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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.

肝素结合表皮生长因子样生长因子（HB-EGF）是一种Ⅰ型质膜蛋白（proHB-EGF），含有EGF样结构域、跨膜区和胞质短尾。通过金属蛋白酶活化（胞外域脱落）切割proHB-EGF产生EGF受体的可溶性配体和跨膜-胞质片段我们先前表明HB-EGF的胞质结构域（HB-EGF-cyto）与转录阻遏物相互作用并影响其活性。本研究试图揭示HB-EGF-cyto在脱落刺激后的转录调控及其时空信息，发现另一个转录抑制因子Bcl 6与HB-EGF-cyto结合。荧光素酶分析表明，脱落的proHB-EGF逆转Bcl 6介导的转录抑制。此外，染色质免疫沉淀表明，在脱落刺激HB-EGF-cyto废除Bcl 6结合细胞周期蛋白D2启动子，这是抑制Bcl 6在稳态。结论：内源性cyclin D2蛋白水平的增加与proHB-EGF的脱落平行。这些观察结果表明，HB-EGF-cyto与Bcl 6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。免疫荧光显微镜观察显示，HB-EGF-cyto在脱落刺激后靶向于核膜。HB-EGF-cyto的14个氨基酸区域显示出核膜靶向活性。毛地黄皂苷透化的细胞表明HB-EGF-cyto靶向细胞核内，即，内核膜。此外，过量表达Rab 11抑制了HB-EGF-cyto的核膜靶向，这表明再循环内体参与了HB-EGF-cyto的再定位。总的来说，这些数据指向一种新的转录调控通过核膜靶向质膜生长因子。