Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes
© Lilienthal et al; licensee BioMed Central Ltd. 2010
Received: 4 December 2009
Accepted: 20 May 2010
Published: 20 May 2010
Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources.
Phosphorylation-site specific antibodies can be used to monitor the accumulation of the phosphorylated product in kinase assays. We present a modified configuration of an enzyme-linked immunosorbent assay (ELISA)-based kinase assay by using the phosphospecific antibody as the capture antibody. This assay format allowed the detection of small amounts of phosphopeptide in mixtures with an excess of the unphosphorylated substrate peptide (10 fmol phosphorylated peptide over a background of 50 pmol unphosphorylated peptide). Consequently, low substrate turnover rates can be determined. We applied this method to the measurement of endogenous DYRK1A activity in mouse heart tissue by immunocomplex kinase assay. Furthermore, we detected DYRK1-like kinase activity in Xenopus laevis oocytes and identified this kinase as a DYRK1 isoform distinct from the Xenopus DYRK1A ortholog.
We present a non-radioactive and highly sensitive method for the measurement of endogenous activities of DYRKs in biological samples. Xenopus laevis oocytes contain an active DYRK1-related protein kinase more similar to mammalian DYRK1B than DYRK1A.
Most cellular processes are controlled by protein phosphorylation, and aberrant kinase activity has been implicated in the etiology of a wide spectrum of diseases, including cancer, chronic inflammatory disorders and neurodegeneration. Studies on protein kinases are important not only to elucidate molecular mechanisms of signal transduction, but also for drug development. Therefore, methods for measuring kinase activity and for the identification of kinase inhibitors have become increasingly important in biomedical research [1, 2].
A widely employed type of assay is based on the use of radioactively labelled ATP as phosphate donor and subsequent detection of phosphate incorporation into a protein or peptide substrate that contains the respective kinase recognition motif [3, 4]. This radiometric technique is simple and suitable for detection of protein kinase activity with high sensitivity but depends on the use of radioactive isotopes (32P or 33P). Use of radioactivity requires special handling, is associated with inherent high costs of waste disposal, and restricts the flexibility because of the short half life of 32P and 33P. Furthermore, these assays are carried out at subphysiological levels of ATP owing to the necessity of keeping ATP levels, and thus the usage of radioisotopes, within reasonable limits.
To circumvent these drawbacks, a wide variety of non-radiometric techniques have been developed to measure kinase activity, particularly for use in high throughput screening of kinase inhibitors (for recent reviews see [1, 2]. Several non-radiometric methods rely on antibodies that can distinguish phosphorylated from unphosphorylated forms of the kinase substrates . Such phosphorylation state-specific antibodies were first used by Yano et al.  to measure protein kinase activity by an ELISA method. In the original format, the in vitro-kinase reaction takes place in the wells after coating of the substrate to the surface of the microplate wells, and the phosphorylated molecules are detected with a phosphospecific antibody [6–8]. The use of biotinylated peptides allows the reaction to be performed in solution before the substrate captured on streptavidin coated plates [9, 10]. An inherent drawback of the existing ELISA-based assays is that in case of low enzymatic turnover, the large amount of unphosphorylated substrate will outcompete the phosphorylated substrate for binding to the surface of the wells. This decreases the overall sensitivity of the assay, and radiometric assays are generally preferred for detecting endogenous kinase activity.
Protein kinases of the DYRK family have been implicated in a number of important biological processes in diverse eukaryotic organisms, e.g. Pom1p in cell morphogenesis and mitotic entry in S. pombe [11, 12], MBK2 in oocyte maturation in C. elegans  and a DYRK1 isoform in Xenopus laevis oocyte maturation , minibrain (MNB) in neurogenesis in Drosophila , and DYRK1A in mammalian brain development and in neurodegeneration [16, 17]. Interestingly, alterations in neuronal development were observed in mouse models both with a selective gain or partial loss of function of Dyrk1A (for recent reviews see [17, 18]). This gene dosage effect implies that subtle changes in the activity of this DYRK family kinase can have severe consequences.
Many investigators are characterising the role of DYRKs in various biological processes or their involvement in human diseases [19–22]. For measuring the activity of DYRKs, radiometric assays are presently the standard in laboratory practice. We aimed at developing a non-radiometric assay sufficiently sensitive to measure kinase activity of endogenous DYRKs. By a modification of the existing ELISA configurations, we accomplished to reach a detection limit in the range of radiometric assays. The sensitivity of the assay was sufficient to measure the activity of DYRK1A in mouse heart. Moreover, we used the new method to characterize the activity of a DYRK1 isoform expressed in Xenopus laevis oocytes.
Development and characterization of the assay
We performed a titration experiment to determine the key parameters of the assay, i.e. the power to discriminate between the phosphorylated and unphosphorylated biotinylated tau peptide (hereafter referred to as tau207-219), the absolute lower detection limit, and the linear range of the assay. As shown in Figure 1B, the detection limit was about 5-10 fmol phosphopeptide per well. A comparable signal was obtained with 320 pmol of the unphosphorylated peptide, indicating that this combination of phosphospecific antibody and substrate peptide offers excellent selectivity for the detection of the phosphorylated substrate (~ 105-fold discrimination). Saturation of the assay was reached at about 1 pmol of phosphopeptide, but the linear plot (Figure 1C) illustrates that the useful measuring range was between 0.01 pmol and 0.1 pmol. Thus, the sensitivity of the ELISA compares well with radiometric assays, where about 0.1 pmol of phosphopeptide can be routinely detected. For comparison, we analysed the same concentrations of phosphorylated and unphosphorylated tau207-219 in the inverse configuration, in which the phosphospecific antibody was used to detect the biotinylated peptide after binding to streptavidin-coated plates (Figure 1D). This assay was much less sensitive (detection limit of 1 pmol phosphorylated peptide) and suffered from crossreaction of the antibody with the unphosphorylated peptide at concentrations greater than ~ 40 pmol per well.
Next we tested whether small amounts of the phosphopeptide can be detected in mixtures with a large excess of the unphosphorylated peptide. Although the experiment shown in Figure 1B suggested that 50-100 pmol of unphosphorylated peptide should only marginally contribute to the total signal, these concentrations reduced signal intensities obtained with 10-1000 fmol phosphopeptide (Figure 1E). We decided to apply a maximum of 50 pmol total peptide per well in subsequent experiments, and to keep this amount constant in all samples of an assay (including the standards).
Measurement of DYRK1A kinase activity
DYRK1A kinase activity in mouse heart
Phosphorylated peptide (pmol)
Activity measurements of a DYRK1-related kinase in Xenopus laevis oocytes
Characteristics of the two Xenopus DYRK1 isoforms
XenBasea gene symbol
Expression clones used
Sequence identity with hDYRK1A
Sequence identity with hDYRK1B
xDYRK1B kinase activity in Xenopus laevis oocytes
Phosphorylated peptide (pmol)
ELISA-based assay (n = 3)
Radiometric assay (n = 1)
Mean ± S.D.
0.93 ± 0.55
8.57 ± 2.60
In the present report we describe a simple and non-radiometric assay sufficiently sensitive to measure endogenous activities of DYRKs in cell and tissue lysates. We demonstrate the usefulness of the method by measuring the activity of DYRK1A in mouse heart lysate and of a novel DYRK1B-related kinase in Xenopus laevis oocytes.
The novel assay relies on the detection of the phosphorylated substrate peptide by a phosphospecific antibody in a direct sandwich ELISA configuration. Existing phospho-ELISA procedures for in vitro- kinase assays have been designed mainly for screening application of protein kinase inhibitors, where recombinant kinases are used and sensitivity is not a major issue (e.g. [7, 8, 10, 27], as well as many commercial assays). In our ELISA configuration, we use the phospho-specific antibody as a capturing antibody. This set-up has already been utilized for the detection of phosphoproteins in cellular lysates, where the sensitive detection of a small fraction of phosphoprotein is a key issue . Here we show that this configuration allows the detection of small amounts (10-100 fmol) of phosphopeptide in reaction mixtures containing a great excess of the unphosphorylated substrate. The inverse configuration of the ELISA, in which the biotinylated peptide was bound to streptavidin-coated wells, was much less sensitive with a detection limit of about 1 pmol phosphorylated peptide (Figure 1D). We have not further investigated the reason for this difference, but we speculate that the high density of the biotinylated peptide that is immobilized by the streptavidin, as compared with the few phosphorylated molecules that are captured by the phosphoantibody, impedes the binding of the detection antibody to the epitope. Further analysis of other peptide/antibody pairs will be necessary to determine whether this difference between the two configurations of the ELISA is generally valid.
Advantages of our method in comparison with other kinase assays are i) the high sensitivity ii) the possibility to use high ATP levels, iii) the avoidance of radioisotopes and iv) the simple set-up, i.e. the independence of specialized reagents (such as labelled tracers) or instruments. Apart from the plate reader, the method basically requires a high-quality phosphospecific antibody and a biotinylated substrate peptide. A drawback of ELISAs in comparison with radiometric assays is the semiquantitative nature with respect to the determination of the amount of phosphate incorporated into substrate. However, even radiometric assays of kinase activities are only rarely evaluated in a quantitative manner, because they frequently aim at the comparison of kinase activities in different samples analysed in parallel. In addition, the ELISA allows for the quantification of the DYRK activity in a given lysate by relating samples to a phosphopeptide standard. A minor limitation of the ELISA is the narrow linear range of detection, which can make it necessary to determine a suitable dilution of the reaction mixes.
We show that the present assay with tau207-219 as the substrate peptide can also be employed for measuring the activity of DYRK2, a kinase with 46% sequence identity with DYRK1A in the catalytic domain. Thr212 in tau has also been reported to be phosphorylated by other kinases such as protein kinase A (PKA) and cyclin-dependent kinase 5 (CDK5) [28, 29]. This lack of specificity precludes the use of this particular substrate for assays of a specific kinase in cell lysates, but on the other hand should allow its use for PKA and CDK5 in immunocomplex kinase assays. Obviously, the method should be applicable for other protein kinases, provided that suitable substrate peptide sequences are known and that the respective phosphoepitope-specific antibodies are available. It should be noted that the quality of the phosphospecific antibody is a critical issue, because any binding of the unphosphorylated peptide will directly affect the background and thereby limit sensitivity.
The evolutionary conservation of the DYRK family encouraged us to assess whether the ELISA could be used for measuring the activity of non-mammalian DYRKs. Given that in many species DYRKs are involved in differentiation processes [11–13, 30–33], early Xenopus development is an interesting model system to study the role of DYRKs in vertebrate embryogenesis. Here we have for the first time determined the activity of a kinase related to mammalian DYRK1B (designated xDYRK1B) in Xenopus laevis oocytes. In a previous report, an mRNA for a DYRK1-releated kinase has been identified in a microarray experiment with mRNA from Xenopus laevis oocytes and been designated "DYRK1A" . However, the probe on the array was derived from a Xenopus tropicalis expressed sequence tag (TNeu006j18, GenBank acc. AL635205) that is derived from the Dyrk1a.2 gene, which is more closely related to DYRK1B (see Table 2). Surprisingly, Qu et al.  used a polyclonal antibody directed against the C-terminus of human DYRK1A to detect a 90-kD band in lysates of matured oocytes. These results may suggest that both xDYRK1A and xDYRK1B are expressed in Xenopus oocytes. Our attempts to immunoprecipitate xDYRK1A from the oocyte lysates remained unsuccessful (data not shown), but it is possible that the monoclonal anti human DYRK1A antibody that we used (from Abnova, Taipei, Taiwan) does not recognize xDYRK1A. Further studies will be necessary to dissect the roles of xDYRK1A and xDYRK1B in Xenopus development.
The use of a phosphospecific capture antibody in ELISA-based kinase assays allows for the sensitive measurement of endogenous DYRK activity in diverse biological samples. With the help of this method, kinase activity of DYRK1B was detected in Xenopus laevis oocytes. We expect that this assay should prove useful for the study of kinase functions in various model systems.
Peptides and antibodies
A biotinylated substrate peptide mimicking the sequence around Thr212 of the tau protein (Biotin-Ttds-GSRSRT PSLPTPP-NH2, Thr212 is underlined, where Ttds is a proprietary linker) was selected from the Single Kinase Substrate Peptides commercially available from JPT Peptide Technologies (Berlin, Germany). For use in standard curves, a phosphorylated version was custom synthesized by Eurogentec (Seraing, Belgium). The optimized substrate peptide DYRKtide and the rabbit antibody directed against the C-terminus of human DYRK1B have been described previously [26, 34]. The following antibodies were obtained from commercial sources: rabbit anti TAU(pT212) antibody (Cat. No. 44-740G; Invitrogen, Carlsbad, CA), mouse monoclonal antibodies for phosphotyrosine (PY99, Santa Cruz Biotechnology, Santa Cruz, CA), FLAG epitope (FLAG-M2, Sigma, Munich, Germany) and DYRK1A (clone 7D10; Abnova, Taipei City, Taiwan), and goat GFP-specific antibody (Rockland, Gilbertsville, PA).
Preparation of mouse heart and Xenopus oocyte lysates
Hearts from TW-104 mice were weighed and 2 ml of lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% NP-40, 15% glycerol, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/mL pepstatin, 10 μg/mL leupeptin, 1 μL PMSF) were added per 400 mg of tissue. The hearts were cut into small pieces, subjected to 10 strokes of medium rpm (stage 6 out of 10) in a Potter Elvehjem homogenizer, and the homogenates were then sonified for 20 s. After centrifugation (21,000 g, 4°C, 10 minutes), the supernatant was taken and stored at -80°C. The lysates were cleared by pre-incubation for 30 minutes with 30 μL protein G agarose (EZview™ Red Protein G Affinity Gel, Sigma, Munich, Germany) and subsequent centrifugation before the supernatant was taken for immunoprecipitations. Follicle cell-free oocytes of oogenesis stages V or VI from Xenopus laevis  were kindly provided by the members of the Schmalzing group in the Institute of Pharmacology. The oocytes were lysed in 4 volumes of lysis buffer by up-and-down pipetting, and the lysates were cleared by repeated centrifugation to remove lipids and insoluble debris.
For the immunoprecipitation from heart lysates, 1 μg monoclonal anti DYRK1A or 1 μg anti-FLAG antibody were added to a volume of lysate containing 1 mg of total protein. For the oocyte experiments, the amount of lysate derived from a volume of 100 μl settled oocytes was used for immunoprecipitation with either 1 μg anti DYRK1A antibody, 1 μl anti DYRK1B serum  or 1 μl non-reacting serum (preimmune serum of the same rabbit used to raise the DYRK1B-specific serum).
Immuncomplexes were captured by over-night incubation with protein G agarose at 4°C in an end-over-end rotator. After washing the agarose beads twice with TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA) supplemented with 0.1% Igepal CA-630, twice with TBS without Igepal and once with kinase buffer, bound immunocomplexes were assayed for in vitro-kinase activity (see below). In some experiments, the affinity matrix was divided equally between two test tubes during the last washing step to allow for the direct comparison of radioactive and non-radioactive assay.
Cell culture and transfection
HeLa cells were cultured in Quantum 101 Medium for HeLa cells (PAA Laboratories, Pasching, Austria) and COS7 cells in DMEM medium supplemented with 10% fetal calf serum (PAA) at 37°C in a humidified 5% CO2 atmosphere. Transient transfections were performed using the FuGENE HD transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany). Expression clones for GFP-DYRK1A and GFP-DYRK1B (p69 splicing variant) have been described previously [26, 36]. An expression clone for untagged full length xDYRK1B (IMAGE clone 4724858) was obtained from Imagenes (Berlin, Germany).
After washing the immunoprecipitates with TBS, bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE (7%) and blotted onto nitrocellulose membranes. Immunodetection with commercial antibodies was performed according to the supplier's instructions. The DYRK1B antiserum was used at a dilution of 1:5000 for 2 h at room temperature, and the TBS for the washes following incubation with the primary antibody was supplemented with 0.1% SDS to reduce non-specific binding . Western blots were developed using horseradish peroxidase-coupled secondary antibodies and chemiluminescence detection. The Clean-Blot IP Detection Reagent (Thermo Scientific, Rockford, IL) was used as a secondary antibody when proteins were immunodetected with the same antibody used for immunoprecipitation.
Recombinant glutathione S-transferase (GST) fusion proteins of rat DYRK1A-ΔC and human DYRK2 were prepared as described previously [34, 36]. One unit of kinase activity was defined as the amount of enzyme that catalyzed the phosphorylation of 1 nmol DYRKtide per min at 30°C in kinase buffer (25 mM Hepes, pH 7.0, 5 mM MgCl2, 0.5 mM dithiothreitol).
To determine the assay sensitivity (Figure 2), reactions were performed in a total volume of 10 μl with variable amounts of the recombinant kinase, 50 μM tau207-219 and 100 μM ATP at 30°C for 30 min. Reactions were stopped by addition of 10 μl 10 mM EDTA and stored at -20°C until analysis by ELISA.
To prepare phosphorylated tau207-219 for the use in the ELISA (Figure 1), we aimed to achieve maximal phosphorylation by incubating 2.5 nmol tau207-219 with an excess of active kinase (4 mU/μL GST-DYRK1A-ΔC) in the presence of 0.82 mM ATP. The reaction mixture was incubated for 2 hours, and the same amount of kinase was supplemented after one hour. Total incorporation of phosphate was determined in a parallel reaction that was incubated in the presence of [γ-33P]ATP under identical conditions. All other kinase assays were incubated for 30 min at 30°C.
Immunocomplex kinase assays were carried out in a total reaction volume of 50 μl in kinase buffer, 100 μM ATP and 50 μM tau207-219 at 30°C for 30 min and stopped by addition of 50 μL 10 mM EDTA. Samples were stored at -20°C until evaluation by ELISA. Radiometric assays were run in the presence of [γ-33P]ATP (~ 2000 cpm/pmol). Incorporation of 33P into the biotinylated tau peptide was determined by spotting aliquots of the reaction mix onto a streptavidin membrane (SAM2 Biotin Capture Membrane, Promega, Mannheim, Germany), which was then washed as described by the manufacturer and evaluated by scintillation counting. Radiometric immunocomplex kinase assays with the substrate peptide DYRKtide (at 100 μM) were run with 10 μM ATP as described before .
Direct Sandwich ELISA
MaxiSorp F96 MicroWell™ Plates (Nunc, Langenselbold, Germany) were coated with 100 ng/well TAU(pT212) antibody at 4°C overnight. To keep the antibody consumption low, we used 100 ng/well of capturing antibody for coating in the standard protocol, although 200 ng/well gave a slightly higher readout (1.2-fold). After blocking for 1 hour with 200 μl/well 2% (w/v) bovine serum albumin (fraction V, modified Cohn, Calbiochem) in phosphate buffered saline (PBS) (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), the antigen was added in a volume of 100 μL/well and the plates were incubated for 2 h at room temperature. Bound antigen was detected with streptavidin-HRP conjugate (Pierce, Rockford, IL; 100 μL/well, 2 h at room temperature; 25 ng/ml) and subsequent incubation with 100 μL/well TMB (3,3',5,5'-tetramethylbenzidine) substrate solution until colour development was observed. Other detection reagents (HRP-linked anti-biotin antibody from CST; strepavidin-HRP from R&D systems) were less sensitive, and higher concentration of the streptavidin-HRP conjugate resulted in higher background in empty wells. The reaction was then stopped by adding 50 μl/well 1 M HCl, and plates were read at 450 nm (reference wavelength 620 nm). After each incubation step, plates were washed 4 times with PBS, pH 7.4, 0.1% Tween-20. Antibody and antigen were diluted in PBS.
ELISA format based on streptavidin-coated plates
Reacti-Bind™ Streptavidin High Binding Capacity Coated 96-Well Plates (Pierce; Rockford, IL, USA) were loaded with the indicated concentrations of the biotinylated peptides (100 μL/well) and incubated at RT for 2 h. After washing, bound phosphopeptide was detected with rabbit anti TAU(pT212) antibody (1:16.000) and HRP-linked goat anti rabbit IgG (Fc) (Pierce) (1:40.000) and TMB substrate as described above. PBS with 2% BSA and 0.1% Tween-20 was used for plate washing and for dilution of antigen and antibodies.
Data evaluation and curve fitting
Standard curves of phosphorylated tau peptide and the graphical representation of the time course shown in Figure 4 were generated by non-linear curve fitting using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
We are grateful to the members of the Schmalzing group for the gift of the Xenopus oocytes and to Ulf Soppa for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Be 1967/2-2 and Be 1967/3-1).
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