RESUMO
Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353-38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/fisiologia , Vaccinia virus/enzimologia , Replicação Viral/fisiologia , Células 3T3/enzimologia , Células 3T3/virologia , Actinas/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Replicação do DNA/genética , DNA Viral/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Viral da Expressão Gênica/genética , Genes Precoces/genética , Proteínas Imediatamente Precoces/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptídeos/fisiologia , Fosforilação , Biossíntese de Proteínas/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Células Vero/virologia , Proteínas Elk-1 do Domínio etsRESUMO
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.
Assuntos
Células 3T3/enzimologia , Actinas/fisiologia , Cloranfenicol O-Acetiltransferase/metabolismo , Echinococcus/genética , Proteínas de Helminto/fisiologia , Regiões Promotoras Genéticas/fisiologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Genes Reporter , Proteínas de Helminto/química , Proteínas de Helminto/genética , Camundongos , Regiões Promotoras Genéticas/genética , Relação Estrutura-AtividadeRESUMO
We have developed a protocol that reveals two antagonistic effects of phorbol-12-myristate-12-acetate (PMA) on the G0-->G1-->S transition of mammalian cell cycle. Balb-3T3 (Clone A31) cells arrested in G0 by serum starvation can be stimulated to traverse the G1 phase and initiate DNA synthesis 12 h later by a 2-h pulse with PMA. In contrast with this early stimulatory effect, PMA has an inhibitory effect when presented to the cells during the last 6 h of G1. PMA is able to inhibit DNA synthesis initiation irrespective of the triggering agent, i.e., serum, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, or PMA itself (presented as an early pulse). We have established that the critical period for the PMA inhibitory effect is between 6 and 8 h after cell stimulation. This dual effect of PMA is not a peculiarity of Balb-3T3 (clone A31) cells because it is also observed with other fibroblastic cell lines, namely, SWISS 3T3, NIL 8, and RAT 1, and also with the epithelial Y-1 adrenocortical cell line. Treatment with PMA for 0.5 or 2 h activates protein kinase C (PKC) in Balb-3T3-A31 cells, but is not sufficient to down-regulate the enzyme because a second 30-min PMA pulse applied between 6 and 6.5 h activates PKC again. On the other hand, a continuous 6.5-h PMA treatment causes PKC down-regulation; therefore, the inhibitory effect of PMA could be mediated by PKC. Growth factor early response proto-oncogenes c-myc, c-fos, and c-jun are induced transiently by both early and late PMA pulses, suggesting that these genes are not involved in the PMA inhibitory effect.