RESUMO
The Caenorhabditis elegans anteroposterior axis is established in response to fertilization by sperm. Here we present evidence that RhoA, the guanine nucleotide-exchange factor ECT-2, and the Rho guanosine triphosphatase-activating protein CYK-4 modulate myosin light-chain activity to create a gradient of actomyosin, which establishes the anterior domain. CYK-4 is enriched within sperm, and paternally donated CYK-4 is required for polarity. These data suggest that CYK-4 provides a molecular link between fertilization and polarity establishment in the one-cell embryo. Orthologs of CYK-4 are expressed in sperm of other species, which suggests that this cue may be evolutionarily conserved.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/embriologia , Polaridade Celular , Embrião não Mamífero/citologia , Proteínas Ativadoras de GTPase/fisiologia , Actomiosina/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/genética , Citocinese , Citoesqueleto/fisiologia , Embrião não Mamífero/química , Embrião não Mamífero/fisiologia , Feminino , Fertilização , Proteínas Ativadoras de GTPase/análise , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Masculino , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/análise , Organelas/química , Proteínas/análise , Interferência de RNA , Espermatozoides/química , Espermatozoides/ultraestrutura , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
BACKGROUND: The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. RESULTS: We report that the C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf. Here, we show that TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase and that phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. CONCLUSIONS: The C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Western Blotting , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Complementar/genética , Imuno-Histoquímica , Imunoprecipitação , Fosforilação , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: The Tousled kinases comprise an evolutionarily conserved family of proteins that have been previously implicated in chromatin remodeling, DNA replication, and DNA repair. Here, we used RNA mediated interference (RNAi) to determine the function of the C. elegans Tousled kinase (TLK-1) during embryonic development. RESULTS: TLK-1-deficient embryos arrested with a phenotype reminiscent of embryos that are broadly defective in transcription, and the expression of several reporter genes was dramatically reduced in tlk-1(RNAi) embryos. Furthermore, posttranslational modifications of RNA polymerase II (RNAPII) and histone H3 that have been correlated with transcription elongation, phosphorylation of the RNAPII CTD at Serine 2, and methylation of histone H3 at Lysine 36 were found at significantly reduced levels in tlk-1(RNAi) embryos as compared to wild-type. CONCLUSIONS: These results reveal a surprising requirement for a Tousled-like kinase in transcriptional regulation during development, likely during the elongation phase. In addition, our results confirm that the link between RNAPII phosphorylation and histone H3 methylation previously observed in budding yeast is functionally conserved in metazoans.