Ravid Tommer, Heidinger Jill M, Gee Peter, Khan Elaine M, Goldkorn Tzipora
Signal Transduction, Department of Internal Medicine, University of California, School of Medicine, Davis, CA 95616, USA.
J Biol Chem. 2004 Aug 27;279(35):37153-62. doi: 10.1074/jbc.M403210200. Epub 2004 Jun 21.
Epidermal growth factor receptor (EGFR) controls cell growth and has a key role in tumorigenic processes. The extent of EGFR signaling is tightly regulated by post-transcriptional modifications leading to down-regulation of the levels of the receptor. Previous studies from our laboratory demonstrated that the reactive oxidant hydrogen peroxide activates the EGFR, yet, without down-regulation of the receptor levels, which results in prolonged receptor signaling. In the present study we examined the role of the E3 ligase c-Cbl, as a possible link between oxidative stress, EGFR signaling, and tumorigenic responses. First, we ectopically expressed a mutant EGFR (Tyr-1045 --> Phe) in cells lacking endogenous receptor, to determine whether the lack of phosphorylation at this site is the cause for EGFR retention at the membrane under oxidative stress, as we have previously suggested. Our findings suggest that abrogation of tyrosine 1045 phosphorylation alone is not enough to retain the EGFR at the plasma membrane under oxidative stress. Second, through the use of the Src inhibitor PP1, our findings establish EGFR movement out of the early endosomes as the exact location where c-Cbl-mediated ubiquitinylation is essential for EGFR trafficking. Finally, our studies substantiate the findings that c-Cbl-mediated ubiquitinylation is needed for degradation, but not for internalization of the EGFR in both transfection-dependent Chinese hamster ovary cells and transfection-independent A549 lung epithelial cells. These findings only begin to explain the features seen under oxidative stress, but they yield a greater understanding of the role of c-Cbl in EGFR trafficking.
表皮生长因子受体（EGFR）控制细胞生长，在肿瘤发生过程中起关键作用。EGFR信号传导的程度受到转录后修饰的严格调控，导致受体水平下调。我们实验室之前的研究表明，活性氧化剂过氧化氢可激活EGFR，但不会使受体水平下调，从而导致受体信号延长。在本研究中，我们研究了E3连接酶c-Cbl的作用，它可能是氧化应激、EGFR信号传导和肿瘤发生反应之间的一个联系。首先，我们在缺乏内源性受体的细胞中异位表达了一种突变型EGFR（Tyr-1045→Phe），以确定如我们之前所提出的，该位点磷酸化的缺失是否是氧化应激下EGFR保留在细胞膜上的原因。我们的研究结果表明，仅酪氨酸1045磷酸化的缺失不足以在氧化应激下将EGFR保留在质膜上。其次，通过使用Src抑制剂PP1，我们的研究结果确定EGFR从早期内体中移出是c-Cbl介导的泛素化对EGFR转运至关重要的确切位置。最后，我们的研究证实了在依赖转染的中国仓鼠卵巢细胞和不依赖转染的A549肺上皮细胞中，c-Cbl介导的泛素化是EGFR降解所必需的，但不是其内化所必需的。这些发现只是开始解释氧化应激下所观察到的特征，但它们使我们对c-Cbl在EGFR转运中的作用有了更深入的了解。
