TY - JOUR
T1 - A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks
T2 - application to sepsis-apoptosis.
AU - Janes, Kevin A.
AU - Albeck, John
AU - Peng, Lili X.
AU - Sorger, Peter K.
AU - Lauffenburger, Douglas A.
AU - Yaffe, Michael B.
PY - 2003/1/1
Y1 - 2003/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0842320458&partnerID=8YFLogxK
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U2 - 10.1074/mcp.M300045-MCP200
DO - 10.1074/mcp.M300045-MCP200
M3 - Article
C2 - 12832460
AN - SCOPUS:0842320458
VL - 2
SP - 463
EP - 473
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 7
ER -