Monitoring Protein Phosphorylation

Four Common Methods of Detecting Protein PhosphorylationClick for larger version (44K) Signal transduction pathways comprise the cell's communications system; they transmit cues--from hormones, growth factors, or cytokines, for instance--that tell the cell to proliferate, differentiate, activate (or deactivate) a gene, or even die. As with any critical communications network, problems can arise when the lines go down or the signals get crossed, and studies have linked perturbations in signal transduction pathways with illnesses such as cancer and diabetes. Researchers therefore need reliable ways to monitor signaling cascades. The most widely studied signaling event is phosphorylation, a sort of protein on/off switch, as it were. Addition (by kinases) or removal (by phosphatases) of one or more phosphate groups to serine, threonine, or tyrosine residues can profoundly change a protein's function, modulating its activity, for instance, or causing it to interact with other proteins. But Joe Duffy, assistant professor of biology, Indiana University, Bloomington, cautions that protein phosphorylation does not automatically imply function. Turning on MAPK (mitogen-activated protein kinase), for instance, requires both phosphorylation and nuclear translocation. Historically, researchers investigated phosphorylation events biochemically, by measuring the incorporation of radio-labeled inorganic phosphate into proteins. But more recently, with dozens of biotech companies jumping into the signaling field, tedious and hazardous radioactive protocols have given way to safer and simpler alternatives--everything from phosphorylation-specific gel stains to mass spectrometry. Many of these stem from the development of an ever-increasing assortment of phospho-specific antibodies, affording researchers several angles from which to probe the modification state of specified proteins both in vitro and in vivo. METHOD MADNESS Perhaps the simplest way to observe phosphorylated proteins is to stain for them directly, using, for example, the GelCode® Phosphoprotein Staining Kit from Pierce Biotechnology, Rockford, Ill., or the Pro-Q™ Diamond Phosphoprotein Gel Stain from Molecular Probes of Eugene, Ore. Both systems obviate the need for a Western blot, but provide only a global overview of phosphorylation, as they illuminate all phosphorylated proteins. To study specific phosphoproteins, researchers rely on modification state- specific antibodies. These reagents, says Duffy, have become the workhorse of signaling laboratories, finding use in such techniques as Western blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), multiplexed bead assays, and flow cytometry. A huge and growing number of companies now supply the necessary phosphoprotein-specific antibodies (see Article Extras, at right), sometimes in conjunction with control modification-insensitive antibodies, purified enzymes, and artificial substrates. Most antibody-based approaches limit researchers to analyzing one phosphoprotein at a time. But bead assay systems, in which "sandwich" immunoassays are performed on the surface of micro-spheres rather than in microtiter plates, can be highly multiplexed. Key to all such systems is a collection of discretely identifiable beads, each of which can capture a specific protein. A number of companies, among them Bio-Rad Laboratories in Hercules, Calif., and Upstate in Waltham, Mass., offer phosphoprotein assays based on Luminex's xMAP™ bead set, a collection of up to 100 individually addressable beads. To read the results, the bead "array" is interrogated sphere by sphere in Luminex's proprietary reader, but other companies, such as BD Biosciences in San Diego, offer multiplexed bead systems that can run on any flow cytometer. Flow cytometers also can be used to monitor signaling events in vivo. "Flow cytometry offers the unique ability to rapidly analyze and correlate multiple cellular markers in individual cells within a hetero-geneous population," explains Eric Scharf, director of marketing for Cell Signaling Technology, Beverly, Mass., whose phosphospecific antibodies are sometimes used for this purpose. Some scientists forego phosphospecific antibodies in favor of mass spectrometry-based methods. Comparison of a peptide sequence (determined by peptide fragmentation during tandem mass spectrometry) in the presence and absence of phosphatase treatment, for instance, can reveal whether the peptide is phosphorylated, and on which residues. An alternative approach is to monitor the downstream effects of signaling events--the alterations in gene expression. The ultimate consequence of most signaling cascades is an altered gene expression profile, which can be studied globally, using DNA microarrays, for instance. Researchers also can use reporter genes to investigate the activation of specific pathways by focusing on the phosphorylation of specific transcription factors. Stratagene in La Jolla, Calif., has a series of PathDetect® kits, for example, that enables researchers to assess the impact of drugs, growth factors, or genes of interest on a variety of signaling pathways. Such approaches, says Duffy, often find use in drug development labs, as they can provide a quick screen of signaling activity and are amenable to automation. To be on the cutting edge of signaling research, says John Scott, professor of biochemistry at Oregon Health Sciences University in Portland, scientists must complement some or all of these techniques, with genetic, knockout, or gene expression data. In this way, they can develop a comprehensive picture of signaling pathways, and their consequences. Christine Yanicek (yanicek@optonline.net) is a freelance writer in Manville, NJ. Jeffrey M. 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Monitoring Protein Phosphorylation

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