Abstract
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 [gamma-33P]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.
Highlights
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 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
Kinome profiling could be a realistic possibility and especially interesting as the current metabolomics effort has been hampered by the lack of techniques that allow high-throughput analysis of the flow of cellular metabolism
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. 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. The above-mentioned considerations prompted us to test the usefulness of peptide arrays containing spatially addressed mammalian kinase substrates for studying the kinome in a cellular context We show that such peptide arrays allow us to make a comprehensive description of the phosphorylation events induced by lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells. The peptide array technology enabled us to identify the first example of p21Ras activation by a member of the toll receptor family
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