Analysis of protein phosphorylation in human neutrophils.
Polymorphonuclear neutrophils play a key role in host defense and inflammation. Neutrophils can be activated by a variety of soluble and particulate factors, leading to an increase in the phosphorylation of numerous proteins on tyrosines, serines and threonines. Upon covalent binding of phosphates to these amino acids, the charge and conformation of the corresponding proteins are modified, generally leading to changes in protein functions such as protein-protein interactions and enzymatic activity. Protein phosphorylation in neutrophils can be studied by following protein incorporation of radiolabeled inorganic phosphate. This technique is based on labeling the intracellular ATP pool with 32P prior to applying a stimulus that induces changes in protein phosphorylation status. The proteins are extracted from the cells, immunoprecipitated or not, resolved on polyacrylamide gel electrophoresis, then transferred onto nitrocellulose membranes and visualized by means of autoradiography. Phosphorylated sites of a multisite-phosphorylated protein can be analyzed by using two-dimensional tryptic phosphopeptide mapping. This chapter describes a phosphorylation protocol and the analysis of the phosphorylation of a neutrophil protein, p47phox, by two-dimensional tryptic phosphopeptide mapping.
El-Benna
Jamel
J
INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon, Faculté de Medecine, Paris, France.
Dang
Pham My-Chan
PM
D001671:Biochemistry / Q000379:methods*; D001781:Blood Component Removal / Q000379:methods; D015153:Blotting, Western; D002469:Cell Separation; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007531:Isoflurophate / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism*; D010766:Phosphorylation; D013194:Staining and Labeling
D010750:Phosphoproteins; D007531:Isoflurophate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1