by Jeffrey M. Perkel
There are 518 protein kinases in the human kinome, and each one tells a story. They are stories of cellular communication and developmental checkpoints, of chromatin remodeling and cell fate decisions—stories, in short, of keen interest to the scientific community.
Needless to say, then, protein kinases—enzymes that catalyze the ATP-dependent addition of phosphate groups to proteins on serine, threonine, or tyrosine amino acids—are a well-studied class of proteins. And it is one for which there exists a rich diversity of tools and reagents. Whether you work in academia or a drug development lab, you're bound to find an assay that suits your needs.
The classic, "gold standard" assay of kinase activity is perhaps the simplest: feed cells a radioactive phosphorus-containing compound (such as gamma-32P-ATP), apply a stimulus, and monitor changes in the phosphorylation state of your molecule of interest by, for instance, counting the radioactivity of immunoprecipitated protein. In vitro, using radiolabeled ATP, purified enzyme, and putative substrate, researchers can determine whether (and to what extent) a specific enzyme modifies a particular protein—and, from the drug development perspective, whether specific compounds inhibit that reaction.
The advantage of such an approach is it requires no specialized reagents (other than radiolabeled ATP); in other words, it is applicable to any kinase regardless of substrate. Millipore even offers a pharma-oriented Kinase Profiling Service based on this technology, measuring the specificity of inhibitor compounds against some 300 protein kinases.
Yet the method also is relatively difficult to scale for high-throughput work. Promega's SignaTECT assay circumvents that problem. Using biotinylated substrates and streptavidin-coated filters sized to fit pharma-friendly microtiter plates, SignaTECT facilitates the washing steps required to remove unincorporated label from the reaction prior to counting. Best of all, it is a broadly applicable assay, says Product Manager Mike Curtin, because it requires no specialized reagents; if you can biotinylate it, you can measure its phosphorylation.
"You can make biotinylated peptides, lipids, sugars—there is no limitation on the substrate, it is universal," says Curtin.
Nevertheless, many researchers are reluctant to use 32P (or even the lower energy 33P), both out of safety and waste disposal considerations. For these researchers, a number of non-radioactive alternatives exist.
One popular approach relies on modification of state-specific antibodies (i.e., phosphospecific antibodies). Applicable to the full range of antibody-based assays, from western blots, to immunohistochemistry, to ELISA, state-specific antibodies are just what they sound like: antibodies that specifically recognize the phosphorylated, but not the unphosphorylated, form of a protein. These antibodies can very specifically recognize a unique phosphorylation event in a particular protein, immunoreacting with the phosphorylated residues only in the context of a specific amino acid sequence.
Millipore's STAR ELISA and Cell Signaling Technologies' (CST) PathScan ELISA kits use the common sandwich assay ELISA format to measure the extent of protein phosphorylation via phosphospecific antibodies.
"It's a great way to study in vivo kinase activity, because you are measuring the actual result of kinase activity in the context of its natural environment," says Ze'ev Gechtman, group leader for molecular assays at CST.
In the case of CST's PathScan ELISA kits, says Gechtman: "We have antibodies coated on the bottom of 96-well plates. They serve to capture the target from complex mixtures like cell extracts. Unbound material is washed away, and bound material is detected with a different detection antibody." The two-antibody format "gives a very high specificity to the assay," he adds.
Similarly, LI-COR Biosciences' In-Cell Western™ Assays can detect protein phosphorylation (and quantify that event relative to total protein concentration) in a two-color immunocytochemistry assay employing both generic and phosphospecific antibodies. Cells adhered to the bottom of 96-well plates are fixed and stained with one or two primary antibodies, followed by one (or two) IRDye®-tagged secondary antibodies. These IRDye infrared dyes, in turn, are detected in one of LI-COR Biosciences' proprietary Odyssey or Aerius infrared imaging systems.
"You could do this in a western blot," concedes Harry Osterman, principal scientist at LI-COR, "but doing it in a plate offers higher throughput and increased reproducibility." And, he adds, the InCell Western assay format eliminates the need for making cell extracts, and running and blotting gels.
Some researchers prefer to run kinase reactions in vitro using recombinant kinases, for instance, to confirm that a specific enzyme acts upon a particular substrate, or for drug development screens; CST offers nearly 85 recombinant kinases, and Millipore has more than 350. "We span across the human kinome, every major family is represented," says Jeff Till, marketing director for drug discovery at Millipore.
The output of such reactions, of course, can be measured by any phosphorylation-state detection assay, whether radioactive or not. Promega's ProFluor kits fall into the latter category. ProFluor uses a fluorescent dye (rhodamine-110) covalently coupled to two kinase peptide substrates. In such a configuration, the dye is quenched. But, if the peptides are digested away with an amino-exoprotease, the dye unquenches in a burst of fluorescence. It is "a very neat technology," says Curtin: In the absence of phosphorylation, the peptides completely degrade; if the substrates are phosphorylated, the dye remains dark.
"If you have high enzyme activity, you get low fluorescence," says Curtin.
Some kinase assays measure the concentration of ATP or ADP following the reaction. Promega's Kinase-Glo kits, for example, monitor ATP concentration in vitro, while its ADP-Glo kit measures the appearance of ADP. Both quantify the end products of the reaction using a luciferase assay, in which light generation is proportional to ATP concentration.
As Curtin explains, though the two kits work in fundamentally the same manner, ADP-Glo is especially useful for relatively weak enzyme preparations, because when measuring the loss of ATP, small changes are hard to observe. "It's much easier to measure a difference of increasing from zero to one than a decrease from 100 to 99," he says.
Though amenable to basic researchers, Kinase-Glo and ADP-Glo are both one-pot in vitro assays that require at least semi-purified enzymes; thus, they are especially useful in drug development. So is Millipore's KinEASE kit, which uses phosphospecific antibodies to change the fluorescence polarization properties of a fluorescently labeled peptide substrate.
"You measure something that's tumbling in solution," says Till. "When you add an antibody, that slows the tumbling and that affects its fluorescence polarization signature."
Researchers who want a wider perspective on cell signaling can use systems such as Roche's xCELLigence RTCA (Real-Time Cell Analyzer) or MDS Analytical Technologies' CellKey. Both are label-free platforms that measure the electrical impedance—that is, disruption in current—induced by changes in cell number, migration, and morphology, many of which are induced by the action of receptor tyrosine kinases (RTKs) on the cell surface.
Though not specifically kinase assays, such systems can be used to detect the signatures of kinase activation, if you know what to look for; they are especially applicable to inhibitor studies. "RTK activation can induce both short-term cellular responses (minutes to hours), such as adhesion or cytoskeletal rearrangement, and long-term cellular responses (several hours to days), like migration/invasion or proliferation," says Shelli Kirstein, Roche field marketing manager. "These responses can easily be monitored using the xCELLigence RTCA System."
Alternatively, researchers can probe the gene expression changes that underlie observed variations in kinase activity, for instance using Applied Biosystems' TaqMan® Arrays, or SABiosciences' RT2 Profiler PCR Array system.
Applied Biosystems, part of Life Technologies Corp., has some 5,000 TaqMan®-based kinase assays available (including species other than humans), 96 of which have been bundled into its TaqMan Array Human Protein Kinase Plate and TaqMan Array Human Protein Kinase Micro Fluidic Card. The panels, says Jon Sherlock, product manager of Applied Biosystems TaqMan Arrays and Gene Signature Plates at Life Technologies, "Provide a different way to look at protein kinases." For example: "Many kinases have overlapping activities. You want to figure out which of many possible genes has a modification in mRNA expression to account for differences in protein activity."
There are other ways to monitor kinase activity, of course—from protein microarrays to cell-based imaging—and other types of kinases, too. "Lipid kinases seem to be the next big thing," says Till. Whatever your enzyme or target, one thing is certain: there's an assay format out there to test it.