Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

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 initiation of primary tumor growth and 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 gene 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, and deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2. Moreover, acute induction of IRS2 degradation reduces CSC function, similar to the constitutive knockout of IRS2. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to elucidate IRS2 localization and function.