Jo Minjeong, Lee Ji-Sun, Tocheny Claire E, Lero Michael W, Bui Quyen Thu, Morgan Jennifer S, Shaw Leslie M
Department of Molecular, Cell & Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.
Department of Molecular, Cell & Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.
J Biol Chem. 2024 Nov;300(11):107796. doi: 10.1016/j.jbc.2024.107796. Epub 2024 Sep 19.
Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both the initiation of primary tumor growth and the establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin-inducible degradation (AID) sequence, 3x-FLAG tag, and mNeon-green was introduced at the N-terminus of the IRS2 protein to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.
胰岛素受体底物2（IRS2）是胰岛素（IR）和胰岛素样生长因子1（IGF-1R）受体的信号转导衔接蛋白。在乳腺癌中，IRS2通过调节癌症干细胞（CSC）功能和侵袭，促进原发性肿瘤生长的起始和继发性转移的建立。然而，IRS2如何介导其多种功能尚不清楚。我们使用CRISPR/Cas9介导的基因编辑来修饰内源性IRS2，以研究这种衔接蛋白的表达、定位和功能。在IRS2蛋白的N端引入一个包含生长素诱导降解（AID）序列、3x-FLAG标签和mNeon-绿色荧光蛋白的盒式结构，以提供对IRS2蛋白降解的快速可逆控制，并对内源性IRS2的表达和定位进行分析。对这些细胞的实时荧光成像显示，IRS2以PI3K依赖的方式响应生长调节信号在细胞质和细胞核之间穿梭。抑制核输出或删除C末端尾巴中的假定核输出序列可促进IRS2在细胞核中的保留，这表明核输出参与了IRS2细胞内定位的调节机制。此外，IRS2降解的急性诱导降低了肿瘤细胞的侵袭能力，证明了靶向这种衔接蛋白进行治疗的潜力。我们的数据突出了我们构建的内源性标记IRS2模型作为研究IRS2定位和功能工具的价值。
