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Kinome Profiling for Studying Lipopolysaccharide Signal Transduction in Human Peripheral Blood Mononuclear Cells
The DNA array technique allows comprehensive analysis of the genome and transcriptome, but the high throughput array-based assessment of intracellular signal transduction remains troublesome. The goal of this study was to test a new peptide array technology for studying the activity of all kinases o...
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Published in: | The Journal of biological chemistry 2004-11, Vol.279 (47), p.49206-49213 |
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Main Authors: | , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The DNA array technique allows comprehensive analysis of the genome and transcriptome, but the high throughput array-based
assessment of intracellular signal transduction remains troublesome. The goal of this study was to test a new peptide array
technology for studying the activity of all kinases of whole cell lysates, the kinome. Cell lysates from human peripheral
blood mononuclear cells before and after stimulation with lipopolysaccharide were used for in vitro phosphorylation with [γ- 33 P]ATP arrays consisting of 192 peptides (substrates for kinases) spotted on glass. The usefulness of peptide arrays for studying
signal transduction was demonstrated by the generation of the first comprehensive description of the temporal kinetics of
phosphorylation events induced by lipopolysaccharide stimulation. Furthermore analysis of the signals obtained suggested activation
of p21Ras by lipopolysaccharide, and this was confirmed by direct measurement of p21Ras GTP levels in lipopolysaccharide-stimulated
human peripheral blood mononuclear cells, which represents the first direct demonstration of p21Ras activation by stimulation
of a Toll receptor family member. Further confidence in the usefulness of peptide array technology for studying signal transduction
came from Western blot analysis of lipopolysaccharide-stimulated cells, which corroborated the signals obtained using peptide
arrays as well as from the demonstration that kinase inhibitors effected peptide array phosphorylation patterns consistent
with the expected action of these inhibitors. We conclude that this first metabolic array is a useful method to determine
the enzymatic activities of a large group of kinases, offering high throughput analysis of cellular metabolism and signal
transduction. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M405028200 |