by Caitlin Smith
Signaling pathways are everywhere. We couldn't live without them: the intracellular pathways that govern the interior workings of cells, the pathways involved in intercellular communication, nor the pathways that govern the communication of cells with their physical environment. But these nicely organized terms quickly become more complex when you look at the bigger picture. None of these pathways exists in isolation, and indeed, pathway is not the best term: “web” would be better, as many of these signaling events are connected to others – for example, the signaling cascades triggered by the activation of kinases that phosphorylate multiple proteins. This spotlight will focus on some new developments in kinase assay technology, and some areas of kinase research that are making use of these types of technology.
Kinases...
“Every day, new disorders and diseases are being linked to kinases,” says Krista Steger, director of marketing in the US for Cisbio. “Cisbio recognized the increasing interest in kinases in drug discovery efforts. Thus we greatly expanded our line of kinase kits and assay reagents in 2007 and will continue this expansion throughout 2008.”
Other companies are doing the same to meet the increasing demand for reagents and kits as kinases are increasingly shown to be involved in, or directly responsible for, major diseases such as cancer. For example, Huaibin Chen and colleagues1 showed in a recent paper in Molecular Cell that mutations in receptor tyrosine kinases (RTKs) cause various human cancers by disengaging a molecular brake that inhibits RTK activation. They found that a triad of amino acids normally constitutes a molecular brake that inhibits RTK activation, and that pathogenic mutations involving increased RTK activity result in this molecular brake being disengaged. Working with the RTK fibroblast growth-factor receptor-2 (FGFR2), which contains several pathogenic mutations, they found that the molecular brake consists of a network of hydrogen bonds that inhibits a molecular rotation, and that this inhibition keeps the kinase in an autoinhibited conformation that results from autophosphorylation. This molecular brake also appears to be conserved, as they found this same inhibitory hydrogen-bond network in many other RTKs, including all FGFRs, platelet-derived growth-factor receptors, and vascular endothelial growth-factor receptors.
Every kinase researcher must choose a method to measure kinase activity. One of the unique things about Cisbio’s assays is that they are all based on HTRF (homogeneous time resolved fluorescence), which “inherently has a number of benefits over other technologies, such as the ability to miniaturize to 1536-well, is easily automated, and robust,” adds Steger. In addition, HTRF assays are not as sensitive to buffer additives as other methods, and can be used with whole cells or complex biological samples. Cisbio’s KinEASE STK assay is a serine/threonine kinase assay that “is compatible with a wide range of ATP concentrations,” says Steger. “This is very important to researchers who want to run their kinase assays at ATP Km values.” According to Steger, the KinEASE STK is unique because “no one else has a universal serine/threonine kinase assay that measures phosphorylated substrate.” Similarly, Cisbio’s KinEASE TK assay is also compatible with a wide range of ATP concentrations, and Steger says that “no one else has a universal tyrosine assay that uses a universal substrate with a single phosphorylation site, which is key for accurate kinetics. Everyone else uses poly GT or poly GAT as a substrate.”
Cisbio also offers a new ADP assay based on HTRF to measure kinase activity. Steger explains that “most other ATP/ADP kits use luminescent-based technologies. These use the luciferase enzyme to produce light, [so] a compound that inhibits this enzyme (but not the kinase) will still look like a hit.” However, this leads to false positives that need to be eliminated or validated, which can be a long and expensive process. Most ADP assays measure ATP depletion, but Cisbio’s kit measures ADP production. “It is much easier and more sensitive to measure the increase of something (ADP) rather than the depletion of something (ATP),” says Steger, adding that “other ADP assays based on fluorescence have limited sensitivity and thus have not been widely used. Our HTRF-based kit has far better sensitivity.”
... and their targets
As important as studying the activity of kinases, is learning about their phosphoprotein targets, which can be difficult if you have to isolate phosphoproteins from complex samples. PerkinElmer’s line of Phos-tools is designed to facilitate this. Phos-tag gel and blot stains use a novel phosphosensitive chelator, which may be coupled to fluorophores for subsequent imaging. For enrichment, the Phos-trap system consists of magnetic beads coated with titania, which is selective for phosphopeptides. For some applications, Phos-trap has an advantage over other enrichment methods, such as immobilized metal ion affinity chromatoagraphy (IMAC). Phos-trap surfaces require no pre-activation, the metal oxide surfaces are stable, and there are fewer non-selective interactions with the titania (IMAC systems can have significant non-selective enrichment of highly acidic proteins). Phos-trap is particularly well-suited for challenging samples like serum.
Interactions hold the key
Studying multiple cell signaling molecules in a pathway, or even multiple pathways, immediately complicates matters; yet because most pathways are connected to others, this is not uncommon. For example, Jayne Stommel and colleagues 2 found that multiple RTKs are co-activated in glioblastoma multiforme (GBM) brain tumors. Targeting these tumors with individual RTK inhibitors shows little or no therapeutic value, but this recent paper in Science suggests that combinations of RTK inhibitors might successfully treat them. In addition, profiles of untreated primary GBM tumors using antibody arrays showed co-activation of different RTKs, suggesting that profiling individual tumors to determine which RTKs are activated might speed therapy with inhibitors.
Future directions
Like many subdisciplines of cell signaling, kinase research is moving forward at a fast pace, revealing areas of weakness that need shoring up. “In the area of kinases, there is a huge demand for intracellular kinase assays,” says Steger. “All assays are currently biochemical enzyme assays. These are challenging for a number of reasons. It is inherently difficult to work with cells due to the complex nature of the cells and the huge level of proteins that can non-specifically interfere with assay technologies. Also, kinases use a signal cascade, one acting upon another with cross-talk between pathways. If researchers are looking early on for compounds which inhibit a specific kinase, it requires creating an artificial/engineered system (overexpressing the kinase and/or substrate) for each kinase they want to look at and then hoping that the baseline level of kinase activity in the cell doesn’t swamp the assay.” Clearly there are obstacles to overcome in the study of kinases and other areas of cell signaling, but the the field continues to move forward in the lab and in the clinic. For example, clinical trials of the RTK inhibitor combination treatments are already underway, with hopes of a new successful treatment for refractory brain tumors.
References
1Chen, H. et al. “A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases.” Mol Cell 27, 717–730, 2007.
2 Stommel, J. M. et al. “Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies.” Science 318, 287–290, 2007.
