Discovery and identification of serine and threonine phosphorylated proteins in activated mast cells: implications for regulation of protein synthesis in the rat basophilic leukemia mast cell line RBL-2H3.
Mast cells are important in allergic inflammation and innate immunity. Antigen-induced activation via cell-surface receptors initiates a series of intracellular signaling events, leading to the secretion of inflammatory mediators. While many of the kinases involved in this process have been defined, their substrates are generally unknown. This study aimed to identify proteins phosphorylated by serine or threonine kinases in the early stages of mast cell activation, using the rat basophilic leukemia cell line RBL-2H3 as a model system. Cells were activated via FcepsilonRI cross-linking, and lysed at different time points between 1-10 min. A novel, specific mixture of serine and threonine phospho-specific antibodies was utilized, and was shown to selectively detect proteins that were phosphorylated upon cell activation. The mixture of antibodies was used to immunoprecipitate such regulated phosphoproteins from cell lysates enriched in phosphoproteins by phospho-affinity chromatography. Immunoprecipitated proteins were analyzed by SDS-PAGE, Western blotting and liquid chromatography/mass spectrometry. With this approach, we highlighted a number of phosphoproteins, demonstrated differences in the phosphorylation/dephosphorylation rates among the regulated proteins, and identified eleven serine- or threonine-phosphorylated proteins that are substrates of kinases involved in mast cell intracellular signaling. Among these were proteins with functions in protein metabolism, including elongation factor 2, calnexin and heat shock proteins; and cell structure, including moesin, tubulin and actin. The novel approach applied in this study proved useful for the identification of kinase substrates, and can readily be extended for use in similar phosphoproteomic studies.
Olson
Fredrik J
FJ
Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia.
Ludowyke
Russell I
RI
Karlsson
Niclas G
NG