Luciferase assay optimization
To start luciferase activity assay optimization, we first expressed and purified red luciferase protein (Additional file 1: Figure S2A). After this we determined the ideal ATP concentration and pH for reaction (Fig. 1a and b), as well as the luciferin concentration (Fig. 1c) as described in methods section. As shown in in the graph in Fig. 1a, concentrations above 500 μM of ATP saturated the reaction. Based on this, we choose 1 μM ATP for all assay because it is a concentration that does not saturate the reaction, except to CK consumption assays where we used 50 μM ATP to improve the signals in the reaction.
In relation to pH titration, as shown in Fig. 1b the maximum activity of luciferase was obtained at pH 8.0, as previously reported [6, 7]. However, it still shows high intense luminescence in the whole range from pH 9 to 7, indicating that this luciferase can be used in experiments using different pHs.
Next, we tested the luciferin concentration and found that concentrations above 100 μM were saturating (Fig. 1c). Since we want to focus on measuring ATP, the substrate luciferin should not be at limiting levels and on the other hand the cost factor is also relevant. Therefore, we used 100 μM of luciferin in the other assays (Fig. 1c).
Since the emission of light by luciferase is very fast and follows a flash-like kinetics, we also analyzed the time of decay of light emission. As shown in Fig. 1d, we verified that the decay of light emission is very fast and that after 15 min no more light emission was detectable. Therefore, the measurement of the ATP concentration must be performed immediately without any pre-incubation period.
The purity of luciferase used in this method must be very high, because any contamination by proteins that consume ATP will give false positive background result. To test if there was any contamination of this kind we pre-incubated the purified luciferase for 30 min with ATP but without luciferin and compared the intensity of emitted luminescence with a reaction without previous incubation (Additional file 1: Figure S1). Had there been a contamination, the intensity of luminescence would be lower in the condition with a 30 min pre-incubation. However, both test conditions resulted in very similar luminescence intensities, thereby indicating that the purified luciferase was not contaminated with ATP-consuming proteins (see also Additional file 1: Figure S2A for purity of protein samples).
Together, these experiments demonstrate optimization of the assay conditions for red luciferase-based detection of ATP, for screening substrates and inhibitors of kinases.
Applications: A luciferase assay to detect kinase activity and to screen for kinase inhibitors
Railroad worm red-emitting luciferase, similar to firefly luciferases, emits light dependent on ATP, magnesium and luciferin. This assay is based on luminescence intensity, where the luminescence produced is directly proportional to the ATP concentration. Therefore, this assay can be used to measure the activity of any protein that consumes or produces ATP. To test this, we employed two kinases. One is the kinase NEK7, that modifies other proteins by covalently adding phosphate groups to them (phosphorylation), and the other one is creatine kinase (CK), which can transfer a phosphate group from ATP to creatine, to form phosphocreatine.
CK has four isoforms (uMtCK, sMtCK, CKB and CKM) and all of them were tested for ATP consumption. In Fig. 2 we can observe that in the absence of any kinase there is no ATP consumption and hence no decrease in luciferase light emission, even in the presence of creatine (columns 1 and 2).
In the absence of ATP, there is no luminescence emission, since luciferase requires ATP for bioluminescence (columns 3, 4; and also 7, 8, 11, 12, 15, 16, 19, 20). When we incubated CK protein isoforms with ATP but without creatine there was no decrease in light emission, indicating no variation in ATP concentration (columns 6, 10, 14, 18). However, when creatine was added, the light emission was significantly decreased to 20–35% of the initial values (compare columns 5, 9, 13, 17 to column 1). These results show that all CKs were active. Therefore, in principle this method can be efficiently used to measure ATP consumption by all four CK isoforms through a decrease of light emission in the coupled red luciferase assay.
The other kinase used to test this method was the serine/threonine protein kinase NEK7, which has roles in mitotic spindle formation, cytokinesis, and centrosome duplication, and which has been found over-expressed in breast, colorectal, laryngeal and lung cancer as well as in non-Hodgkin lymphoma. To identify substrates of NEK7 kinase [13] and to identify inhibitors, we previously performed screening assays with commercial kinase assays and kinase inhibitor libraries [14]. Here, we were interested to develop a sensitive but less costly assay, using red-emitting luciferase as a reporter enzyme in competitor methods according to the methods section above.
In Fig. 3a we can observe a significant decrease in the emitted bioluminescence intensity when we added three different substrate proteins to the NEK7 kinase in the reaction (columns 2–4), relative to the condition without the presence of protein substrate (column 1). This is a consequence of the ATP consumption by NEK7, due to its phosphorylation of substrate proteins/domains: NEK9-regulatory domain(764–976), Mat1(Full- length protein) and CC2D1A(501–940).
After establishing the assays functionality, the next step was to test previously identified inhibitors of NEK7 kinase activity [14], including Aminopurvanalol A (A9), GSK-3b inhibitor VIII (D8) and GSK-3 inhibitor XIII (E3) as well as new inhibitors, not previously tested for NEK7, such as a ATM kinase inhibitor (A3) and a ATM/ATR kinase inhibitor (A4). The reactions were performed as in the previous assay, with the addition of protein NEK9(764–976) as a confirmed protein substrate of NEK7 [13], and 100 μM of each of the different inhibitors.
As shown in Fig. 3b we can see an increase in luminescence intensity in the presence of inhibitors (columns 5–8) relative to the condition with the absence of inhibitor (column 3). These results show that it was possible to optimize the activity assays using luciferase as competitor and that this might be a new method of quantifying kinase activity with different substrates and for performing screening tests for inhibititors.
Human NEKs are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. Our new methodology can be very useful for finding promising general and specific candidate inhibitors for any kind of kinase, which then may function as scaffolds to design more potent and selective inhibitors for the treatment of different diseases.