RESUMO
Many bacteria possess 2 or more genes for the chaperonin GroEL and the cochaperonin GroES. In particular, rhizobial species often have multiple groEL and groES genes, with a high degree of amino-acid similarity, in their genomes. The Rhizobium leguminosarum strain A34 has 3 complete groE operons, which we have named cpn.1, cpn.2 and cpn.3. Previously we have shown the cpn. 1 operon to be essential for growth, but the two other cpn operons to be dispensable. Here, we have investigated the extent to which loss of the essential GroEL homologue Cpn60.1 can be compensated for by expression of the other two GroEL homologues (Cnp60.2 and Cpn60.3). Cpn60.2 could not be overexpressed to high levels in R. leguminosarum, and was unable to replace Cpn60.1. A strain that overexpressed Cpn60.3 grew in the absence of Cpn60.1, but the complemented strain displayed a temperature-sensitive phenotype. Cpn60.1 and Cpn60.3, when coexpressed in Escherichia coli, preferentially selfassembled rather than forming mixed heteroligomers. We conclude that, despite their high amino acid similarity, the GroEL homologues of R. leguminosarum are not functionally equivalent in vivo.
Assuntos
Chaperonina 60/genética , Chaperonina 60/metabolismo , Rhizobium leguminosarum/genética , Chaperonina 60/química , Escherichia coli , Teste de Complementação Genética , Filogenia , Estrutura Quaternária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, we show that hSpry2 is a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Proteínas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Quinases da Família src/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Linhagem Celular , DNA Complementar/metabolismo , Ativação Enzimática , Humanos , Imunoprecipitação , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Células NIH 3T3 , Peptídeos/química , Fosfoproteínas/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Sulfonamidas/farmacologia , Fatores de Tempo , Transfecção , Tirosina/química , Tirosina/metabolismoRESUMO
Ligand-dependent signalling pathways have been characterised as having morphogen properties where there is a quantitative relationship between receptor activation and response, or threshold characteristics in which there is a binary switch in response at a fixed level of receptor activation. Here we report the use of a bacterial artificial chromosome (BAC)-based transgenic system in which a hypermorphic mutation has been introduced into the murine Fgfr1 gene. These mice exhibit cranial suture and sternal fusions that are exacerbated when the BAC copy number is increased. Surprisingly, increasing mutant BAC copy number also leads to the de novo appearance of digit I polydactyly in the hind limb and transformations of the vertebrae. Polydactyly is accompanied by a reduction of programmed cell death in the developing hind limb. Candidate gene analysis reveals downregulation of Dkk1 in the digit I field and upregulation of Wnt5a and Hoxd13. These findings show that Fgfr1-mediated developmental pathways exhibit differing signalling dynamics, whereby development of the cranial sutures and sternum follows a morphogen mode, whereas development of the vertebral column and the hind limbs has threshold signalling properties.
Assuntos
Desenvolvimento Ósseo/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Padronização Corporal/genética , Desenvolvimento Ósseo/genética , Cromossomos Artificiais Bacterianos/genética , Suturas Cranianas/anormalidades , DNA Complementar/genética , Dosagem de Genes , Membro Posterior/anormalidades , Hibridização in Situ Fluorescente , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Polidactilia/genética , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais , Coluna Vertebral/anormalidades , Esterno/anormalidadesRESUMO
Fibroblast growth factor receptors (FGFRs) are a family of transmembrane tyrosine kinases involved in signaling via interactions with the family of fibroblast growth factors. Alternative splicing of the juxtamembrane region of FGFR1-3 leads to the inclusion or exclusion of two amino acids, valine and threonine, the VT site. The presence or absence of VT (VT+ or VT-, respectively) affects the signaling potential of the receptor. The VT+ receptor isoform is required for Erk2 phosphorylation, a component of the mitogen-activated protein kinase signaling pathway. FRS2 is an adaptor protein that links FGFRs to the mitogen-activated protein kinase signaling pathway. FRS2 interacts with a region of the juxtamembrane domain of FGFR1 that includes the alternatively spliced VT site. We investigated the interaction of FRS2 with murine Fgfr1 juxtamembrane domain. We showed the alternatively spliced VT motif, at the juxtamembrane domain of Fgfr1 is required for FRS2 interaction with Fgfr1. Activation of signaling pathways from FRS2 is likely to be regulated by controlling the Fgfr1/FRS2 interaction through alternative splicing of the VT motif of Fgfr1.
