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

Kevin A. Janes, John Albeck, Lili X. Peng, Peter K. Sorger, Douglas A. Lauffenburger, Michael B. Yaffe

Research output: Contribution to journalArticle

84 Scopus citations

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 languageEnglish (US)
Pages (from-to)463-473
Number of pages11
JournalMolecular & cellular proteomics : MCP
Volume2
Issue number7
DOIs
StatePublished - Jan 1 2003
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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