Arany Istvan, Faisal Amir, Nagamine Yoshikuni, Safirstein Robert L
Department of Internal Medicine, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, AR 72205, USA.
J Biol Chem. 2008 Mar 7;283(10):6110-7. doi: 10.1074/jbc.M708799200. Epub 2008 Jan 3.
The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.
在适度氧化应激下，完全激活的表皮生长因子受体（EGFR）/Ras/MEK/ERK信号通路对肾上皮细胞具有促生存作用。然而，我们和其他人已经证明，在严重氧化应激期间，在培养的肾近端小管细胞以及缺血/再灌注损伤后的肾近端小管中，激活的EGFR与ERK激活脱节，导致坏死性死亡。研究表明，衔接蛋白ShcA家族的酪氨酸磷酸化p46/52亚型将激活的EGFR与Ras和ERK的激活联系起来，而p66（shc）亚型可以抑制这种p46/52（shc）功能。在这里，我们确定严重氧化应激（在短暂激活后）会终止Ras/MEK/ERK信号通路的激活，这与ERK/JNK依赖的p66（shc）的Ser（36）磷酸化相吻合。p66（shc）的亚型特异性敲低或Ser（36）突变为Ala（而非Asp）可减轻体外严重氧化应激介导的ERK抑制和细胞死亡。此外，严重氧化应激（与支持生存作用的配体刺激和适度氧化应激不同）增加了p66（shc）与激活的EGFR和Grb2的结合。这种结合使SOS1衔接蛋白从EGFR招募的信号复合物中解离，导致Ras/MEK/ERK激活终止。值得注意的是，在体内缺血/再灌注损伤后的肾脏中也发生了p66（shc）的Ser（36）磷酸化及其与EGFR结合的增加。同时，SOS1与EGFR的结合下降，类似于体外研究结果。因此，我们在体外提出的机制提供了一种通过在体内抑制p66（shc）的Ser（36）磷酸化或敲低p66（shc）表达来减轻氧化应激诱导的细胞损伤的方法。
