RESUMO
Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. Here we identify such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require wallenda.
Assuntos
Sistema Nervoso Central/embriologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Cones de Crescimento/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Sinapses/enzimologia , Animais , Diferenciação Celular/genética , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Mapeamento Cromossômico , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Cones de Crescimento/ultraestrutura , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Hidrolases/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/isolamento & purificação , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sinapses/ultraestrutura , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.