Following ligand binding, receptors activate one or more signal transduction proteins that, in turn, can affect the activity of multiple target effector proteins (see Figures 15-1 and 15-7). Understanding a signaling cascade requires the researcher to be able to quantitatively measure the activity of these signal transduction proteins. Kinases and GTP-binding proteins are found in many signaling cascades; in this section, we describe several assays used for measuring their activities.

Immunoprecipitation of Kinases Kinases function in virtually all signaling pathways. Typical mammalian cells contain a hundred or more different types of kinases, each of which is highly regulated and can phosphorylate many target proteins. Immunoprecipitation assays, a type of antibody-affinity chromatography (see Figure 3-40c), are frequently used to measure the activity of a particular kinase in a cell extract. In one version of this method, an antibody specific for the desired kinase is first incubated with small beads coated with protein A; this causes the antibody to bind to the beads via its Fc segment (see Figure 4-33). The beads are then mixed with an extract of the whole cell or of an organelle, such as the nucleus, then recovered by centrifugation and washed extensively with a salt solution to remove weakly bound proteins that are unlikely to be binding specifically to the antibody. Thus only cell proteins that specifically bind to the antibody—the kinase itself and proteins tightly bound to the kinase—are present on the beads. The beads are then incubated in a buffered solution with a substrate protein and γ-[³²P]ATP, in which only the γ phosphate of the ATP is radiolabeled. The amount of [³²P] transferred to the substrate protein is a measure of kinase activity and can be quantified either by polyacrylamide gel electrophoresis followed by autoradiography (see Figure 3-38) or by immunoprecipitation with an antibody specific for the substrate followed by measurement of the radioactivity in the immunoprecipitate. By comparing extracts from cells before and after ligand addition, for example, one can readily determine whether or not a particular kinase is activated in the signal transduction pathway triggered by that ligand.

Western Blotting with Monoclonal Antibodies Specific for a Phosphorylated Peptide We noted above that many proteins can be phosphorylated by several different kinases, usually on different serine, threonine, or tyrosine residues. Thus it is important to measure the extent of phosphorylation of a single amino acid side chain in a specific protein, usually before and after ligand addition. Antibodies play a crucial role in detecting such phosphorylation events. To generate an antibody that can recognize a specific phosphorylated amino acid in a specific protein, one first chemically synthesizes an approximately 15-amino-acid peptide that has the amino acid sequence surrounding the phosphorylated amino acid of the specific protein, with a phosphate group chemically linked to the desired serine, threonine, or tyrosine. After this peptide is coupled to an adjuvant (see Chapter 23) to increase its immunogenicity, it is used to generate a set of monoclonal antibodies (see Figure 4-6). One then selects a particular monoclonal antibody that reacts only with the phosphorylated, but not the nonphosphorylated, peptide; such an antibody generally will bind to the parent protein only when the same amino acid is phosphorylated. This specificity is possible because the antibody binds simultaneously to the phosphorylated amino acid and to side chains of adjacent amino acids. As an example of the use of such antibodies, Figure 15-10 shows that three signal transduction proteins in erythroid progenitors become phosphorylated on specific amino acid residues within 10 minutes of stimulation by varying concentrations of the hormone erythropoietin; phosphorylation, which is the first step in triggering the differentiation of these cells into red blood cells, increases with Epo concentration.

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Pull-Down Assays of GTP-Binding Proteins We’ve seen that the GTP-binding switch proteins of the GTPase superfamily cycle between an active (“on”) form with bound GTP, which modulates the activity of specific target proteins, and an inactive (“off”) form with bound GDP. The principal assay for measuring activation of this class of proteins takes advantage of the fact that each such switch protein has one or more targets to which it binds only when it has a bound GTP; the target protein usually has a specific binding domain that binds to the switch segments of that GTP-binding protein. Pull-down assays used to quantify the activation of a specific GTP-binding protein are similar to immunoprecipitation assays, except that the specific binding domain of the target protein is immobilized on small beads (Figure 15-11a). The beads are mixed with a cell extract and then recovered by centrifugation; the amount of the GTP-binding protein on the beads is quantified by Western blotting. Figure 15-11b shows that the fraction of the small GTPase Rac that has a bound GTP increases markedly after stimulation by the hormone platelet-derived growth factor (PDGF), indicating that Rac is a signal transduction protein activated by the PDGF receptor.