B and T cell antigen receptors, certain receptors for immunoglobulin Fc regions and some viral proteins contain one or more copies of a approximately 26 amino acid motiftermed immunoreceptor-based tyrosine activation motif (ITAM) utilized for communication with cytoplasmic effectors during signal transduction. We propose to use matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry to directly map the sites and determine the stoichiometry of phosphorylation of the ITAM-containing transducer components of the B cell receptor complex, Ig-alpha and Ig-beta. The studies will extend preliminary findings that the tyrosines in Ig-alpha and Ig-beta ITAMS are asymmetrically phosphorylated by various Src-family kinases in vitro and findings that suggest the asymmetry results in unique biological responses of B cells. The studies will assess whether asymmetrical ligand-induced tyrosine phosphorylation occurs as a function of B cell maturity and/or antigen affinity and valency. Immature and mature B cells from transgenic 3-83 mice will be stimulated with antigen, lysed and ITAMs immunoprecipitated using anti-Ig-alpha. Following SDS-PAGE separation and electroblotting onto nitrocellulose, strips representing Ig-alpha and Ig-beta bands will be excised, in situ enzymatic digests will be performed, and peptides extracted. MALDI-TOF win be used to map phosphorylation sites by postsource decay (PSD) analysis. Protein purification and mass spectrometric parameters will be optimized using synthetic ITAM peptides and chimeric mutants.
| Farren, Matthew R; Carlson, Louise M; Netherby, Colleen S et al. (2014) Tumor-induced STAT3 signaling in myeloid cells impairs dendritic cell generation by decreasing PKC?II abundance. Sci Signal 7:ra16 |
| Koorella, Chandana; Nair, Jayakumar R; Murray, Megan E et al. (2014) Novel regulation of CD80/CD86-induced phosphatidylinositol 3-kinase signaling by NOTCH1 protein in interleukin-6 and indoleamine 2,3-dioxygenase production by dendritic cells. J Biol Chem 289:7747-62 |
