Analyzing Protein Phosphorylation

Abstract

Protein phosphorylation is a ubiquitous modification used by eukaryotic cells to alter the function of enzymes, ion channels, and other proteins in response to extracellular stimuli, or mechanical or metabolic change within the cell. In many instances, phosphorylation results in a change in the catalytic activity of the phosphoprotein, which influences one particular aspect of cellular physiology, thereby allowing the cell to respond to the initiating stimulus. A number of different residues within a protein can be modified by phosphorylation. Serine, threonine, and tyrosine residues can be phosphorylated on the side chain hydroxyl group (o-phosphoamino acids), whereas others become phosphorylated on nitrogen atoms (N-phosphoamino acids, lysine, histidine, and arginine). The former group are involved in dynamic “regulatory” functions and have been studied extensively (1), whereas the latter group may perform both structural/catalytic roles and signaling functions, the study of which has occurred more recently (2). The disparity in our understanding of the role of o- and N-phosphoamino acids is in part a consequence of the acid lability of N-phosphoamino acids, which leads to their destruction during the analysis of many phosphorylation experiments.