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Probing the Kinome Researchers Are Eager to Map out All the Signaling Pathways Regulated by Protein Kinases

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Probing the Kinome Researchers Are Eager to Map out All the Signaling Pathways Regulated by Protein Kinases TECHNOLOGY FEATURE Identifying in vivo substrates 226 Screening libraries 228 Laying down the chips 229 Box 1: Phosphospecific antibodies 227 Box 2: Scansite 228 Probing the kinome Researchers are eager to map out all the signaling pathways regulated by protein kinases. Many new methods protocols and reagents are available to tackle this formidable task. Laura Bonetta reports. Phosphorylation is the main device a cell for these tests, including off-the-shelf and uses to control the function of its proteins. custom-made kinase kits, phosphospecific .com/nature e The responsible enzymes, protein kinases, antibodies, purified kinases, and specific regulate a myriad of processes from cell substrates and inhibitors. .natur w division in development to neuronal com- The initial kinase assays used radioac- munication in memory. In addition, many tive ATP in the reaction mixture. But the of them have been implicated in disease. trend in recent years has been to move http://ww The notorious Novartis drug Gleevec, away from using radioactivity. A research- which has had great success in treating er can now choose from a wide variety of oup chronic myeloid leukemia and gastroin- fluorescence- and luminescence-based r G testinal stromal tumors, is a kinase inhibi- assays. Molecular Devices, for example, tor. It is no surprise then that academic has a fluorescence polarization–based labs and pharmaceutical companies are kinase assay that uses its proprietary The IMAP technology from Molecular Devices lishing hot on the trail of understanding kinase IMAP technology. When fluorescently b is a homogeneous fluorescent kinase (or function. labeled peptides are incubated with a phosphatase) assay. Phosphorylation of Pu Kinases impart their signal by transfer- kinase, the phosphorylated peptides bind a fluorescent substrate is detected by its ring the γ-phosphate group from ATP to to the IMAP reagent—a trivalent metal binding to trivalent metals on nanoparticles and measuring the resulting increase in Nature a recipient serine, threonine or tyrosine with high affinity for phosphates—caus- fluorescence polarization. (Courtesy of 5 residue on a protein. The choice of target ing an increase in the polarization of the Molecular Devices.) 200 is decided by the catalytic domain of the fluorescence. “The assay is highly resistant © kinase, which usually recognizes a docking to interference and gives a strong signal,” domain on the substrate and a specific, says Richard Sportsman, vice president phosphorylated peptides causing them to short sequence of amino acids (the con- of reagents development. For a new lose the FRET signal. “We have developed sensus motif) that surround the residue to kinase of unknown specificity, Molecular a portfolio of substrates that have been be phosphorylated. Each kinase can have Devices provides the IMAP Substrate validated with over 130 kinases and have several substrates and be itself a substrate Finder products, consisting of arrays of the potential to be used to assay 85% of for other kinases. 55 different peptides in microwell-plate all kinases in Z-LYTE format,” says Chris To start to delineate the complex net- formats, which can be tested against the Armstrong, business area manager for work of pathways involving kinases, kinase. “The substrates are picked from drug discovery solutions. researchers want to match every kinase in all relevant regions of the kinome,” says Invitrogen also markets another assay a cell, representing the so-called kinome, Sportsman. “Typically customers will get based on time resolved (TR)-FRET called to specific phosphorylation sites on target 3–4 hits. They can then order the best sub- LanthaScreen, which can be used to study proteins. Products and assays to accom- strates for further testing.” not only phosphorylation by kinases but a plish this ambitious task are making their Another platform offered by a number whole variety of protein interactions. An way into research labs. of companies is based on fluorescence antibody labeled with a donor terbium resonance energy transfer (FRET)— fluorophore associates specifically with Monitoring kinase activity a detection method that relies on the phosphorylated peptide substrates labeled Not every kinase will work in an in vitro proximity of two fluorescent dye mol- with the acceptor fluorophore, resulting in assay and a peptide that is phosphorylated ecules (fluorophores) to allow the trans- an increased TR-FRET value in the reaction by a kinase in vitro does not necessarily fer of excitation from donor to acceptor mixture. “We have tried to focus on generic reflect that kinase’s physiological target. moiety. Invitrogen’s Z-LYTE kinase assay reagents, so that a researcher can build his However, kinase kits provide an efficient uses synthetic peptides labeled with both or her own assays.” says Armstrong. way to monitor the activity of a kinase donor and acceptor fluorophores. When Promega markets a luminescence- and screen compounds that will affect this a site-specific protease is added to the based system, Kinase-Glo, which mea- function. Many companies offer reagents phosphorylation reaction, it cleaves non- sures changes in ATP levels in a kinase NATURE METHODS | VOL.2 NO.3 | MARCH 2005 | 225 TECHNOLOGY FEATURE Dual laser detection Identifying in vivo substrates using the Beadlyte The holy grail of kinome analysis is to technology. As determine the physiological substrates of each bead flows all kinases to begin to map out signaling through a cuvette in a single file, a pathways. The KESTREL (kinase sub- red laser detects strate tracking and elucidation) method the identity of the offered by Kinasource identifies specific bead and a green sequences that are phosphorylated in laser quantifies vivo. Cell lysates from many different tis- the fluorescence sues are incubated with a purified active associated with kinase and the resulting phosphoproteins the analyte-bound are separated by gel electrophoresis. Any methods reporter. (Courtesy of Upstate Group LLC.) leads identified in this initial screen are then pursued in a scaled-up protocol to isolate selected proteins from the gel and determine their identity by mass spec- .com/nature e trometry. “Most researchers ask us to purify all the strong signals from the ini- .natur w tial screen,” says Axel Knebel, managing reaction. ATP from a completed kinase with a broad range of purified kinase-sub- director of Kinasource. The initial screen assay drives a reaction with luciferin and strate combinations” says Michael Curtin, typically requires 2–3 weeks and the puri- http://ww luciferase to produce oxyluciferin and light; product manager for cellular analysis. “The fication an additional month on average. the amount of light produced is inversely substrate can be a peptide, protein, lipid, “In theory the experiment is quite easy to oup correlated with kinase activity. “The nice sugar—just about anything a kinase nor- do, but, like mass spectrometry, you need r G thing about this assay is that it works mally uses as a substrate”. to accumulate the experience and skill to lishing b Pu Nature 5 200 © 226 | VOL.2 NO.3 | MARCH 2005 | NATURE METHODS TECHNOLOGY FEATURE do it,” says Knebel. Kinasource provides To enrich for proteins that are phos- offers “a simple procedure to selectively KESTREL as a service to pharmaceutical phorylated on tyrosine—the minority enrich for the tyrosine phosphopro- companies and as a mix of collaboration of all phosphorylated proteins in a cell— teome,” says Christopher Bunker, director and service to academic labs. “Academic researchers in Arthur Salomon’s group of new business development at CST. “It researchers get a much better price than the at Brown University added an immuno- avoids the limitations of IMAC methods pharmaceutical companies, but we end up precipitation step using an antibody that such as charge bias, enrichment based on on the published paper,” says Knebel. specifically recognizes proteins phos- abundance and multistep procedures.” According to Knebel the method is suc- phorylated on a tyrosine residue (Box 1) According to Bunker, the company has cessful in finding physiologically relevant before purification by IMAC1. “The goal identified nearly 4,000 phosphotyrosine substrates 80% of the time. “Some kinases is not to find the most phosphorylation sites, of which almost 70% are new. don’t bind ATP that well and cannot be sites. It is to find those associated with To make these procedures amenable to used in the assay,” he says. Another limita- signaling pathways and disease in the large-scale screens, Salomon has automat- methods tion is that KESTREL does not work well shortest amount of time,” says Salomon. ed many of the steps involved in processing for scarce or rare substrates because the “One-third of all proteins in a cell are samples and gathering data. “In one hour technique suffers from a relatively high phosphorylated, but most are not relevant we can get 300 phosphopeptide mass spec- level of background contamination. to signaling. We tried to design a method tra,” says Salomon. The bottleneck, how- .com/nature e In an effort to get around some of these to efficiently extract the most meaningful ever, is having to interpret the information. problems other researchers are finessing data by focusing on proteins phosphory- A number of algorithms are available to .natur w methods that do not require gel electro- lated on tyrosine.” match mass spectra with protein sequences phoresis. One such technique uses immo- As an alternative, the
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