A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks - Application to sepsis-apoptosis
A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks - Application to sepsis-apoptosis
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DOI：
10.1074/mcp.m300045-mcp200
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发表时间：
2003-07-01
影响因子：
7
通讯作者：
Yaffe, MB
中科院分区：
文献类型：
--
作者：
Janes, KA;Albeck, JG;Yaffe, MB
为了有效地治疗复杂的人类疾病，需要一种系统水平的方法来了解疾病状态下的环境线索，细胞内信号和细胞行为的相互作用。这种方法需要高通量，多重技术，测量细胞内信号网络中多种蛋白质活性的定量时间变化。在这里，我们描述了一个单一的微量滴定为基础的格式，同时定量的磷脂酰肌醇3-激酶途径（Akt），核因子-κ B途径（IKK），和三个核心丝裂原活化蛋白激酶途径（ERK，JNK 1，MK2）的蛋白激酶活性。与经典的低通量技术相比，这些平行的高通量检测具有严格的线性、冗余的特异性、可重复性和灵敏度。当应用于脓毒症诱导的结肠上皮细胞凋亡的模型时，该方法确定了Akt活性的晚期作为细胞存活的关键介质，其定量地有助于胰岛素作为抗凋亡线索的功效。因此，对细胞内信号网络中的平行节点进行采样，确定了胰岛素治疗人脓毒症疗效的分子机制的一部分。
To treat complex human diseases effectively, a systems-level approach is needed to understand the interplay of environmental cues, intracellular signals, and cellular behaviors that underlie disease states. This approach requires high-throughput, multiplex techniques that measure quantitative temporal variations of multiple protein activities in the intracellular signaling network. Here, we describe a single microtiter-based format that simultaneously quantifies protein kinase activities in the phosphatidylinositol 3-kinase pathway (Akt), nuclear factor-kappaB pathway (IKK), and three core mitogen-activated protein kinase pathways (ERK, JNK1, MK2). These parallel high-throughput assays are stringently linear, redundantly specific, reproducible, and sensitive compared with classical low-throughput techniques. When applied to a model of sepsis-induced colon epithelial apoptosis, this approach identified a late phase of Akt activity as a critical mediator of cell survival that quantitatively contributed to the efficacy of insulin as an anti-apoptotic cue. Thus, sampling parallel nodes in the intracellular signaling network identified part of the molecular mechanism underlying the efficacy of insulin in the treatment of human sepsis.