Wide-scale and quantitative phosphoproteomic method for analysis of bidirectional signaling within interacting B and T cells (58.11)

Abstract

Abstract Physiological interactions between T cells and antigen presenting cells (APC) induce the formation of the immunological synapse. The subsequent reversible tyrosine phosphorylation of a variety signaling proteins plays a pivotal role in regulation of T cell activation. Novel phosphoproteomic methods in mass spectrometry can facilitate and accelerate elucidation of cell signaling networks by providing a global picture of cellular phosphorylation. In this study a B and T cell line were labeled with stable isotopes of amino acids in cell culture, using the SILAC method. This labeling helped to differentiate and quantitate phosphopeptides from either type of cell through a timecourse of their interaction. Quantitative phosphoproteomic analysis revealed a wide array of newly discovered phosphorylation sites that changed significantly during B/T cell interaction. Phosphorylation sites were observed and quantified on the earliest upstream signaling components and the following downstream target proteins in both T cell and B cells (T cells: TCR-CD3 subunits, CD28, Erk, Fyn, Lck, P38, PI3K, PLCγ2, Zap-70 et al; B cells: B cell antigen receptor complex, liner protein, CD19, Fyn, Jnk, Lck, Lyn, P38, PI3K, PLCγ2, et al) .