RESUMO
The TATA-binding protein (TBP)-associated factor TAF(II)250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAF(II)250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G(1). At the nonpermissive temperature (39.5 degrees C), transcription from only a subset of protein encoding genes, including the G(1) cyclins, is dramatically reduced in the mutant cells. Here we demonstrate that the ability of the ts13 allele of TAF(II)250 to acetylate histones in vitro is temperature sensitive suggesting that this enzymatic activity is compromised at 39.5 degrees C in the mutant cells. Mutagenesis of a putative acetyl coenzyme A binding site produced a TAF(II)250 protein that displayed significantly reduced histone acetyltransferase activity but retained TBP and TAF(II)150 binding. Expression of this mutant in ts13 cells was unable to complement the cell cycle arrest or transcriptional defect observed at 39.5 degrees C. These data suggest that TAF(II)250 acetyltransferase activity is required for cell cycle progression and regulates the expression of essential proliferative control genes.
Assuntos
Acetiltransferases/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Acetiltransferases/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Cricetinae , Ciclina A/genética , Ciclina D1/genética , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Histona Acetiltransferases , Humanos , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Especificidade por Substrato , Temperatura , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.
Assuntos
Ciclo Celular , Divisão Celular , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Animais , Células CHO , Caseína Quinase II , Tamanho Celular , Cricetinae , Ciclina D1/metabolismo , Replicação do DNA/genética , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Mitose/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.