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Molecular & Cellular Proteomics 2:463-473, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A High-throughput Quantitative Multiplex Kinase Assay for Monitoring Information Flow in Signaling Networks

Application to Sepsis-Apoptosis^*

Kevin A. Janes, John G. Albeck, Lili X. Peng, Peter K. Sorger^,,¶, Douglas A. Lauffenburger^,,¶ and Michael B. Yaffe^,,¶,||

From the Biological Engineering Division, Department of Biology, and ^¶ Center for Cancer Research, Massachusetts Institute of Technology, 400 Main Street, Cambridge, MA 02139

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-B 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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