TC Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland, United States of America.
PLoS One. 2012;7(8):e39093. doi: 10.1371/journal.pone.0039093. Epub 2012 Aug 2.
The Notch signaling pathway is an intercellular communication network vital to metazoan development. Notch activation leads to the nuclear localization of the intracellular portion (NICD) of the Notch receptor. Once in the nucleus, NICD binds the transcription factor CSL through a bivalent interaction involving the high-affinity RAM region and the lower affinity ANK domain, converting CSL from a transcriptionally-repressed to an active state. This interaction is believed to directly displace co-repressor proteins from CSL and recruit co-activator proteins. Here we investigate the consequences of this bivalent organization in converting CSL from the repressed to active form. One proposed function of RAM is to promote the weak ANK:CSL interaction; thus, fusion of CSL-ANK should bypass this function of RAM. We find that a CSL-ANK fusion protein is transcriptionally active in reporter assays, but that the addition of RAM in trans further increases transcriptional activity, suggesting another role of RAM in activation. A single F235L point substitution, which disrupts co-repressor binding to CSL, renders the CSL-ANK fusion fully active and refractory to further stimulation by RAM in trans. These results suggest that in the context of a mammalian CSL-ANK fusion protein, the main role of RAM is to displace co-repressor proteins from CSL.
Notch 信号通路是细胞间通讯网络，对后生动物的发育至关重要。Notch 激活导致 Notch 受体的细胞内部分（NICD）核定位。一旦进入细胞核，NICD 通过涉及高亲和力 RAM 区域和低亲和力 ANK 结构域的二价相互作用与转录因子 CSL 结合，将 CSL 从转录抑制状态转变为激活状态。这种相互作用被认为直接将共抑制蛋白从 CSL 上置换，并募集共激活蛋白。在这里，我们研究了这种二价组织将 CSL 从抑制形式转变为激活形式的后果。RAM 的一个提议功能是促进弱的 ANK:CSL 相互作用；因此，CSL-ANK 的融合应该绕过 RAM 的这个功能。我们发现 CSL-ANK 融合蛋白在报告基因检测中具有转录活性，但转染的 RAM 的加入进一步增加了转录活性，这表明 RAM 在激活中具有另一个作用。单个 F235L 点取代破坏了共抑制蛋白与 CSL 的结合，使 CSL-ANK 融合蛋白完全激活，并对转染的 RAM 的进一步刺激产生抗性。这些结果表明，在哺乳动物 CSL-ANK 融合蛋白的背景下，RAM 的主要作用是将共抑制蛋白从 CSL 上置换。
