Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1^−/− and IRS-2^−/− mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins.

胰岛素/ IGF-1的作用是由一个复杂且高度集成的信号网络驱动的。失功能研究表明，主要的胰岛素/ IGF-1受体底物（IRS）蛋白质，IRS-1和IRS-2，介导不同的生物学功能的体外和体内尽管他们高度的同源性，表明特定的信令特性。为了确定在胰岛素/ IGF-1作用的介导中IRS-1和IRS-2的差异信号传递特性的贡献机制，我们对来自野生型IRS-1的棕色前脂肪细胞进行了基于质谱（MS）的全面质谱分析^-/-和IRS-2 ^-/-处于基础和IGF-1刺激状态的小鼠。我们对细胞培养物中的氨基酸进行了稳定同位素标记（SILAC），以准确定量蛋白质磷酸化的变化。我们发现，检测到的6262个独特的磷酸化位点中约有10％受IGF-1调控。这些受调控的位点包括先前报道的胰岛素/ IGF-1信号通路的底物，以及包括核因子IX和Semaphorin-4B在内的新型底物。在计算机上预测表明蛋白激酶B（PKB），蛋白激酶C（PKC）和细胞周期蛋白依赖性激酶（CDK）是这些磷酸化事件的主要介质。重要的是，我们根据IRS-1或IRS-2的存在找到了优先的磷酸化模式，IRS-1或IRS-2与参与这些底物下游的信号转导的特定激酶组有关，例如IRS-1的PDHK1，MAPK3和PKD1，以及IRS-2的PIN1和PKC beta。总体而言，通过响应IGF-1刺激从棕色前脂肪细胞生成全面的磷酸化蛋白质组图谱，我们揭示了由特定IRS蛋白介导的常见和不同的胰岛素/ IGF-1信号事件。