Preparation and analysis of phosphorylated proteins.
The function of cells of the immune system is regulated in part by engagement of specific receptors on the cell surface by soluble ligands or ligands presented on the surface of other cells. One of the major mechanisms by which cell-surface receptor engagement influences cellular function is by activating protein kinases and increasing the phosphorylation of critical cellular proteins. There are two major categories of protein kinases: serine/threonine kinases which phosphorylate both serine and threonine residues; and tyrosine kinases which phosphorylate tyrosine residues exclusively. By metabolically labeling cells with (32)P, as described in this unit, the phosphorylation state of cellular proteins can be analyzed and changes occurring with receptor stimulation can be examined. Phosphoamino acid analysis is also described to determine whether serine, threonine, or tyrosine residues are phosphorylated, thereby indicating which category of protein kinase is responsible. A protocol for phosphopeptide mapping is provided to allow the investigator to produce a "fingerprint" of the peptides that are phosphorylated, providing information regarding the sites of phosphorylation.
Siegel
Jeffrey N
JN
Naval Medical Research Institute, Bethesda, Maryland.
D000818:Animals; D001665:Binding Sites / Q000276:immunology; D002855:Chromatography, Thin Layer / Q000295:instrumentation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D051379:Mice; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D008024:Ligands; D010748:Phosphopeptides; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases