Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Texting has become a commonplace communication channel for sharing information globally and in real time with a large community. In cells, phosphorylation events mirror this technique. A large number of interconnected cell signaling pathways rely on kinases (and phosphatases) to relay key information to a series of messengers, sometimes all the way from a membrane-bound surface receptor to a transcription factor.
Many of these messengers are themselves kinases, which may have several serine, threonine and tyrosine residues that can reversibly accept an inorganic phosphate (Pi). Detecting their phosphorylation states—including changes in response to disease or other perturbations, for example, and how changes may be altered by small molecules—is an important component of drug-screening programs and basic research alike.
Broadly speaking, querying cell signaling often means looking at phosphorylation or its proxy—by looking for the presence of an “activated” kinase or the result of its activity (the phosphorylated substrate) or even for a by-product of that activity (like ADP). Here is a sampling of some ways researchers are examining cell signaling by monitoring kinase activity.
Specifically?
Many screens used in pharma and biotech are biochemical, relying on a single purified or recombinant kinase to phosphorylate a substrate. A typical tool might be a time-resolved fluorescence resonance energy transfer (TR-FRET) assay that looks for changes in phosphorylation activity under different conditions, says Kurt Vogel, associate director of external R&D at Thermo Fisher Scientific. Here, “the substrate is typically a small peptide that’s labeled with a fluorophore on the end. There’s another fluorophore on the phospho-specific antibody. So when that antibody binds to the phospho-peptide, these two fluorescent labels are now in proximity to one another and can undergo FRET.”
A counter screen can be performed—essentially reversing the situation by screening panels of different kinases with a single compound—after compounds of interest are identified.
But for biomedical research directed at understanding cell signaling systems and how they’re affected by different perturbations—growth factors, toxins or (other) drugs, for example—“you need to measure the enzyme activities of signaling proteins from crude cell lysates, and that’s what’s most challenging right now,” says Steven Pelech, president and chief scientific officer of Kinexus.
The problem “is that today we still don’t actually have reliable assays for measuring the activities of specific protein kinases from crude lysates made from cells, or from tissues,” Pelech explains. Because most peptide and protein substrates are promiscuous—they can be phosphorylated by a variety of endogenous kinases—“you’re actually measuring several different protein kinases at that same time. Lots of companies sell kinase-assay kits, but they’re not actually that specific.”
Glowing reports
It used to be that lysates would be biochemically fractionated and assayed by measuring the incorporation of radioactive Pi into the cells. The assumption was that experimental and control samples could be fractionated to the level that the kinase responsible for the difference could be distinguished. Such assays are now far less popular thanks to cost, safety, disposal and regulatory considerations.
Assay formats using radioactivity have largely been replaced by luminescent assays such as Promega’s ADP-Glo™ Kinase Assay, and fluorescent assays like Thermo Fisher Scientific’s Adapta® Universal Kinase Assay and DiscoveRx’s ADP Hunter™, which detect ADP, a by-product of phosphorylation. Affinity reagent-based assays that use generic phospho-site-specific antibodies to identify phosphorylated substrate are also popular, and Thermo Fisher Scientific’s Omnia® kinase assay uses a substrate that fluoresces when phosphorylated, enabling kinetic assays.
Yet it is well known that even column-fractionated lysates typically contain kinases with overlapping specificities, Pelech points out. One work-around is to use a selective inhibitor for the kinase(s) you’re interested in, says Said Goueli, senior research fellow at Promega. It’s important to establish that the inhibitor itself is not promiscuous for the kinases you’re trying to distinguish among, and thus the difference in activity seen between before and after adding the selective inhibitor is primarily due to the targeted enzyme. Kinase promiscuity is less of an issue when working with purified materials.
Pull it down, pull it out
Most kinase assays rely on antibodies in one form or another—if not for detection and visualization then at least for capture or isolation of a specific protein for further study. The best known of these technologies are the classical Western blots and ELISAs, with “room for more advanced techniques, such as HTRF [Homogeneous Time-Resolved Fluorescence] and other TR-FRET- or bead-based technologies, to be recognized in the field of cell signaling,” says Fabienne Charrier-Savournin, head of the cell signaling group at Cisbio Bioassays. These include platforms such as LanthaScreen and AlphaLISA.
The use of antibodies for sample-population enrichment prior to mass-spectrometry analysis is another widely used methodology. Cell Signaling Technology’s (CST’s) PTMScan products and services, for example, use motif antibodies that “recognize particular post-translational modifications [PTMs] like phospho-tyrosine, phospho-serine/threonine and other PTMs to immunoprecipitate broad classes of peptide from a single sample,” notes Matt Stokes, principal scientist at CST. Some vendors instead use metal (like titanium dioxide or iron-based) affinity beads to enrich for phosphorylations.
Kinexus’ KinexTM KAM-880 antibody microarray uses 877 pan- and phosphosite-specific antibodies, in many cases enabling discrimination between the phosphorylated and nonphosphorylated states. “We sell those chips in kits for about $750—so for the cost of maybe three antibodies, you can actually get everything you need to assay 877 [epitopes],” says Pelech. Each quadruplicate-printed array is designed to query two samples in parallel, in duplicate. Antibody- and protein-based arrays are available from other vendors, as well.
Many technologies are available to dissect the nodes of a cell signaling pathway, search for mechanistic clues as to where they fit into the larger network or examine how they may be perturbed or rescued. Assays may be activity- or binding-based, cell-based or biochemical, low- or high-throughput, universal or specific, suitable for an academic lab or best outsourced to a contract provider. Chances are you can’t (or won’t want to) use just one.