Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors
Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors
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DOI：
10.1073/pnas.0409143102
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发表时间：
2005-01-18
影响因子：
11.1
通讯作者：
Gygi, SP
中科院分区：
文献类型：
--
作者：
Ballif, BA;Roux, PP;Gygi, SP
可逆的蛋白质磷酸化是一种重要的细胞调控机制。许多蛋白质整合并受来自复杂信号信号的多个磷酸化事件的调节。同时检测和量化蛋白质所有磷酸化位点的时间变化不仅可以提供对已知生化活性的即时评估，还可以提供对分子信号机制的重要见解。在这里，我们展示了使用基于稳定同位素的定量MS来全局监控典型的丝裂原活化蛋白激酶信号通路中蛋白质玩家上复杂、有序的磷酸化事件的动力学。与活性分析和相同样本的磷酸特异性免疫印迹很好地一致，我们量化了表皮生长因子诱导的细胞外信号调节激酶(ERK)/P90核糖体S6激酶信号盒中九个磷酸化位点的变化。此外，我们监测了佛波酯刺激后ERK/P90核糖体S6激酶信号靶标结节性硬化症复合体(TSQ)肿瘤抑制因子TSC1和TSC2中14个以前未描述的事件和6个已知的磷酸化事件。通过结合使用定量磷酸化图谱和药理激酶抑制剂，我们发现了TSC2磷酸化的一条不依赖ERK、依赖蛋白激酶C的途径。这些结果建立了定量磷酸化图谱，作为一种同时识别、量化和描绘给定蛋白质上有序的磷酸化事件的动力学变化的手段，并为快速发现体内重要的磷调节机制确定了参数。
Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein kinase signaling pathway. in excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 ribosomal S6 kinase-signaling targets, the tuberous sclerosis complex (TSQ tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological kinase inhibitors we uncovered a ERK-independent, protein kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.