Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.
J Biol Chem. 2021 Jan-Jun;296:100739. doi: 10.1016/j.jbc.2021.100739. Epub 2021 May 13.
Insulin-like growth factor (IGF) is a potent mitogen that activates the IGF receptor (IGFR)/insulin receptor substrate (IRS) axis, thus stimulating growth in normal cells and uncontrolled cell proliferation in cancer. Posttranslational modifications of IRS such as ubiquitination tightly control IGF signaling, and we previously identified IRS-1 as a potential substrate for the E3 ubiquitin ligase TRAF4 using an unbiased screen. Here we provide evidence that TRAF4-mediated ubiquitination of IRS-1 is physiologically relevant and crucial for IGF signal transduction. Through site-directed mutagenesis we found that TRAF4 promotes an atypical K29-linked ubiquitination at the C-terminal end of IRS-1. Its depletion abolishes AKT and ERK phosphorylation downstream of IGF-1 and inhibits breast cancer cell proliferation. Overexpression of TRAF4 enhances IGF1-induced IGFR-IRS-1 interaction, IRS-1 tyrosine phosphorylation, and downstream effector protein activation, whereas mutation of IRS-1 ubiquitination sites completely abolishes these effects. Altogether, our studies demonstrate that nonproteolytic ubiquitination of IRS-1 is a key step in conveying IGF-1 stimulation from IGFR to IRS-1.
胰岛素样生长因子 (IGF) 是一种有效的有丝分裂原，可激活 IGF 受体 (IGFR)/胰岛素受体底物 (IRS) 轴，从而刺激正常细胞的生长和癌症中不受控制的细胞增殖。IRS 的翻译后修饰（如泛素化）可严格控制 IGF 信号，我们之前使用无偏筛选方法鉴定了 IRS-1 是 E3 泛素连接酶 TRAF4 的潜在底物。在这里，我们提供了证据表明 TRAF4 介导的 IRS-1 泛素化在生理上是相关的，并且对于 IGF 信号转导至关重要。通过定点突变，我们发现 TRAF4 促进 IRS-1 羧基末端的非典型 K29 连接泛素化。其耗竭会阻止 IGF-1 下游 AKT 和 ERK 的磷酸化，并抑制乳腺癌细胞的增殖。TRAF4 的过表达增强了 IGF1 诱导的 IGFR-IRS-1 相互作用、IRS-1 酪氨酸磷酸化和下游效应蛋白的激活，而 IRS-1 泛素化位点的突变完全消除了这些效应。总之，我们的研究表明 IRS-1 的非蛋白水解泛素化是将 IGF-1 刺激从 IGFR 传递到 IRS-1 的关键步骤。
