélites/genética , Hibridização de Ácido Nucleico , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Fatores Sexuais
8.
Biol Psychol ; 26(1-3): 299-306, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3207788

RESUMO

Relationships between the P300 component of the event-related potential (ERP) and processes of evaluation and categorization of events have been demonstrated in numerous investigations. On the other hand, these same processes have also been studied extensively by means of rating scale procedures within the framework of the psychological Adaptation-Level Theory. Recently, it has been suggested that the P300 amplitude reflects the distance between the adaptation level and the event that elicits the ERP. In this study we explored this suggestion by examining the effects of stimulus categorization on P300. Seven healthy adult subjects had to judge the length of five letter strings which were presented equiprobably in a randomized sequence. The ERPs (Fz, Cz, Pz derivations) elicited by string presentation were selectively averaged according to the five categories of string length. U-shaped trends of P300 amplitude changes were found. Minimum amplitudes were elicited by strings of medium length consistent with the inferred adaptation level. The data provide further evidence that P300 amplitude reflects the mental distance to adaptation level.


Assuntos
Adaptação Psicológica , Potenciais Evocados Visuais , Percepção Visual/fisiologia , Adaptação Fisiológica , Adulto , Feminino , Humanos , Masculino , Teoria Psicológica
9.
Int J Psychophysiol ; 10(1): 33-8, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2269645

RESUMO

The present study investigates whether the amplitude of the P300 in 'normal' subjects adapts to unexpected changes in global stimulus probability. The adaptation level (AL) metrics (in terms of Helson's [1964] theory) was applied to explain the variations in the P300 amplitude. In an oddball paradigm the probability of the deviant tone (target 1000 Hz, 80 dB, 50 ms) occurring randomly in a train of standard ones (non-target 1100 Hz, 80 dB, 50 ms) was changed in a stepwise way from 0.5 to 0.3 and then to 0.1 in 3 blocks without interruption of the stimulus presentation. Two experiments were performed. In the first one the subjects did not receive any prior information about the changes in the stimulus probability, whereas in the second experiment they were aware of such changes. The task always required the subject to silently count the deviants. EEG was recorded monopolarly from Fz, Cz and Pz in a total of 15 subjects. The block averaged P300 increased monotonically with probability, the increase being larger in Expt. II. In contrast to the block averaged P300, the variations in the P300 amplitude following the abrupt change in probability showed a non-linear dependency. On the basis of the AL a simulation of the time course of the P300 variations with probability was performed and a clear similarity between the simulated P300 and the experimental data was found. Some properties of the AL model are discussed.


Assuntos
Percepção Auditiva/fisiologia , Eletroencefalografia , Potenciais Evocados Auditivos , Estimulação Acústica , Adaptação Fisiológica , Adulto , Atenção/fisiologia , Humanos , Modelos Neurológicos , Aprendizagem por Probabilidade , Enquadramento Psicológico
10.
Int J Psychophysiol ; 5(2): 145-9, 1987 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3610730

RESUMO

Recently, it has been shown that P300 measures depend on stimulus evaluation processes. This paper explores the effect of messages informing about the degree of difficulty of subsequent tasks on the P300 component. Ten healthy volunteers were asked to solve mental arithmetic tasks of graduated difficulty. The degree of difficulty was announced by messages which preceded the tasks and which were used as P300 eliciting stimuli. Measurements of the amplitude and latency of P300 components from 3 brain regions (Fz, Cz, Pz) were collected selectively for each category of task difficulty announced. Subjective ratings of difficulty and the frequency of errors confirmed the monotonic increase of task difficulty from categories I to VI. The mean P300 amplitude in categories I-VI showed a U-shaped trend, i.e. the highest amplitude occurred in the extreme categories 'extremely easy' and 'very difficult'. No significant effect of stimulus categories on P300 peak latencies was found. It is concluded that the P300 amplitude reflects an evaluation in the sense of distance judgements on an internal scale which in this study concerned the task difficulty.


Assuntos
Nível de Alerta , Atenção , Eletroencefalografia , Adulto , Potenciais Evocados , Feminino , Humanos , Masculino
11.
Percept Mot Skills ; 70(2): 543-8, 1990 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2342853

RESUMO

This study was aimed at testing a new approach for examination of functional laterality based on hemispheric specialization. The subjects had to perform verbal (words/nonwords) and nonverbal (similar/different patterns) discrimination. The separation of the two hemispheres during information processing was realized by requiring a simultaneous response of both index fingers. The obtained over-all reaction times (RT) were faster for verbal than for pattern tasks. Considering the RTs for solely the particular, faster response of one or the other index finger, the right index finger turned out to be faster on verbal tasks whereas the left one dominated on pattern tasks. According to the hypothesis that the faster hand indicates the more active (contralateral) hemisphere, it can be assumed that words are responded to more quickly when processed in the left hemisphere. On the other hand, patterns are responded to more quickly when the right hemisphere is active. These results suggest that each hemisphere may be capable of processing verbal and nonverbal material; the speed of information processing, however, is faster in the more adept one.


Assuntos
Atenção , Dominância Cerebral , Percepção de Forma , Lateralidade Funcional , Reconhecimento Visual de Modelos , Desempenho Psicomotor , Tempo de Reação , Leitura , Adulto , Aprendizagem por Discriminação , Humanos , Masculino
17.
Draper Fund Rep ; (13): 3-5, 1984 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12339637

RESUMO

PIP: Many of the 135 countries participating in the 1974 UN World Population Conference were far from accepting the basic human right to decide freely and responsibly the number and spacing of their children and to have the information, education, and means to do so. Considerable progress has been made since then, and the number of developing countries that provide direct government support for family planning has increased to over 60%. Many have liberalized laws and regulations which restricted access to modern contraceptive methods, and a growing number provide family planning services within their health care programs. A few have recognized the practice of family planning as a constitutional right. In late 1983 at the Second African Population Conference, recognition of family as a human right was strongly contested by several governments, particularly those of West Africa. in developed countries most of the women at risk of unwanted pregnancy are using contraceptives. Of the major developing regions the highest use level is in Latin America, wherein most countries 1/3 to 1/2 of married women are users. Levels in Asian countries range from up to 10% in Afghanistan, Nepal, and Pakistan to up to 40% in the southeastern countries. China, a special case, now probably exceeds an overall use level of 2/3 of married women. Contraceptive use is lowest in Africa. There is room for improvement even among many of the successful family planning programs, as access to contraceptives usually is not sufficient to overcome limiting factors. To ensure the individual's free choice and strengthen the acceptability and practice of family planning, all available methods should be provided in service programs and inluded in information and education activities. Family planning programs should engage local community groups, including voluntary organizations, in all aspects of planning, management, and allocation of resources. At the government level a clear political commitment to family planning and population policies is essential as is administrative support. All government agencies concerned with socioeconomic benefit to the family and the community need to be involved in the coordinated planning and implementation.^ieng


Assuntos
Atitude , Comportamento , Comportamento Contraceptivo , Atenção à Saúde , Países em Desenvolvimento , Serviços de Planejamento Familiar , Planejamento em Saúde , Acessibilidade aos Serviços de Saúde , Marketing de Serviços de Saúde , Comportamento Sexual , África , América , Ásia , Região do Caribe , América Central , Anticoncepção , Demografia , Países Desenvolvidos , Economia , Fertilidade , Saúde , Direitos Humanos , América do Norte , Organização e Administração , Política , População , Dinâmica Populacional , Psicologia , América do Sul , Direitos da Mulher
18.
Popul Bull UN ; (19-20): 129-38, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-12314719

RESUMO

PIP: The Population Commission was originally charged with providing information and advice to the Economic and Social Council on population trends and issues, not direct technical assistance to governments: the needed factual basis was lacking and technical assistance was not yet a major activity of the United Nation (UN). By the 1950s, a technical assistance program focusing on data collection and analytical studies had been adopted. The 1st assistance request in population policy and action programs came from the Indian Government in 1952, followed by requests from Indonesia, Thailand, and Brazil. In 1952 the 1st 2 UN-supported regional demographic centers were founded. After the 1960 censuses, the emphasis of UN technical assistance in the population field shifted from statistical activities and training to developing methods for dealing with population problems. The early 1960s saw confrontation on whether technical cooperation should be provided by the UN for population action programs. In 1965 a high-level UN expert group was sent to India to make recommendations for the national FP program, and an ad hoc expert group recommended to the Commission that the UN respond to requests for assistance on all aspects of population, including FP. In 1966 the General Assembly unanimously adopted a resolution calling on the UN and its agencies to provide population technical assistance, and in 1967 the commission voted to give high priority to research and technical assistance in the fertility area. To finance this expanded role, the Secretary-General established, in 1967, a special UN Trust Fund for Population Activities, to be managed by the UN Secretariat. A Population Program and Projects Office was established in the Population Division and by 1969 Population Program Officers were stationed in developing countries to assess needs and assist in formulating population assistance requests. The assistance demang grew rapidly and the Fund reference terms were expanded, responsibililty for its administration being transferred to the UN Devleopment Program.^ieng


Assuntos
Órgãos Governamentais , Agências Internacionais , Cooperação Internacional , Organizações , Controle da População , Mudança Social , Planejamento Social , Nações Unidas , Economia , Política Pública
19.
EMBO J ; 10(6): 1579-84, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2026151

RESUMO

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT-rich 13mers which have been shown to become single-stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single-stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT-rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation.


Assuntos
Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/genética , Compostos de Manganês , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Cátions Bivalentes , DNA Bacteriano/ultraestrutura , Manganês/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óxidos/química , Plasmídeos , Sequências Reguladoras de Ácido Nucleico
20.
J Biol Chem ; 274(31): 22033-40, 1999 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-10419529

RESUMO

The involvement of Ras in the activation of multiple early signaling pathways is well understood, but it is less clear how the various Ras effectors interact with the cell cycle machinery to cause G(1) progression. Ras-mediated activation of extracellular-regulated kinase/mitogen-activated protein kinase has been implicated in cyclin D(1) up-regulation, but there is little extracellular-regulated kinase activity during the later stages of G(1), when cyclin D(1) expression becomes maximal, implying that other effector pathways may also be important in cyclin D(1) induction. We have addressed the involvement of Ras effectors from the phosphatidylinositol (PI) 3-kinase and Ral-GDS families in G(1) progression and compared it to that of the Raf/mitogen-activated protein kinase pathway. PI 3-kinase activity is required for the expression of endogenous cyclin D(1) and for S phase entry following serum stimulation of quiescent NIH 3T3 fibroblasts. Activated PI 3-kinase induces cyclin D(1) transcription and E2F activity, at least in part mediated by the serine/threonine kinase Akt/PKB, and to a lesser extent the Rho family GTPase Rac. In addition, both activated Ral-GDS-like factor and Raf stimulate cyclin D(1) transcription and E2F activity and act in synergy with PI 3-kinase. Therefore, multiple cooperating pathways mediate the effects of Ras on progression through the cell cycle.


Assuntos
Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclina D1/genética , Fase G1/fisiologia , Proteínas Ativadoras de GTPase , Regulação da Expressão Gênica , Genes Reporter , Humanos , Cinética , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fase S , Transfecção , Proteínas Ativadoras de ras GTPase
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