A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks: application to sepsis-apoptosis.
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.
Janes
Kevin A
KA
Biological Engineering Division, Massachusetts Institute of Technology, 400 Main Street, Cambridge, MA 02139, USA.
Albeck
John G
JG
Peng
Lili X
LX
Sorger
Peter K
PK
Lauffenburger
Douglas A
DA
Yaffe
Michael B
MB
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology / Q000627:therapeutic use; D047908:Intracellular Signaling Peptides and Proteins; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000188:drug therapy / Q000503:physiopathology; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000099190:MAP-Kinase-Activated Kinase 2
D051550:I-kappa B Kinase; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D048055:Mitogen-Activated Protein Kinase 8; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D014409:Tumor Necrosis Factor-alpha; D000099190:MAP-Kinase-Activated Kinase 2