Tamura K, Taniguchi Y, Minoguchi S, Sakai T, Tun T, Furukawa T, Honjo T
Department of Medical Chemistry, Kyoto University Faculty of Medicine, Japan.
Curr Biol. 1995 Dec 1;5(12):1416-23. doi: 10.1016/s0960-9822(95)00279-x.
The mammalian transcription factor RBP-J kappa binds to the DNA sequence motif CGTGGGAA and is involved in the regulation of gene expression; for example, it plays a part in the transactivation of viral and cellular genes by Epstein-Barr virus nuclear antigen-2. The Drosophila homologue of RBP-J kappa is the product of the Suppressor of Hairless (Su(H)) gene. Su(H) is a neurogenic gene that acts downstream of Notch, which encodes a cell-surface receptor. Furthermore, in the mouse, the phenotypes of homozygous mutant Notch1 embryos are very similar to those of homozygous mutant RBP-J kappa embryos. Recent studies, using the yeast two-hybrid system, have led to the suggestion that the CDC10/ankyrin-like repeats of the Drosophila Notch protein interact with the Su(H) protein.
We searched for proteins that interact with mouse RBP-J kappa using the yeast two-hybrid system, and in this way identified a short intracellular region (mRAM23) of the mouse Notch1 protein that lacks any known sequence motif. In vitro interaction studies, using proteins fused to glutathione-S-transferase, showed that RBP-J kappa and Su(H) bind directly to the RAM23 regions of mouse Notch1 and Drosophila Notch, respectively. Immunoprecipitation analysis showed that RBP-J kappa and the mRAM23 region of mouse Notch1 also interact in vivo. Further studies, including site-directed mutagenesis experiments, narrowed down the region of mouse Notch1 that interacts with RBP-J kappa. The results indicate that this region is less than 50 amino-acid residues in length, and lies immediately downstream of the transmembrane region.
We show that the transcription factor RBP-J kappa/Su(H) interacts directly with a novel intracellular domain of the cell-surface receptor Notch. RBP-J kappa/Su(H) does not appear to interact with Notch via the CDC10/ankyrin repeats implicated in previous studies.
哺乳动物转录因子RBP-Jκ与DNA序列基序CGTGGGAA结合，并参与基因表达的调控；例如，它在爱泼斯坦-巴尔病毒核抗原-2对病毒和细胞基因的反式激活中发挥作用。RBP-Jκ的果蝇同源物是无翅抑制因子（Su(H)）基因的产物。Su(H)是一个神经发生基因，在Notch下游起作用，Notch编码一种细胞表面受体。此外，在小鼠中，纯合突变型Notch1胚胎的表型与纯合突变型RBP-Jκ胚胎的表型非常相似。最近使用酵母双杂交系统的研究表明，果蝇Notch蛋白的CDC10/锚蛋白样重复序列与Su(H)蛋白相互作用。
我们使用酵母双杂交系统寻找与小鼠RBP-Jκ相互作用的蛋白质，并以此鉴定出小鼠Notch1蛋白的一个短的细胞内区域（mRAM23），该区域缺乏任何已知的序列基序。使用与谷胱甘肽-S-转移酶融合的蛋白质进行的体外相互作用研究表明，RBP-Jκ和Su(H)分别直接与小鼠Notch1和果蝇Notch的RAM23区域结合。免疫沉淀分析表明，RBP-Jκ和小鼠Notch1的mRAM23区域在体内也相互作用。包括定点诱变实验在内的进一步研究缩小了小鼠Notch1与RBP-Jκ相互作用的区域。结果表明，该区域长度小于50个氨基酸残基，位于跨膜区域的紧邻下游。
我们表明转录因子RBP-Jκ/Su(H)直接与细胞表面受体Notch的一个新的细胞内结构域相互作用。RBP-Jκ/Su(H)似乎不是通过先前研究中涉及的CDC10/锚蛋白重复序列与Notch相互作用。
