Masson N, Hurst H C, Lee K A
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Nucleic Acids Res. 1993 Jun 11;21(11):1163-9. doi: 10.1093/nar/21.5.1163.
The mammalian transcription factor CREB is thought to activate cAMP-inducible genes in a variety of differentiated cell types and is probably involved in other signalling pathways. Undifferentiated F9 embryonal carcinoma (UF9) cells are refractory to cAMP and become cAMP-responsive following differentiation to endoderm like cells. It has been proposed that UF9 cells contain a negative regulator(s) of the cAMP-response that might act through direct interaction with CREB. We have used a protein blotting assay and 32P-labelled CREB to probe for CREB-binding proteins in nuclear extracts from F9 cells and to examine their abundance during differentiation. We find that ATF1 (a protein that is highly homologous to CREB) and a novel polypeptide(s) of approximately 100 kDa (CBP100) are the major CREB-binding proteins in extracts from UF9 cells. As expected ATF1 is detected due to leucine zipper-dependent heterodimerisation with CREB. In contrast CBP100 interacts with CREB independently of the leucine zipper. The total amount of ATF1 and the amount of ATF1 that is complexed with CREB are substantially reduced following differentiation. In addition, ATF1 mRNA levels are lower in differentiated F9 cells indicating that a pretranslational mechanism contributes to the decreased ATF1 protein levels observed. CBP100 levels are also reduced or CBP100 is modified upon differentiation. We discuss the potential roles of ATF1 and CBP100 in regulating CREB activity during differentiation of F9 embryonal carcinoma cells.
哺乳动物转录因子CREB被认为可在多种分化细胞类型中激活cAMP诱导基因，并且可能参与其他信号通路。未分化的F9胚胎癌细胞对cAMP不敏感，在分化为内胚层样细胞后变得对cAMP有反应。有人提出，UF9细胞含有cAMP反应的负调节因子，可能通过与CREB直接相互作用发挥作用。我们使用蛋白质印迹分析和32P标记的CREB来探测F9细胞核提取物中的CREB结合蛋白，并检测它们在分化过程中的丰度。我们发现ATF1（一种与CREB高度同源的蛋白质）和一种约100 kDa的新型多肽（CBP100）是UF9细胞提取物中的主要CREB结合蛋白。正如预期的那样，由于与CREB的亮氨酸拉链依赖性异源二聚化，检测到了ATF1。相比之下，CBP100与CREB的相互作用不依赖于亮氨酸拉链。分化后，ATF1的总量以及与CREB复合的ATF1量大幅减少。此外，分化的F9细胞中ATF1 mRNA水平较低，表明转录前机制导致了观察到的ATF1蛋白水平降低。分化后CBP100水平也降低或CBP100被修饰。我们讨论了ATF1和CBP100在F9胚胎癌细胞分化过程中调节CREB活性的潜在作用。
