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 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.
Diks
Sander H
SH
Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Kok
Klaartje
K
O'Toole
Tom
T
Hommes
Daan W
DW
van Dijken
Peter
P
Joore
Jos
J
Peppelenbosch
Maikel P
MP
D000595:Amino Acid Sequence; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D040081:Protein Array Analysis; D020543:Proteome; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors
D008070:Lipopolysaccharides; D010455:Peptides; D020543:Proteome; D006160:Guanosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)