RESUMO
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
Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimologia , Clonagem Molecular , Ativação Enzimática , Teste de Complementação Genética , Glutationa Transferase/genética , Humanos , Fases de Leitura Aberta , Fenótipo , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Especificidade por Substrato , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Ciclo Celular , Sequência Conservada , Ciclina H , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Drosophila , Ativação Enzimática , Dosagem de Genes , Genes Supressores , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
We have proved the presence of (2'-5')oligoadenylates [(2'-5')An] and oligoadenylate synthetase [(2'-5')An synthetase] in the marine sponge Geodia cydonium. (2'-5')An isolated from sponge crude extract competed with authentic (2'-5')An for binding to polyclonal antiserum against (2'-5')An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2'-5')A oligomers. The biological activity of sponge (2'-5')An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2'-5')An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon-treated cell extract. The (2'-5')An synthetase from sponge extract binds to poly(I).poly(C) as does the mammalian enzyme. Western blot analysis with antibodies to recombinant rat 43-kDa (2'-5')An synthetase revealed in sponge immunologically related proteins with molecular masses of approximately 110, 65, 61 and 34 kDa. We conclude, that the (2'-5')An system has evolved from receptors and enzymes involved in cell adhesion and/or growth control.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Poríferos/enzimologia , 2',5'-Oligoadenilato Sintetase/química , 2',5'-Oligoadenilato Sintetase/imunologia , Nucleotídeos de Adenina/isolamento & purificação , Nucleotídeos de Adenina/metabolismo , Nucleotídeos de Adenina/farmacologia , Animais , Evolução Biológica , Proteínas Sanguíneas/biossíntese , Western Blotting , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Imunoquímica , Técnicas In Vitro , Camundongos , Peso Molecular , Oligorribonucleotídeos/isolamento & purificação , Oligorribonucleotídeos/metabolismo , Oligorribonucleotídeos/farmacologia , Poríferos/metabolismo , Inibidores da Síntese de Proteínas/isolamento & purificação , Inibidores da Síntese de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Coelhos , Ratos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Especificidade da EspécieRESUMO
A new 9,11-secosterol, 24-nor-9,11-seco-11-acetoxy-3 beta,6 alpha-dihydroxycholest-7,22(E)-dien-9-one, was found to exhibit growth inhibitory (IC50 below 10 microM) and cytotoxic activities against human leukemia K562, human cervical cancer HeLa, and Ehrlich ascites tumor cells in vitro. The cytostatic concentrations of the compound generally caused the G2/M block in the cell cycle progression, but differences between the three tumor cell lines in the events leading to cell death were remarkable. While inhibiting cell proliferation, 9,11-secosterol caused accumulation of HeLa and K562 cells in the metaphase of mitosis. So, abnormal mitosis can play an important role in the cytotoxicity of 9,11-secosterol in these cell lines. In the Ehrlich ascites tumor cell line the increasing concentrations of the drug (up to 40 microM) did not cause an immediate cell killing. Instead, due to continued DNA synthesis without entry into mitosis, cells with high DNA ploidy were produced. It was shown that the cytoskeletal systems such as microtubules and microfilaments were not damaged by the action of 9,11-secosterol. Further studies are necessary to elucidate the mechanism of the cytotoxic effect of 9,11-secosterol.
Assuntos
Antineoplásicos/farmacologia , Colestenonas/farmacologia , Cnidários/química , Inibidores do Crescimento/farmacologia , Animais , Antineoplásicos/isolamento & purificação , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Colestenonas/isolamento & purificação , Cromossomos Humanos/efeitos dos fármacos , Inibidores do Crescimento/isolamento & purificação , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Estrutura Molecular , Células Tumorais CultivadasRESUMO
Nerve growth factor (NGF) rapidly increases the cyclic GMP (cGMP) level about 2-3-fold and enhances the cGMP phosphodiesterase (PDE) activity about 2-fold in rat pheochromocytoma PC12 cells. No changes in the level of cyclic AMP (cAMP) and in the activity of cAMP PDE were found. GTP and a nonhydrolysable analog of GTP, GMP-PCP, at 100 microM, were able to mimic the effect of NGF on the cGMP PDE activity. These results suggest that the cGMP system may be one of the second messengers of NGF action in PC12 cells.