RESUMO
The LKB1 tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine/threonine kinase of unknown function. Here we show that mice with a targeted disruption of Lkb1 die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor (VEGF) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1(-/-) fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.
Assuntos
Vasos Sanguíneos/anormalidades , Embrião de Mamíferos/metabolismo , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linfocinas/genética , Linfocinas/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Fatores de Transcrição , Proteínas Quinases Ativadas por AMP , Animais , Vasos Sanguíneos/embriologia , Morte Celular , Hipóxia Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário e Fetal , Endotélio Vascular/anormalidades , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Marcação de Genes , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Hibridização In Situ , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/anormalidades , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Defeitos do Tubo Neural/embriologia , Proteínas Nucleares/metabolismo , Fenótipo , Placenta/irrigação sanguínea , Placenta/embriologia , Placenta/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The trimeric Cdk7-cyclin H-Mat1 complex comprises the kinase subunit of basal transcription factor TFIIH and has been shown to function as a cyclin-dependent kinase (Cdk)-activating kinase. Herein we report that disruption of the murine Mat1 gene leads to peri-implantation lethality coincident with depletion of maternal Mat1 protein. In culture, Mat1(-/-) blastocysts gave rise to viable post-mitotic trophoblast giant cells while mitotic lineages failed to proliferate and survive. In contrast to wild-type trophoblast giant cells, Mat1(-/-) cells exhibited a rapid arrest in endoreduplication, which was characterized by an inability to enter S phase. Additionally, Mat1(-/-) cells exhibited defects in phosphorylation of the C-terminal domain (CTD) of RNA polymerase II on both Ser5 and Ser2 of the heptapeptide repeat. Despite this, Mat1(-/-) cells demonstrated apparent transcriptional and translational integrity. These data indicate an essential role for Mat1 in progression through the endocycle and suggest that while Mat1 modulates CTD phosphorylation, it does not appear to be essential for RNA polymerase II-mediated transcription.
Assuntos
Blastocisto/citologia , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição TFII , Animais , Sítios de Ligação , Blastocisto/fisiologia , Feminino , Morte Fetal , Genótipo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Gravidez , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/deficiência , Sequências Repetitivas de Aminoácidos , Fase S , Serina , Fator de Transcrição TFIIH , Fatores de Transcrição/metabolismo , Transcrição Gênica , Trofoblastos/citologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1 to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II. Here, we report a novel interaction between Cdk7 and a histidine triad (HIT) family protein, Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation and subcellular localization studies, where overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The physical association is independent of cyclin H binding or Cdk7 kinase activity and is conserved between the related Sacharomyces cerevisiae CTD kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption of HNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly elongated cell morphology and reduced colony formation, indicating a genetic interaction between KIN28 and HNT1. The physical and genetic interactions of Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine triad proteins in the regulation of Cdk7 and Kin28 functions.