Characterization of a high-molecular-weight notch complex in the nucleus of Notchic-transformed RKE cells and in a human T-cell leukemia cell line
Characterization of a high-molecular-weight notch complex in the nucleus of Notchic-transformed RKE cells and in a human T-cell leukemia cell line
复制标题
DOI：
10.1128/mcb.22.11.3927-3941.2002
复制
发表时间：
2002-06-01
影响因子：
5.3
通讯作者：
Capobianco, AJ
中科院分区：
文献类型：
--
作者：
Jeffries, S;Robbins, DJ;Capobianco, AJ
Notch基因编码的跨膜蛋白家族参与许多细胞过程，如分化、增殖和凋亡。已经确定所有四种Notch基因都可以作为致癌基因;然而，Notch蛋白转化细胞的机制仍然未知。以前，我们报告说，核定位和转录激活所需的肿瘤转化的RKE细胞。此外，我们确定细胞周期蛋白D1作为组成型活性Notch分子的直接转录靶点。为了理解Notch在细胞核中发挥作用的机制，我们试图使用尺寸排阻色谱法确定Notch是否形成稳定的复合物。在此，我们报告的Notch细胞内结构域（N-IC）形成不同的高分子量复合物在转化的RKE细胞的细胞核。最大的复合物约为1.5 MDa，含有内源性CSL（CBF 1、无毛抑制因子和Lag-1）和Mastermind-Like-1（Mam 1）。不具有CSL（RAM）的高亲和力结合位点的N-ic分子保留通过涉及Maml的相互作用在稳定复合物中与CSL缔合的能力。然而，Maml不直接结合CSL。此外，Maml可以拯救CSL依赖性启动子上的DeltaRAM转录活性。这些结果表明RAM结构域的缺失不等同于CSL非依赖性信号传导。此外，在SUP-T1细胞中，N-ic仅存在于最大的含N-ic的复合物中。SUP-T1细胞来源于携带t（7;9）（q34; q34.3）易位并组成性表达N-ic的T细胞白血病。综上所述，我们的数据表明，复合物的形成可能是由Notch（ic）肿瘤转化所需的。
Notch genes encode a family of transmembrane proteins that are involved in many cellular processes, such as differentiation, proliferation, and apoptosis. It is well established that all four Notch genes can act as oncogenes; however, the mechanism by which Notch proteins transform cells remains unknown. Previously, we reported that both nuclear localization and transcriptional activation are required for neoplastic transformation of RKE cells. Furthermore, we identified cyclin D1 as a direct transcriptional target of constitutively active Notch molecules. In an effort to understand the mechanism by which Notch functions in the nucleus, we sought to determine if Notch formed stable complexes using size exclusion chromatography. Herein, we report that the Notch intracellular domain (N-ic) forms distinct high-molecular-weight complexes in the nuclei of transformed RKE cells. The largest complex is approximately 1.5 MDa and contains both endogenous CSL (for CBF1, Suppressor of Hairless, and Lag-1) and Mastermind-Like-1 (Maml). N-ic molecules that do not have the high-affinity binding site for CSL (RAM) retain the ability to associate with CSL in a stable complex through interactions involving Maml. However, Maml does not directly bind to CSL. Furthermore, Maml can rescue DeltaRAM transcriptional activity on a CSL-dependent promoter. These results indicate that deletion of the RAM domain does not equate to CSL-independent signaling. Moreover, in SUP-T1 cells, N-ic exists exclusively in the largest N-ic-containing complex. SUP-T1 cells are derived from a T-cell leukemia that harbors the t(7;9)(q34; q34.3) translocation and constitutively express N-ic. Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notch(ic).