Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, 171 76 Stockholm, Sweden.
Department of Experimental Oncology, European Institute of Oncology, 20142 Milan, Italy.
Free Radic Biol Med. 2014 Mar;68:268-77. doi: 10.1016/j.freeradbiomed.2013.12.022. Epub 2013 Dec 27.
Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFβR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.
生长因子受体与受体结合后，会诱导活性氧（ROS）水平短暂增加，以促进通过氧化蛋白酪氨酸磷酸酶（PTP）进行信号转导。大多数研究都集中在 NADPH 氧化酶作为诱导 PTP 氧化的主要 ROS 来源。生长因子诱导的 PTP 氧化的一个潜在的额外调节剂是 p66Shc，它可以刺激线粒体 ROS 的产生。本研究通过分析 p66Shc-KO 成纤维细胞和使用 siRNA 介导的 p66Shc 下调的细胞，探讨了 p66Shc 诱导的 ROS 对 PTP 氧化和生长因子受体诱导的信号转导和迁移的贡献。在两个独立的细胞系统中分析 PDGFβR 磷酸化，发现 p66Shc 缺失或下调后 PDGFβR 磷酸化减少，这是一种部分位点选择性和抗氧化敏感的方式。p66Shc 的缺失也降低了 PDGF 诱导的 Erk、Akt、PLCγ-1 和 FAK 的下游信号转导的激活。重要的是，PDGF 刺激后，p66Shc 水平降低导致 DEP1、PTP1B 和 SHP2 的氧化减少。在 PDGF 刺激的 p66Shc-KO 成纤维细胞中，迁移反应明显减少，与 PDGFβR-Y1021 和 PLCγ-1 磷酸化减少一致，证明了这些发现的细胞生物学相关性。p66Shc 的下调也降低了 EGFR 的磷酸化和信号转导，表明 p66Shc 在受体酪氨酸激酶信号中的积极作用可能是普遍的。此外，下调线粒体过氧化氢清除剂过氧化物酶 3 增加了 PDGFβR 磷酸化，表明一般来说，线粒体 ROS 促进 PDGFβR 信号转导。因此，本研究确定了 p66Shc 在调节控制生长因子诱导的信号转导和迁移的 PTP 氧化中的先前未被认识的作用。更一般地说，该研究表明线粒体衍生的 ROS 在控制 PTP 氧化影响生长因子信号转导方面发挥了调节作用。
