A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks - Application to sepsis-apoptosis

被引:81
作者
Janes, KA
Albeck, JG
Peng, LLX
Sorger, PK
Lauffenburger, DA
Yaffe, MB
机构
[1] MIT, Ctr Canc Res, Cambridge, MA 02139 USA
[2] MIT, Dept Biol, Cambridge, MA 02139 USA
[3] MIT, Biol Engn Div, Cambridge, MA 02139 USA
关键词
INTENSIVE INSULIN THERAPY; CRITICALLY-ILL PATIENTS; ACTIVATED PROTEIN-KINASE; KAPPA-B KINASE; SEPTIC SHOCK; TRANSDUCTION; INHIBITOR; CELLS; ALPHA; JNK;
D O I
10.1074/mcp.M300045-MCP200
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:463 / 473
页数:11
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