Flores A M, Kassatly R F, Cress W D
H Lee Moffitt Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612, USA.
Oncogene. 1998 Mar 12;16(10):1289-98. doi: 10.1038/sj.onc.1201633.
E2F is a complex family of transcription factors which appears to regulate the transcription of genes required for the S phase of the mammalian cell cycle. In the present work, we have examined the mechanisms regulating E2F-3 accumulation in mouse fibroblasts. We have determined that E2F-3 DNA binding activity is restricted to the G1/S transition and S phase in both normal BALB/c-3T3 fibroblasts and in an SV40 virus-transformed BALB/c-3T3 derivative. Immunoblot analysis indicates that G0 and G1 cells have little or no E2F-3 polypeptide and that the increase in the DNA binding activity of E2F-3 at the G1/S boundary is reflected by an increase in total E2F-3 protein. In contrast to the E2F-3 polypeptide, RNAse protection assays demonstrate that the E2F-3 mRNA is clearly present in G0 and G1 cells. Finally, pulse/chase experiments indicate that the half-life of E2F-3 is approximately 40-fold greater in cells blocked in S phase relative to asynchronously growing cells. Together, these results indicate that the accumulation E2F-3 at S phase may be regulated, at least in part, at the level of protein stability.
E2F是一个复杂的转录因子家族，它似乎能调控哺乳动物细胞周期S期所需基因的转录。在本研究中，我们检测了小鼠成纤维细胞中调控E2F - 3积累的机制。我们确定，在正常的BALB/c - 3T3成纤维细胞和SV40病毒转化的BALB/c - 3T3衍生物中，E2F - 3的DNA结合活性都局限于G1/S期转换和S期。免疫印迹分析表明，G0期和G1期细胞中几乎没有或不存在E2F - 3多肽，E2F - 3在G1/S边界处DNA结合活性的增加反映在总E2F - 3蛋白的增加上。与E2F - 3多肽不同，核糖核酸酶保护试验表明，E2F - 3 mRNA在G0期和G1期细胞中明显存在。最后，脉冲追踪实验表明，相对于同步生长的细胞，处于S期阻滞的细胞中E2F - 3的半衰期大约长40倍。这些结果共同表明，S期E2F - 3的积累可能至少部分地在蛋白质稳定性水平上受到调控。
