Fingerprinting of signal transduction pathways using a combination of anti-phosphotyrosine immunoprecipitations and two-dimensional polyacrylamide gel electrophoresis.
Virtually all known cellular processes involve modulation of cellular signaling pathways via changes in protein phosphorylation. With genomics efforts more than doubling the number of proteins available for analysis, a major challenge will be to identify unknown phosphoproteins as they exist in the normal or diseased intracellular environment. Recent advances in proteomic technology have made it possible to examine changes in protein expression with much greater resolution than was previously possible. In this report, we describe a rapid and reproducible method for identifying phosphoproteins upregulated in response to activation of cell surface receptors. Phosphotyrosine-containing proteins were immunoprecipitated from IFNalpha- or IL2-treated primary human lymphocyte extracts using a novel anti-phosphotyrosine immunoprecipitation technique. This technique takes advantage of differing antibody affinities for epitopes on native versus denatured proteins. Following separation from the immunopellets, phosphoproteins are resolved by two-dimensional polyacrylamide gel electrophoresis. With this method, we identified known proteins phosphorylated in response to IL2 or IFNalpha using both silver staining and Western blotting for protein detection/identification. The silver-stained immunoprecipitation profile serves as a fingerprint for phosphorylation events that occur in response to cytokine treatment. By merging these techniques with mass spectrometric microsequencing, new capabilities are achieved. It will then be possible to identify novel signaling proteins that are activated in response to a variety of stimuli, including receptor activation, disease progression, etc.
Stancato
L F
LF
Eli Lilly and Company, Research Triangle Park, NC, USA.
Petricoin
E F
EF
D015153:Blotting, Western / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D008214:Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D010449:Peptide Mapping / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology; D011233:Precipitin Tests / Q000379:methods*; D011994:Recombinant Proteins; D015398:Signal Transduction
D007376:Interleukin-2; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D007371:Interferon-gamma