Two-Dimensional Phosphoamino Acid Analysis

Abstract

Signals transmitted between cells activate intracellular signaling pathways that are precisely regulated, controlled, and organized. Ultimately, the intracellular biochemical events culminate in a specific cellular response(s). A major mechanism for intracellular signal transduction in both eukaryotic and prokaryotic cells is protein phosphorylation (1). Beside being fast and reversible, protein phosphorylation is tightly regulated and highly specific, and allows signals to be sustained or attenuated via amplification, feedback, and crosstalk (2). The specificity in signaling is, in part, achieved by two means. Catalytic specificity of protein kinases and phosphatases provides the basis for site-specific phosphorylation and dephosphorylation, respectively (3). Specific tyrosine and serine/threonine phosphorylation sites and their surrounding sequences, in turn, provide selective binding sites for conserved protein modules found in most cytoplasmic signaling molecules (2,4,5). Identifying the sites of protein phosphorylation and the nature of the phosphorylated residue is therefore paramount in the study of signal transduction.