Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 463-473 |
| Number of pages | 11 |
| Journal | Molecular & cellular proteomics : MCP |
| Volume | 2 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jan 1 2003 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Molecular Biology
Cite this
A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks : application to sepsis-apoptosis. / Janes, Kevin A.; Albeck, John; Peng, Lili X.; Sorger, Peter K.; Lauffenburger, Douglas A.; Yaffe, Michael B.
In: Molecular & cellular proteomics : MCP, Vol. 2, No. 7, 01.01.2003, p. 463-473.Research output: Contribution to journal › Article
}
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.
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U2 - 10.1074/mcp.M300045-MCP200
DO - 10.1074/mcp.M300045-MCP200
M3 - Article
VL - 2
SP - 463
EP - 473
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 7
ER -