A tale of two Cbls: Interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation
A tale of two Cbls: Interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation
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DOI：
10.1128/mcb.01809-07
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发表时间：
2008-05-01
影响因子：
5.3
通讯作者：
Wang, Zhixiang
中科院分区：
文献类型：
--
作者：
Pennock, Steven;Wang, Zhixiang
Cbl在表皮生长因子（EGF）受体（EGFR）内吞和运输中的确切作用仍有待完全揭示。在这里，我们表明，突变EGFR 1044，这是残基1044后截短，不与c-Cbl，并没有泛素化最初在响应EGF，但内在动力学类似的野生型EGFR。这一发现表明c-Cbl介导的泛素化不是EGF诱导的EGFR内吞作用所必需的。我们还发现，以前确定的内化缺陷型突变受体EGFR 1010 LL/AA结合到c-Cbl，并在EGF的反应中完全泛素化，这表明c-Cbl结合和泛素化不足以使EGFR内化。我们接下来研究了EGFR内化后的EGFR转运。我们发现c-Cbl与EGFR的解离发生在EGFR降解之前，并且该事件与EGFR在Y1045处的选择性去磷酸化同时发生。这一发现表明，一旦EGFR被泛素化，EGFR降解就不需要持续的Cbl缔合。由于EGFR 1044与野生型EGFR相似地被泛素化和降解，我们研究了另一个突出的Cbl同源物Cbl-b的作用，发现Cbl-b与EGFR和EGFR 1044都相关。进一步的研究表明，Cbl-b在两个区域与EGFR结合：一个在C-末端方向上从残基1044开始，一个在N-末端方向上从残基958开始。此外，Cbl-b与EGFR的相关性在c-Cbl相关性降低后显著升高，对应于EGFR转运后期发生的EGFR泛素化的第二个峰值。使用RNA干扰敲除c-Cbl和Cbl-b，我们能够消除EGFR下调。这种敲除对EGF诱导的EGFR内化速率没有影响。我们发现，这两个Cbls占总受体泛素化，而c-Cbl和Cbl-b是每个单独足以影响EGFR降解，都参与了生理，EGF介导的受体下调过程。此外，这些数据最终揭示了一个以前未承认的时间相互作用的两个主要的Cbl同系物与EGFR的贩运。
The precise role of Cbl in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking remains to be fully uncovered. Here, we showed that mutant EGFR1044, which was truncated after residue 1044, did not associate with c-Cbl and was not ubiquitinated initially in response to EGF but was internalized with kinetics similar to those of wild-type EGFR. This finding indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. We also showed that the previously identified internalization-deficient mutant receptor EGFR1010LL/AA bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. We next investigated EGFR trafficking following EGFR internalization. We found that c-Cbl disassociation from EGFR occurred well in advance of EGFR degradation and that this event was concurrent with the selective dephosphorylation of EGFR at Y1045. This finding suggests that once EGFR is ubiquitinated, continual Cbl association is not required for EGFR degradation. Because EGFR1044 is ubiquitinated and degraded similarly to wild-type EGFR, we examined the role of another prominent Cbl homologue, Cbl-b, and found that Cbl-b was associated with both EGFR and EGFR1044. Further study showed that Cbl-b bound to EGFR at two regions: one in the C-terminal direction from residue 1044 and one in the N-terminal direction from residue 958. Moreover, Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. Using RNA interference to knock down both c-Cbl and Cbl-b, we were able to abolish EGFR downregulation. This knockdown had no affect on the rate of EGF-induced EGFR internalization. We found that the two Cbls accounted for total receptor ubiquitination and that while c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. Furthermore, these data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking of EGFR.