RESUMO
The Dorsal morphogen directs formation of the Drosophila dorsoventral axis by both activating and repressing transcription. It contains an N-terminal Rel homology domain (RHD), which is responsible for DNA binding and regulated nuclear import, and a C-terminal domain (CTD) that contains activation and repression motifs. To determine if the RHD has a direct role in transcriptional control, we analyzed a series of RHD mutations in S2 cells and embryos. Two classes of mutations (termed class I and class II mutations) that alter activation without affecting DNA binding or nuclear import were identified. The two classes appear to define distinct protein interaction surfaces on opposite faces of the RHD. Class I mutations enhance an apparently inhibitory interaction between the RHD and the CTD and eliminate both activation and repression by Dorsal. In contrast, class II mutations result in increased activation in S2 cells but severely decreased activation in embryos and have little effect on repression. Analysis of the cuticles of class II mutant embryos suggests that, in the absence of Dorsal-mediated activation, Dorsal-mediated repression is not sufficient to pattern the embryo. These results provide some of the first evidence that the RHD plays an active role in transcriptional regulation in intact multicellular organisms.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Linhagem Celular , Proteínas de Drosophila/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Fosfoproteínas/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Ativação TranscricionalRESUMO
An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.
Assuntos
Monofosfato de Adenosina/imunologia , Anticorpos/química , Treonina/imunologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Western Blotting , Clonagem Molecular , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Soros Imunes , Processamento de Proteína Pós-Traducional , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Treonina/química , Treonina/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Several reports over the last few years have documented the dose-response curve for steroid hormone induction of gene transcription as a modulated property of a given receptor-agonist complex that varies with the changing concentration of a variety of factors including: homologous receptor, co-activators, co-repressors and selected co-factors. In each report, the dose-response curves are sigmoidal and show an excellent fit with the curve generated by Michaelis-Menten kinetics. In addition, even the overall function of human oestrogen receptors (hERs) can show a similar graph for the determination of sex versus oestrogen compounds in reptiles. Thus, the kinetic properties of the simple bimolecular reaction of A+B-->C appear, surprisingly, to be sufficient to describe the dose-response curve of the multi-step process of steroid-regulated gene induction that involves several molecules. Any advance in explaining why the dose-response curve for steroid-regulated gene expression is sigmoidal would assist in understanding what parameters are key factors of the dose-response curve and can benefit in the design of new oestrogenic substances. We have constructed and analysed a multi-step model of hER-induced gene transcription that explains the multiple forms of a simple dose-response curve in an in vitro transcription system.