Characterization of early autophagy signaling by quantitative phosphoproteomics

Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux. To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction.在营养匮乏条件下，自噬（autophagy）是保障持续生物合成所需底物供应的核心细胞机制。对细胞实施饥饿处理或施加雷帕霉素（rapamycin），可通过抑制MTOR信号通路以增强自噬流，从而高效诱导细胞发生自噬。为阐明自噬诱导后早期信号事件的调控机制，本研究采用定量磷酸化蛋白质组学（quantitative phosphoproteomics）技术，解析了细胞经饥饿处理与雷帕霉素处理后的时序磷酸化动态变化。本研究获取了两种诱导自噬的处理方式下，自噬诱导后前30分钟内的全面磷酸化动力学图谱，且两种处理所调控的细胞过程存在广泛差异。仅在处理2分钟后即可检测到动态磷酸化事件，这表明自噬信号通路的早期事件在诱导后迅速发生。对所得数据开展了深入的生物信息学分析，揭示了广泛参与各类细胞过程的蛋白质上的调控磷酸化位点，以及两种处理对细胞激酶组（kinome）的影响。为探究所鉴定磷酸化位点的潜在功能，我们开展了微管相关蛋白1轻链3（MAP1LC3）相互作用蛋白的筛选实验，鉴定出一组呈现动态磷酸化模式的结合伴侣蛋白。本研究所呈现的数据，为解析自噬早期诱导过程中的磷酸化事件提供了宝贵的研究资源。