Protein Protocols Protein Protocols

Analyzing Protein Phosphorylation 603
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Analyzing Protein Phosphorylation
John Colyer
1. Introduction
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.
In terms of the process of studying an individual phosphoprotein, a number of key
issues can be identified. First, one must demonstrate that phosphorylation of the protein
takes place; then define the number of sites within the primary sequence that can
be phosphorylated and by which protein kinase; identify the individual residue(s) phosphorylated;
the functional implication of phosphorylation of each site; and describe the
use of each site of phosphorylation in vivo. This chapter aims to describe the conduct
of an experiment performed to identify a protein as a phosphoprotein. In the case of
oligomeric enzymes, it will identify the subunit(s) phosphorylated by a particular
kinase. The determination of the stoichiometry of phosphorylation is also described,
which provides the first information concerning the number of phosphorylation sites
within a polypeptide. These procedures are most straightforward if one has access to
the purified protein kinase of interest and the protein substrate. In the case of the kinase,
this can be served in many instances by a number of commercial sources, but the
approach may be limited by the availability of sufficient pure protein substrate. If this
is the case, phosphorylation of a particular target as part of a complex mixture of proteins
(e.g., whole-cell extract) can be performed. Under these conditions, identification
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
603