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

Abstract

To treat complex human diseases effectively, a systemslevel 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 highthroughput 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. Molecular & Cellular Proteomics 2:463-473, 2003.Complex patterns of signal transduction arise when cells are exposed to combinations of extracellular cues that vary in onset, duration, origin, and synchrony. Cells process these cues through an interconnected network of multifunctional, redundant molecules to elicit a set of phenotypic responses that subsequently impact function at the cell, tissue, and organ level. In order to develop a molecular understanding of the complex pathophysiology underlying human diseases and utilize this information for prognosis and therapy, a systemslevel, network-biology approach should be applied to the signaling networks governing the relevant cell responses (1). This approach will require frequent temporal sampling of protein activity at critical nodes within parallel signaling pathways inside the cell in a quantitative manner to characterize the flow of information accurately. Such functional measurements are likely to be as valuable, or more valuable, than measurements of simple protein abundance. By quantitatively exploring the functional response of the signaling network to distinct extracellular cues and correlating these molecular events with phenotypic responses, one can construct predictive models of cue-signal and signal-response relationships.Evolving proteomic approaches to network biology have largely focused on measuring abundances of many proteins at only a few time points or under a limited number of experimental conditions (2). Complementary information on functional protein characteristics, such as enzyme activity, has been lacking in these systematic analyses, in large part because there do not exist quantitatively robust, high-throughput techniques that simultaneously measure multiple protein activities in cells. Initially, this type of dat...

Quantitative analysis of Akt phosphorylation and activity in response to EGF and insulin treatment

“…Kinase activity assays were performed as previously described [16]. Briefly, anti-Akt antibody (Upstate Biotech) was incubated in 96-well protein G-coated plates (Pierce) overnight.…”

“…Reaction mixtures were then transferred to a phosphocellulose filter plate and filter bound [γ 32 -P]-substrate was quantified using a scintillation counter. Linearity of the assay in each cell type has been established ( [16], Supplementary Figure 3). Count per minute readings were normalized to lysate concentrations and then to the 5 minute (for HT-29 cells) or 10 minute (for CHO-EGFR cells) value to produce the time series presented in Figures 1-4.…”

Insulin Regulates Cleavage of Procaspase-9 via Binding of X Chromosome-Linked Inhibitor of Apoptosis Protein in HT-29 Cells

“…Also, our experiments with kinase inhibitors demonstrated that the most likely pathway activated by insulin to inhibit the activation of procaspase-9 is the PI3K/Akt pathway. Janes et al (43) emphasized that in HT-29 cells treated with TNF-␣, Akt was the only kinase significantly activated over a long time period by insulin, compared with other kinases (ERK, I-B kinase, c-Jun NH 2 -terminal kinase 1, MAPKK, and MAPK-associated protein kinase 2). In addition, the activation of Akt by insulin showed a biphasic trend, which consists of early time activation and sustained activation from 4 to 24 hours (43).…”

“…Janes et al (43) emphasized that in HT-29 cells treated with TNF-␣, Akt was the only kinase significantly activated over a long time period by insulin, compared with other kinases (ERK, I-B kinase, c-Jun NH 2 -terminal kinase 1, MAPKK, and MAPK-associated protein kinase 2). In addition, the activation of Akt by insulin showed a biphasic trend, which consists of early time activation and sustained activation from 4 to 24 hours (43). By applying a PI3K inhibitor, Janes et al (43) confirmed that late-phase activation of Akt is important in decreasing apoptosis.…”

Structure‐based systems biology: a zoom lens for the cell

“…Technology platforms suitable for genomewide scale experiments are being designed to provide the time component in protein interactions. Indeed, real-time data are already available for cyclin dependent kinases during normal cell cycle progression and in response to DNA damage [32,33]. This is only the beginning and large-scale technologies will continue producing more and more reliable data in the coming years.…”