An excellent monitoring system for surface ubiquitination-induced internalization in mammals.
An excellent monitoring system for surface ubiquitination-induced internalization in mammals.
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DOI：
10.1371/journal.pone.0001490
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发表时间：
2008-01-30
期刊：
影响因子：
3.7
通讯作者：
Ishido S
中科院分区：
文献类型：
--
作者：
Goto E;Mito-Yoshida M;Uematsu M;Aoki M;Matsuki Y;Ohmura-Hoshino M;Hotta H;Miyagishi M;Ishido S
目前，很难想象哺乳动物细胞膜上发生的泛素化诱导的表面受体的内化。这个问题使得揭示哺乳动物中泛素化介导的内化的分子基础变得困难。为了克服它，我们已经产生了T-REx-c-MIR，一种新的哺乳动物Tet-on B细胞系，使用组成型活性E3泛素连接酶，c-MIR，及其人工靶分子。通过将表面生物素化方法应用于T-REx-c-MIR，我们成功地监测了多西环素（Dox）诱导的c-MIR表达启动泛素化过程后表面靶分子的命运。在诱导c-MIR表达之前预先存在于质膜上的靶分子被寡泛素化并被Dox诱导的c-MIR表达降解。Dox诱导的c-MIR表达启动了表面靶分子的快速内化，并且内化的阻断诱导了新被c-MIR泛素化的表面靶分子的积累。通过下调泛素结合酶E2来抑制表面泛素化，损害了靶分子的内化。最后，在质膜处检测到c-MIR和靶分子的复合物。这些结果表明，在T-REx-c-MIR中，表面靶分子在质膜处被泛素化，然后从质膜内化。因此，T-REx-c-MIR是一个有用的实验工具来分析表面泛素化如何调节哺乳动物的内化。
At present, it is difficult to visualize the internalization of surface receptors induced by ubiquitination that is taken place at the plasma membrane in mammals. This problem makes it difficult to reveal molecular basis for ubiquitination-mediated internalization in mammals. In order to overcome it, we have generated T-REx-c-MIR, a novel mammalian Tet-on B cell line using a constitutively active E3 ubiquitin ligase, c-MIR, and its artificial target molecule. By applying the surface biotinylation method to T-REx-c-MIR, we succeeded to monitor the fate of surface target molecules after initiation of ubiquitination process by doxycycline (Dox)-induced c-MIR expression. Target molecules that pre-existed at the plasma membrane before induction of c-MIR expression were oligo-ubiquitinated and degraded by Dox-induced c-MIR expression. Dox-induced c-MIR expression initiated rapid internalization of surface target molecules, and blockage of the internalization induced the accumulation of the surface target molecules that were newly ubiquitinated by c-MIR. Inhibition of the surface ubiquitination by down-regulating ubiquitin conjugating enzyme E2 impaired the internalization of target molecules. Finally, a complex of c-MIR and target molecule was detected at the plasma membrane. These results demonstrate that in T-REx-c-MIR, surface target molecule is ubiquitinated at the plasma membrane and followed by being internalized from the plasma membrane. Thus, T-REx-c-MIR is a useful experimental tool to analyze how surface ubiquitination regulates internalization in mammals.
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影响因子：
2.7
作者：
BAKER, A;CACHIA, P;PADUA, RA
通讯作者：
PADUA, RA
影响因子：
11.4
作者：
Hewitt, EW;Duncan, L;Lehner, PJ
通讯作者：
Lehner, PJ
DOI：
10.1073/pnas.87.4.1377
发表时间：
1990-02-01
影响因子：
11.1
作者：
RIDGE, SA;WORWOOD, M;PADUA, RA
通讯作者：
PADUA, RA
影响因子：
64.8
作者：
Soubeyran, P;Kowanetz, K;Dikic, I
通讯作者：
Dikic, I
影响因子：
5.4
作者：
Bartee, E;Mansouri, M;Früh, K
通讯作者：
Früh, K