Nuclear translocation of cell-surface transmembrane growth factor HB-EGF

细胞表面跨膜生长因子 HB-EGF 的核转位

基本信息

批准号：
17570163
负责人：
HIEDA Miki
金额：
$ 2.24万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I plasma membrane protein (proHB-EGF) containing of the extracellular EGF-like domain, transmembrane segment and cytoplasmic short tail. Cleavage of proHB-EGF via metalloprotease activation (ectodomain shedding) yields a soluble ligand of EGF receptor and transmembrane-cytoplasmic fragment We previously showed that the cytoplasmic domain of HB-EGF (HB-EGF-cyto) interacts with a transcriptional repressor and effects on its activity. Here we attempt to reveal the transcriptional regulation by and spatio-temporal information of HB-EGF-cyto after shedding stimulation.We found that another transcriptional repressor, Bcl6 associates with HB-EGF-cyto. A luciferase assay showed that shedding of proHB-EGF reversed the Bcl6-mediated transcriptional repression. Moreover chromatin immunoprecipitation indicated that upon shedding stimuli HB-EGF-cyto abolished Bcl6 binding on cyclinlin D2 promoter which is repressed by BcI6 at a steady-state. Consistently the level of endogenous cyclin D2 protein increased in parallel with the shedding of proHB-EGF. These observations indicated that HB-EGF-cyto interacts with Bcl6 and abolishes its repression activity. Next we focus on the localization of HB-EGF-cyto. Immunofluorescent microscopy demonstrated that HB-EGF-cyto targeted to the nuclear envelope after shedding stimulation. A 14 amino acids region in HB-EGF-cyto showed a nuclear envelope targeting activity. Digitonin permeabilized cells suggested that HB-EGF-cyto targeted inside the nucleus, i.e., the inner nuclear membrane. Furthermore over expressed Rab11 suppressed the nuclear envelope targeting of HB-EGF-cyto, suggesting the involvement of recycling endosome for the relocalization of HB-EGF-cyto. Collectively these data point to a novel transcriptional regulation via the nuclear envelope targeting of a plasma membrane growth factor.

肝素结合 EGF 样生长因子 (HB-EGF) 被合成为 I 型质膜蛋白 (proHB-EGF)，包含细胞外 EGF 样结构域、跨膜片段和胞质短尾。通过金属蛋白酶激活（胞外域脱落）裂解 proHB-EGF，产生 EGF 受体的可溶性配体和跨膜胞质片段。它的活动。在这里，我们试图揭示HB-EGF-细胞在脱落刺激后的转录调控和时空信息。我们发现另一种转录抑制因子Bcl6与HB-EGF-细胞相关。荧光素酶检测显示 proHB-EGF 的脱落逆转了 Bcl6 介导的转录抑制。此外，染色质免疫沉淀表明，在脱落刺激后，HB-EGF-细胞消除了 Bcl6 在细胞周期蛋白 D2 启动子上的结合，而细胞周期蛋白 D2 启动子在稳态时被 Bcl6 抑制。内源性细胞周期蛋白 D2 蛋白的水平与 proHB-EGF 的脱落同时增加。这些观察结果表明 HB-EGF-cyto 与 Bcl6 相互作用并消除其抑制活性。接下来我们重点关注 HB-EGF-cyto 的定位。免疫荧光显微镜表明，HB-EGF-细胞在脱落刺激后靶向核膜。 HB-EGF-细胞中的 14 个氨基酸区域显示出核膜靶向活性。洋地黄皂苷透化细胞表明 HB-EGF-细胞靶向细胞核内部，即内核膜。此外，过度表达的 Rab11 抑制了 HB-EGF-细胞的核膜靶向，表明循环内体参与了 HB-EGF-细胞的重新定位。总的来说，这些数据表明通过质膜生长因子的核膜靶向进行新的转录调节。