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.

Identification of an interleukin-15alpha receptor-binding site on human interleukin-15.

To identify the epitopes in human interleukin-15 (IL-15) that are responsible for binding to the interleukin-15 receptor alpha chain, antibody and receptor mapping by peptide scanning and site-directed mutagenesis was used. By using peptide scanning, we identified four regions in IL-15. The first region ((85)CKECEELEEKN(95)) is located in the C-D loop and is recognized by a set of non-inhibitory antibodies. The second region ((102)SFVHIVQMFIN(112)) is located in helix D and is recognized by two antibodies that are inhibitory of IL-15 bio-activity but not of IL-15 binding to IL-15Ralpha. The two remaining regions react with a recombinant soluble form of the IL-15Ralpha; the first ((44)LLELQVISL(52), peptide 1) corresponds to a sequence located in the B-helix and the second ((64)ENLII(68), peptide 2) to a sequence located in helix C. The latter is also contained in the epitope recognized by an antibody (monoclonal antibody B-E29) that prevents IL-15 binding to IL-15Ralpha. By site-directed mutagenesis, we confirmed that residues present in peptide 1 (Leu-45, Glu-46, Val-49, Ser-51, and Leu-52) and peptide 2 (Leu-66 and Ile-67) are involved in the binding of IL-15 to IL-15Ralpha. Furthermore, the results presented indicate that residues in the second peptide (Glu-64, Asn-65, and Ile-68) participate in IL-2Rbeta recruitment. This finding could have implications for the dynamics of receptor assembly. These results also indicate that the modes of interaction of IL-15 and IL-2 with their respective alpha chains are not completely analogous. Finally, some of the IL-15 mutants generated in this study displayed agonist or antagonist properties and may be useful as therapeutic agents.